Rin-like (Rinl) is a novel member of the RIN family of proteins that serve as guanine nucleotide exchange factors (GEFs) for Rab GTPases. Rinl preferentially binds and catalyzes GDP/GTP exchange on Rab5a and Rab22, implicating it in endocytic processes regulated by these Rab proteins. Rinl localizes to neuromuscular synapses and interacts with the receptor tyrosine kinase MuSK, a key regulator of neuromuscular synapse development. Overexpression of Rinl affects both fluid-phase and EGFR receptor-mediated endocytosis. Rinl is closely associated with the actin cytoskeleton and thus may recruit Rab5
Biological crosstalk refers to instances in which one or more components of one signal transduction pathway affects another.
This can be achieved through a number of ways with the most common form being crosstalk between proteins of signaling cascades.
In these signal transduction pathways, there are often shared components that can interact with either pathway.
A more complex instance of crosstalk can be observed with transmembrane crosstalk between the extracellular matrix (ECM) and the cytoskeleton.
This document discusses receptor tyrosine kinases (RTKs) and their role in signal transduction. It notes that RTKs are membrane proteins that transmit signals from outside the cell into the cell. When a ligand binds to an RTK, it causes the receptor to dimerize and phosphorylate itself. This activated RTK then transmits the signal by activating downstream proteins like Ras via GRB2 and Sos. Ras further transduces the signal to serine/threonine kinases and transcription factors, ultimately leading to responses in the cell like growth, differentiation, and metabolism. There are 58 known RTK subfamilies involved in various cellular processes.
Signal transduction is the process of transferring signals from outside the cell to inside the cell. It involves stimuli binding to receptors which then activate primary and secondary messengers. There are three main types of receptors: enzyme-linked receptors which undergo a conformational change to activate enzymes, ion channel-linked receptors which allow ion flow, and G-protein coupled receptors which activate heterotrimeric G proteins. Secondary messengers such as cyclic AMP then carry the signal within the cell to trigger responses like glycogen breakdown.
Signal transduction in plant defense responsesVINOD BARPA
Signal transduction a Process by which a cell converts one kind of signal into another. Plant disease resistance and susceptibility are gov¬erned by the combined genotypes of host and pathogen and depend on a complex exchange of signals and responses occurring under given environmental con¬ditions. During the long process of host-pathogen co-evolution, plants have developed various elaborate mechanisms to ward off pathogen attack. Whereas some of these defense mechanisms are preformed and provide physical and chemical barriers to hinder pathogen infection, others are induced only after pa¬thogen attack. Similar to animal immune responses, induced plant defense responses involve a network of signal transduction and the rapid activation of gene expression following pathogen infection. They do not have immune system and locomotary organs to escape environmental challenges and biotic stresses. In plant, nature has provided them some preformed and inducible defense resistance. Host recognition of invading pathogen is often determined by the so called “gene for gene” interaction between avirulence (avr) gene of pathogen and corresponding resistance (R) gene of host (Flor, 1971) which encode receptor for the recognition of specific elicitor or ligand encoded directly or indirectly by pathogen avr gene. Recent studies have revealed intriguing parallels between animal and plant defense responses as demonstrated by the structural and functional conservation of some of their signal transduction processes. Furthermore, signaling components such as G proteins, NADPH oxidase, H202, salicylic acid (SA, and aspirin), mitogen-activated protein kinases (MAPK), and transcription factors have been shown to be associated with or participate in both animal and plant defense responses, suggesting the presence of con¬served signaling pathways for host defenses in diverse higher eukaryotes.
The document discusses receptor tyrosine kinases (RTKs), a class of cell surface receptors that possess intrinsic tyrosine kinase activity. RTKs are activated through ligand binding and dimerization, which leads to autophosphorylation and downstream signaling. This signaling involves phosphorylation of proteins by RTKs and recruitment of adapter proteins, and results in cellular responses like cell division, differentiation, and motility. Common to all RTKs are an extracellular ligand-binding domain, a transmembrane domain, an intracellular tyrosine kinase domain, and regulatory domains.
This document provides an overview of receptor tyrosine kinases (RTKs) and their signaling pathways. It begins with an introduction to cell signaling and the different types of receptors, focusing on protein tyrosine kinases. RTKs are described as high-affinity cell surface receptors that activate upon ligand binding and recruit signal transduction proteins. The mechanisms of RTK activation and recruitment of proteins like Ras are explained. It further details how RTK activation of Ras leads to the Ras/MAP kinase signaling cascade, which regulates processes like cell division and differentiation. The relationships between different signaling pathways that can activate the Ras/MAP kinase cascade are also noted.
In this presentation we will look the different pathways that initiates and propagates a serial cascade events for prpper cellular response, i hope to be intrested, and please if there any suggest do not hasitate for writting a replay to me, thanks
Receptor tyrosine kinases (RTKs) are cell surface receptors that bind polypeptide growth factors, cytokines, and hormones. They regulate normal cellular processes but also play a critical role in cancer development and progression. There are approximately 20 classes of RTKs that exist as single or multimeric complexes and activate intracellular signaling pathways through autophosphorylation following ligand binding. Mutations in RTKs and their downstream effectors can lead to uncontrolled cell growth by constitutively activating growth signaling pathways. Several RTK inhibitors have been developed for cancer treatment, including those that target specific kinases as well as multi-kinase inhibitors.
Biological crosstalk refers to instances in which one or more components of one signal transduction pathway affects another.
This can be achieved through a number of ways with the most common form being crosstalk between proteins of signaling cascades.
In these signal transduction pathways, there are often shared components that can interact with either pathway.
A more complex instance of crosstalk can be observed with transmembrane crosstalk between the extracellular matrix (ECM) and the cytoskeleton.
This document discusses receptor tyrosine kinases (RTKs) and their role in signal transduction. It notes that RTKs are membrane proteins that transmit signals from outside the cell into the cell. When a ligand binds to an RTK, it causes the receptor to dimerize and phosphorylate itself. This activated RTK then transmits the signal by activating downstream proteins like Ras via GRB2 and Sos. Ras further transduces the signal to serine/threonine kinases and transcription factors, ultimately leading to responses in the cell like growth, differentiation, and metabolism. There are 58 known RTK subfamilies involved in various cellular processes.
Signal transduction is the process of transferring signals from outside the cell to inside the cell. It involves stimuli binding to receptors which then activate primary and secondary messengers. There are three main types of receptors: enzyme-linked receptors which undergo a conformational change to activate enzymes, ion channel-linked receptors which allow ion flow, and G-protein coupled receptors which activate heterotrimeric G proteins. Secondary messengers such as cyclic AMP then carry the signal within the cell to trigger responses like glycogen breakdown.
Signal transduction in plant defense responsesVINOD BARPA
Signal transduction a Process by which a cell converts one kind of signal into another. Plant disease resistance and susceptibility are gov¬erned by the combined genotypes of host and pathogen and depend on a complex exchange of signals and responses occurring under given environmental con¬ditions. During the long process of host-pathogen co-evolution, plants have developed various elaborate mechanisms to ward off pathogen attack. Whereas some of these defense mechanisms are preformed and provide physical and chemical barriers to hinder pathogen infection, others are induced only after pa¬thogen attack. Similar to animal immune responses, induced plant defense responses involve a network of signal transduction and the rapid activation of gene expression following pathogen infection. They do not have immune system and locomotary organs to escape environmental challenges and biotic stresses. In plant, nature has provided them some preformed and inducible defense resistance. Host recognition of invading pathogen is often determined by the so called “gene for gene” interaction between avirulence (avr) gene of pathogen and corresponding resistance (R) gene of host (Flor, 1971) which encode receptor for the recognition of specific elicitor or ligand encoded directly or indirectly by pathogen avr gene. Recent studies have revealed intriguing parallels between animal and plant defense responses as demonstrated by the structural and functional conservation of some of their signal transduction processes. Furthermore, signaling components such as G proteins, NADPH oxidase, H202, salicylic acid (SA, and aspirin), mitogen-activated protein kinases (MAPK), and transcription factors have been shown to be associated with or participate in both animal and plant defense responses, suggesting the presence of con¬served signaling pathways for host defenses in diverse higher eukaryotes.
The document discusses receptor tyrosine kinases (RTKs), a class of cell surface receptors that possess intrinsic tyrosine kinase activity. RTKs are activated through ligand binding and dimerization, which leads to autophosphorylation and downstream signaling. This signaling involves phosphorylation of proteins by RTKs and recruitment of adapter proteins, and results in cellular responses like cell division, differentiation, and motility. Common to all RTKs are an extracellular ligand-binding domain, a transmembrane domain, an intracellular tyrosine kinase domain, and regulatory domains.
This document provides an overview of receptor tyrosine kinases (RTKs) and their signaling pathways. It begins with an introduction to cell signaling and the different types of receptors, focusing on protein tyrosine kinases. RTKs are described as high-affinity cell surface receptors that activate upon ligand binding and recruit signal transduction proteins. The mechanisms of RTK activation and recruitment of proteins like Ras are explained. It further details how RTK activation of Ras leads to the Ras/MAP kinase signaling cascade, which regulates processes like cell division and differentiation. The relationships between different signaling pathways that can activate the Ras/MAP kinase cascade are also noted.
In this presentation we will look the different pathways that initiates and propagates a serial cascade events for prpper cellular response, i hope to be intrested, and please if there any suggest do not hasitate for writting a replay to me, thanks
Receptor tyrosine kinases (RTKs) are cell surface receptors that bind polypeptide growth factors, cytokines, and hormones. They regulate normal cellular processes but also play a critical role in cancer development and progression. There are approximately 20 classes of RTKs that exist as single or multimeric complexes and activate intracellular signaling pathways through autophosphorylation following ligand binding. Mutations in RTKs and their downstream effectors can lead to uncontrolled cell growth by constitutively activating growth signaling pathways. Several RTK inhibitors have been developed for cancer treatment, including those that target specific kinases as well as multi-kinase inhibitors.
secondary messengers and intracellular signalingGHAZALA ZIA
Introduction of different types of primary and secondary messengers including hydrophilic, hydrophobic and gaseous.
it also describes the intracellular pathway of cyclic amp, jak stat and mapk in a very simple language.
1) The study found that IKKα, like IKKβ and NEMO/IKKγ, is required for the activation of NF-κB target genes in response to TNFα and IL-1 stimulation in mouse embryonic fibroblasts.
2) DNA microarray analysis identified many known and novel NF-κB dependent target genes that were regulated by all three subunits of the IKK complex.
3) Some NF-κB target genes were dependent on the IKKs even in the absence of extracellular stimuli, suggesting the IKK complex also regulates basal levels of NF-κB activity.
Functional Analysis Of Heterologous Gpcr Signaling Pathways In Yeastbeneshjoseph
The document discusses using yeast as a model system to study heterologous G protein-coupled receptors (GPCRs). Yeast have signaling pathways that can be exploited to study GPCRs. Chimeric Gα proteins were developed to efficiently couple yeast and mammalian GPCRs. Methods like modifying receptor sequences or co-expressing receptor activity modifying proteins allow functional expression of GPCRs in yeast. This enables large-scale screens that can define receptor-G protein specificity and identify novel ligands, improving drug discovery. Studies in yeast have characterized receptors, G proteins, and their regulators like receptor kinases and RGS proteins. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast are discussed
Cell surface receptors transmit signals from outside the cell via signal transduction pathways. Receptors are divided into classes including ion channel-linked and enzyme-linked receptors. Enzyme-linked receptors contain intrinsic enzyme activity or associate with intracellular enzymes. Upon ligand binding, a conformational change activates the enzyme, initiating signaling cascades. Tyrosine kinase receptors have intrinsic kinase activity that phosphorylates tyrosines, creating docking sites and activating downstream pathways such as MAPK cascades. Mutations in these receptors and associated kinases can cause cancers and developmental disorders.
The document summarizes the MAPK (mitogen-activated protein kinase) signaling cascade downstream of insulin receptor activation. It discusses:
1) Upon insulin binding, the insulin receptor phosphorylates IRS-1, which recruits Grb2 and Sos to activate Ras.
2) Ras activates the Raf kinase, which phosphorylates and activates the MEK kinase.
3) MEK then phosphorylates and activates ERK, a MAP kinase that translocates to the nucleus.
4) Nuclear ERK phosphorylates transcription factors like Elk1, stimulating gene transcription required for cell division.
1. G-proteins bind GTP and control intracellular signaling pathways. They exist in two states - active when bound to GTP and inactive when bound to GDP.
2. G-proteins are tightly regulated by accessory proteins that modulate their cycling between GTP-bound and GDP-bound states.
3. The heterotrimeric G-proteins transmit signals from cell surface receptors to enzymes and channels. They are stimulated by receptors, act on effectors, and are regulated by nucleotide exchange and hydrolysis.
This document discusses enzyme-linked cell surface receptors, specifically receptor tyrosine kinases (RTKs). It classifies RTKs and notes they intrinsically possess tyrosine kinase activity. Upon ligand binding, RTKs dimerize and autophosphorylate, activating downstream signaling pathways like Ras-MAPK. RTK activation leads to cell proliferation, survival, and metabolism. Mutations in RTKs like HER2 and EGFR are implicated in some cancers. The document also outlines RTK signaling, including how binding partners GRB2 and Sos link RTK activation to Ras activation, transmitting the signal inside the cell.
The R T K R A S M E K Signaling Pathway By Lenard TardioDickinson Lab Lab
The RTK RAS MEK signaling pathway begins when a ligand binds to a receptor tyrosine kinase (RTK), causing it to dimerize and autophosphorylate. This creates binding sites for the adaptor protein Grb2 and associated protein SOS. The Grb2-SOS complex activates RAS via catalyzing GDP-GTP exchange. Activated RAS recruits RAF to the membrane where it is activated. RAF then phosphorylates MEK, which phosphorylates ERK. ERK phosphorylates transcription factors like ELK-1, leading to expression of genes involved in growth responses. Activated ELK-1 also stimulates a phosphatase to deactivate ERK.
G protein-coupled receptors (GPCRs) are a large family of transmembrane receptors that sense molecules outside the cell and activate intracellular signal transduction pathways. They have seven transmembrane domains and transmit signals by coupling to heterotrimeric G proteins on the inner cell surface. When an agonist binds to a GPCR, it causes a conformational change that activates the G protein, starting intracellular signaling cascades through second messengers like cAMP or IP3. Approximately half of all drugs target GPCRs, making them an important drug discovery area.
The 5' terminal uracil of let-7a is critical for the recruitment of mRNA to A...David W. Salzman
This document investigates the interaction between let-7a microRNA, Argonaute2 protein, and mRNA targets. It finds that recombinant Argonaute2 is sufficient to direct let-7a-guided cleavage of a fully complementary mRNA target in vitro. Additionally, it determines that the 5' terminal uracil of let-7a is critical for recruitment of the mRNA target to the let-7a-Argonaute2 complex. Mutation of this 5' uracil inhibits formation of the ternary let-7a-Argonaute2-mRNA complex, but does not affect formation of the binary let-7a-Argonaute2 complex. This suggests the 5' urac
1. Cellular signal transduction involves signaling molecules, receptors, and intracellular signal transduction pathways that allow cells to respond to changes in their external environment.
2. Signaling molecules like hormones, neurotransmitters, cytokines, and gas molecules bind to membrane or intracellular receptors to activate downstream signaling pathways.
3. The main intracellular signaling pathways include the cAMP/PKA pathway, Ca2+/PKC pathway, cGMP/PKG pathway, and tyrosine kinase pathways which result in phosphorylation of target proteins and regulation of gene expression.
The document discusses three main types of receptors: ligand-gated receptors, enzyme-linked receptors, and nuclear receptors. Ligand-gated receptors include nicotinic acetylcholine receptors and GABAA receptors, which act as ion channels and mediate fast synaptic transmission. Enzyme-linked receptors include tyrosine kinase receptors, JAK/STAT receptors, Toll-like receptors, and guanylyl cyclase receptors, which activate intracellular enzyme pathways to regulate processes like cell growth and inflammation. Nuclear receptors directly bind to DNA and act as transcription factors to regulate gene expression, responding to ligands like steroids, vitamins, and fatty acids.
Protein phosphorylation via protein kinases and dephosphorylation via protein phosphatases regulates many cellular processes through signal transduction cascades. G-protein coupled receptors (GPCRs) detect extracellular signals and activate intracellular heterotrimeric G proteins, which then modulate the activity of effector proteins like adenylate cyclase. When activated by GTP, Gα subunits dissociate from Gβγ complexes to control downstream signaling events. Effector activation is terminated upon GTP hydrolysis by Gα, allowing it to reassociate with Gβγ.
Signal transduction begins with ligand binding to a receptor on the cell surface. This triggers a series of molecular events within the cell through second messengers like cAMP or IP3. These second messengers activate intracellular pathways that ultimately result in changes in cell function or gene expression. The two major pathways are the cAMP pathway which activates protein kinase A, and the phosphatidylinositol pathway which activates protein kinase C through IP3 and calcium release. These second messenger systems allow cells to respond appropriately to signals from other cells.
Sugars are molecules of fundamental importance for life on earth. Sugars act as primary carriers of captured energy from the sun. Sugars not only fuel cellular carbon and energy metabolism but also pay pivotal role as signaling molecules and sugar status modulates & coordinates internal regulators that govern growth and development. The genes involved in production of carbon from photosynthesis with its utilization, mobilization and allocation in various tissues at different developmental stages are highly regulated by sugars. In most plants, sucrose (Suc) is the end product of photosynthesis for translocation from the source to heterotrophic sinks through the sieve element/companion cell complex of the phloem.
Signal transduction is the process by which a chemical or physical signal is transmitted through a cell as a series of molecular events, most commonly protein phosphorylation catalyzed by protein kinases, which ultimately results in a cellular response. Proteins responsible for detecting stimuli are generally termed receptors, although in some cases the term sensor is used.The changes elicited by ligand binding (or signal sensing) in a receptor give rise to a biochemical cascade, which is a chain of biochemical events as a signaling pathway.When signaling pathways interact with one another they form networks, which allow cellular responses to be coordinated, often by combinatorial signaling events. At the molecular level, such responses include changes in the transcription or translation of genes, and post-translational and conformational changes in proteins, as well as changes in their location. These molecular events are the basic mechanisms controlling cell growth, proliferation, metabolism and many other processes.In multicellular organisms, signal transduction pathways have evolved to regulate cell communication in a wide variety of ways.
1. PrPC associates with a multimolecular complex including LRP1 and the ganglioside GM1 within lipid rafts in the cell membrane of human neuroblastoma SK-N-BE2 cells.
2. Confocal microscopy revealed colocalization of PrPC and GM1, and immunoprecipitation showed their association. PrPC signaling involves this lipid raft-associated complex.
3. LRP1 plays a role in PrP-mediated ERK1/2 phosphorylation and signaling within lipid rafts. The complex, dependent on intact lipid rafts, is involved in neuritogenic signaling and trafficking.
Assignment on Secondary messengers and intracellular signalingDeepak Kumar
Assignment on Secondary messengers: cyclic AMP, cyclic GMP, calcium ion, inositol 1,4,5- trisphosphate, (IP3), NO, and diacylglycerol. Detailed study of following intracellular signaling pathways: cyclic AMP signaling pathway, mitogen-activated protein kinase (MAPK) signaling, Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway.
Este documento anuncia la 4a Cumbre Insurance Tech América Latina, que se llevará a cabo del 22 al 25 de agosto en Miami. La cumbre reunirá a ejecutivos líderes de seguros y proveedores de tecnología para discutir cómo aprovechar la tecnología para mejorar la experiencia del cliente, equilibrar costos y seguridad, y acelerar la capacidad móvil. Los oradores incluirán ejecutivos de Chubb, Generali, QBE, WeSURA y otros que compartirán sus experiencias
Life Sciences De-Mystified - Mark Bünger - PICNIC '10PICNIC Festival
This document provides an overview of synthetic biology and its potential applications presented by Mark Bünger of Lux Research. It begins with a brief introduction of Lux Research and their focus on emerging technologies. It then provides a high-level introduction to biology, including DNA, proteins, and how cells communicate. Applications of synthetic biology discussed include using biomass to replace petroleum products, standardizing biological parts for predictable circuits, and rapidly declining DNA sequencing costs enabling new products. Corporations, venture capital investment, and biohackers participating in synthetic biology are also mentioned. The document concludes by discussing participating in shaping the future of this emerging field through learning, action, and teaching.
secondary messengers and intracellular signalingGHAZALA ZIA
Introduction of different types of primary and secondary messengers including hydrophilic, hydrophobic and gaseous.
it also describes the intracellular pathway of cyclic amp, jak stat and mapk in a very simple language.
1) The study found that IKKα, like IKKβ and NEMO/IKKγ, is required for the activation of NF-κB target genes in response to TNFα and IL-1 stimulation in mouse embryonic fibroblasts.
2) DNA microarray analysis identified many known and novel NF-κB dependent target genes that were regulated by all three subunits of the IKK complex.
3) Some NF-κB target genes were dependent on the IKKs even in the absence of extracellular stimuli, suggesting the IKK complex also regulates basal levels of NF-κB activity.
Functional Analysis Of Heterologous Gpcr Signaling Pathways In Yeastbeneshjoseph
The document discusses using yeast as a model system to study heterologous G protein-coupled receptors (GPCRs). Yeast have signaling pathways that can be exploited to study GPCRs. Chimeric Gα proteins were developed to efficiently couple yeast and mammalian GPCRs. Methods like modifying receptor sequences or co-expressing receptor activity modifying proteins allow functional expression of GPCRs in yeast. This enables large-scale screens that can define receptor-G protein specificity and identify novel ligands, improving drug discovery. Studies in yeast have characterized receptors, G proteins, and their regulators like receptor kinases and RGS proteins. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast are discussed
Cell surface receptors transmit signals from outside the cell via signal transduction pathways. Receptors are divided into classes including ion channel-linked and enzyme-linked receptors. Enzyme-linked receptors contain intrinsic enzyme activity or associate with intracellular enzymes. Upon ligand binding, a conformational change activates the enzyme, initiating signaling cascades. Tyrosine kinase receptors have intrinsic kinase activity that phosphorylates tyrosines, creating docking sites and activating downstream pathways such as MAPK cascades. Mutations in these receptors and associated kinases can cause cancers and developmental disorders.
The document summarizes the MAPK (mitogen-activated protein kinase) signaling cascade downstream of insulin receptor activation. It discusses:
1) Upon insulin binding, the insulin receptor phosphorylates IRS-1, which recruits Grb2 and Sos to activate Ras.
2) Ras activates the Raf kinase, which phosphorylates and activates the MEK kinase.
3) MEK then phosphorylates and activates ERK, a MAP kinase that translocates to the nucleus.
4) Nuclear ERK phosphorylates transcription factors like Elk1, stimulating gene transcription required for cell division.
1. G-proteins bind GTP and control intracellular signaling pathways. They exist in two states - active when bound to GTP and inactive when bound to GDP.
2. G-proteins are tightly regulated by accessory proteins that modulate their cycling between GTP-bound and GDP-bound states.
3. The heterotrimeric G-proteins transmit signals from cell surface receptors to enzymes and channels. They are stimulated by receptors, act on effectors, and are regulated by nucleotide exchange and hydrolysis.
This document discusses enzyme-linked cell surface receptors, specifically receptor tyrosine kinases (RTKs). It classifies RTKs and notes they intrinsically possess tyrosine kinase activity. Upon ligand binding, RTKs dimerize and autophosphorylate, activating downstream signaling pathways like Ras-MAPK. RTK activation leads to cell proliferation, survival, and metabolism. Mutations in RTKs like HER2 and EGFR are implicated in some cancers. The document also outlines RTK signaling, including how binding partners GRB2 and Sos link RTK activation to Ras activation, transmitting the signal inside the cell.
The R T K R A S M E K Signaling Pathway By Lenard TardioDickinson Lab Lab
The RTK RAS MEK signaling pathway begins when a ligand binds to a receptor tyrosine kinase (RTK), causing it to dimerize and autophosphorylate. This creates binding sites for the adaptor protein Grb2 and associated protein SOS. The Grb2-SOS complex activates RAS via catalyzing GDP-GTP exchange. Activated RAS recruits RAF to the membrane where it is activated. RAF then phosphorylates MEK, which phosphorylates ERK. ERK phosphorylates transcription factors like ELK-1, leading to expression of genes involved in growth responses. Activated ELK-1 also stimulates a phosphatase to deactivate ERK.
G protein-coupled receptors (GPCRs) are a large family of transmembrane receptors that sense molecules outside the cell and activate intracellular signal transduction pathways. They have seven transmembrane domains and transmit signals by coupling to heterotrimeric G proteins on the inner cell surface. When an agonist binds to a GPCR, it causes a conformational change that activates the G protein, starting intracellular signaling cascades through second messengers like cAMP or IP3. Approximately half of all drugs target GPCRs, making them an important drug discovery area.
The 5' terminal uracil of let-7a is critical for the recruitment of mRNA to A...David W. Salzman
This document investigates the interaction between let-7a microRNA, Argonaute2 protein, and mRNA targets. It finds that recombinant Argonaute2 is sufficient to direct let-7a-guided cleavage of a fully complementary mRNA target in vitro. Additionally, it determines that the 5' terminal uracil of let-7a is critical for recruitment of the mRNA target to the let-7a-Argonaute2 complex. Mutation of this 5' uracil inhibits formation of the ternary let-7a-Argonaute2-mRNA complex, but does not affect formation of the binary let-7a-Argonaute2 complex. This suggests the 5' urac
1. Cellular signal transduction involves signaling molecules, receptors, and intracellular signal transduction pathways that allow cells to respond to changes in their external environment.
2. Signaling molecules like hormones, neurotransmitters, cytokines, and gas molecules bind to membrane or intracellular receptors to activate downstream signaling pathways.
3. The main intracellular signaling pathways include the cAMP/PKA pathway, Ca2+/PKC pathway, cGMP/PKG pathway, and tyrosine kinase pathways which result in phosphorylation of target proteins and regulation of gene expression.
The document discusses three main types of receptors: ligand-gated receptors, enzyme-linked receptors, and nuclear receptors. Ligand-gated receptors include nicotinic acetylcholine receptors and GABAA receptors, which act as ion channels and mediate fast synaptic transmission. Enzyme-linked receptors include tyrosine kinase receptors, JAK/STAT receptors, Toll-like receptors, and guanylyl cyclase receptors, which activate intracellular enzyme pathways to regulate processes like cell growth and inflammation. Nuclear receptors directly bind to DNA and act as transcription factors to regulate gene expression, responding to ligands like steroids, vitamins, and fatty acids.
Protein phosphorylation via protein kinases and dephosphorylation via protein phosphatases regulates many cellular processes through signal transduction cascades. G-protein coupled receptors (GPCRs) detect extracellular signals and activate intracellular heterotrimeric G proteins, which then modulate the activity of effector proteins like adenylate cyclase. When activated by GTP, Gα subunits dissociate from Gβγ complexes to control downstream signaling events. Effector activation is terminated upon GTP hydrolysis by Gα, allowing it to reassociate with Gβγ.
Signal transduction begins with ligand binding to a receptor on the cell surface. This triggers a series of molecular events within the cell through second messengers like cAMP or IP3. These second messengers activate intracellular pathways that ultimately result in changes in cell function or gene expression. The two major pathways are the cAMP pathway which activates protein kinase A, and the phosphatidylinositol pathway which activates protein kinase C through IP3 and calcium release. These second messenger systems allow cells to respond appropriately to signals from other cells.
Sugars are molecules of fundamental importance for life on earth. Sugars act as primary carriers of captured energy from the sun. Sugars not only fuel cellular carbon and energy metabolism but also pay pivotal role as signaling molecules and sugar status modulates & coordinates internal regulators that govern growth and development. The genes involved in production of carbon from photosynthesis with its utilization, mobilization and allocation in various tissues at different developmental stages are highly regulated by sugars. In most plants, sucrose (Suc) is the end product of photosynthesis for translocation from the source to heterotrophic sinks through the sieve element/companion cell complex of the phloem.
Signal transduction is the process by which a chemical or physical signal is transmitted through a cell as a series of molecular events, most commonly protein phosphorylation catalyzed by protein kinases, which ultimately results in a cellular response. Proteins responsible for detecting stimuli are generally termed receptors, although in some cases the term sensor is used.The changes elicited by ligand binding (or signal sensing) in a receptor give rise to a biochemical cascade, which is a chain of biochemical events as a signaling pathway.When signaling pathways interact with one another they form networks, which allow cellular responses to be coordinated, often by combinatorial signaling events. At the molecular level, such responses include changes in the transcription or translation of genes, and post-translational and conformational changes in proteins, as well as changes in their location. These molecular events are the basic mechanisms controlling cell growth, proliferation, metabolism and many other processes.In multicellular organisms, signal transduction pathways have evolved to regulate cell communication in a wide variety of ways.
1. PrPC associates with a multimolecular complex including LRP1 and the ganglioside GM1 within lipid rafts in the cell membrane of human neuroblastoma SK-N-BE2 cells.
2. Confocal microscopy revealed colocalization of PrPC and GM1, and immunoprecipitation showed their association. PrPC signaling involves this lipid raft-associated complex.
3. LRP1 plays a role in PrP-mediated ERK1/2 phosphorylation and signaling within lipid rafts. The complex, dependent on intact lipid rafts, is involved in neuritogenic signaling and trafficking.
Assignment on Secondary messengers and intracellular signalingDeepak Kumar
Assignment on Secondary messengers: cyclic AMP, cyclic GMP, calcium ion, inositol 1,4,5- trisphosphate, (IP3), NO, and diacylglycerol. Detailed study of following intracellular signaling pathways: cyclic AMP signaling pathway, mitogen-activated protein kinase (MAPK) signaling, Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway.
Este documento anuncia la 4a Cumbre Insurance Tech América Latina, que se llevará a cabo del 22 al 25 de agosto en Miami. La cumbre reunirá a ejecutivos líderes de seguros y proveedores de tecnología para discutir cómo aprovechar la tecnología para mejorar la experiencia del cliente, equilibrar costos y seguridad, y acelerar la capacidad móvil. Los oradores incluirán ejecutivos de Chubb, Generali, QBE, WeSURA y otros que compartirán sus experiencias
Life Sciences De-Mystified - Mark Bünger - PICNIC '10PICNIC Festival
This document provides an overview of synthetic biology and its potential applications presented by Mark Bünger of Lux Research. It begins with a brief introduction of Lux Research and their focus on emerging technologies. It then provides a high-level introduction to biology, including DNA, proteins, and how cells communicate. Applications of synthetic biology discussed include using biomass to replace petroleum products, standardizing biological parts for predictable circuits, and rapidly declining DNA sequencing costs enabling new products. Corporations, venture capital investment, and biohackers participating in synthetic biology are also mentioned. The document concludes by discussing participating in shaping the future of this emerging field through learning, action, and teaching.
The document summarizes the agenda for the 63rd Annual Conference of the World Affairs Council of Northern California on April 2-3, 2009. The conference will address three major challenges facing the Obama administration: the global economy, energy and the environment. Breakout sessions on the first day will discuss developing clean and efficient alternative energy strategies and the impact of the global recession on globalization, trade and protectionism. The second day will focus on strategies to address global poverty, economic development, health, hunger and education, as well as strategies for the greater Middle East. Keynote speakers will discuss crafting solutions to climate change, oil dependence and nuclear proliferation.
Grupo 1 modelo de creación de cursos a distanciaJairo Michél
El documento presenta un modelo de creación de cursos a distancia basado en el diseño de instrucción rápida (Rapid Prototyping Design, RPD). El modelo incluye fases de planificación, diseño, desarrollo, ejecución y evaluación. La planificación implica definir objetivos, estrategias de aprendizaje, necesidades de estudiantes y recursos. El diseño consiste en organizar contenidos y diseñar plantillas. El desarrollo comprende crear elementos multimedia y cargar páginas web. La ejecución implica
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Trends im Monitoring 2016 (Auszug Volker Davids)Volker Davids
Der Bereich Social Media Monitoring hat sich in den letzten Jahren kontinuierlich entwickelt und ist für viele Unternehmen schon zu einer Pflichtdisziplin geworden. Dieser Vortrag beschäftigt sich mit dem Status Quo im Bereich Social Listening und zeigt auf, welche Trends Sie nicht verpassen dürfen.
Es werden spannende Use Cases präsentiert und Ausblicke gegeben, wo die Reise hingeht – Social Selling, Customer Touchpoints, Command Centers & Co.. Sie erhalten Inspirationen, wie Sie Ihr eigenes Social Listening auf ein neues Level heben – oder als Neueinsteiger – direkt mit spannenden Einblicken anfangen können.
This document provides information and guidelines for faculty teaching distance education courses at Houston Community College (HCC). It outlines the responsibilities and requirements for faculty before, during, and at the end of the semester. New distance education faculty must complete four online courses on topics like the learning management system and copyright. Faculty must submit an application to develop new distance courses and get approvals. Syllabi must follow the official template and include key information. Faculty should select textbooks and post syllabi and CVs publicly online. Developing online courses requires organizing content and providing active learning opportunities with feedback. The distance education department provides various services to support faculty.
Gachancipá es un municipio colombiano fundado en 1612 ubicado a 30 km de Bogotá. Su economía se basa principalmente en la producción de flores, ganadería, avicultura y agricultura. Cuenta con excelentes vías de comunicación terrestre como la autopista norte y vía férrea. Algunos de sus principales atractivos turísticos son la Capilla de Santa Bárbara, la Casa Cural y la Estación del Ferrocarril reconocida como patrimonio cultural.
Este documento presenta diferentes métodos para valuar empresas, incluyendo el Cost Approach Method, DCF (Discounted Cash Flow), valuación relativa, diligencia debida, Pentagon Value de McKinsey, Leveraged Buyouts y reestructuración financiera. Explica conceptos clave como flujos de fondos libres, flujos de caja para el accionista, valor residual y horizonte de proyección.
La Unión Europea ha acordado un embargo petrolero contra Rusia en respuesta a la invasión de Ucrania. El embargo forma parte de un sexto paquete de sanciones y prohibirá la mayoría de las importaciones de petróleo ruso en la UE a finales de este año. Algunos estados miembros aún dependen en gran medida del petróleo ruso y se les ha concedido una exención, pero se espera que todo el petróleo ruso quede prohibido para fines de 2023.
El documento habla sobre la propuesta de política educativa del PAN. Propone dar mayor autonomía a las instituciones educativas, implementar un sistema de bonos escolares, e incentivar la competencia entre escuelas públicas y privadas basada en la matrícula para mejorar la calidad educativa.
La Unión Europea ha acordado un embargo petrolero contra Rusia en respuesta a la invasión de Ucrania. El embargo forma parte de un sexto paquete de sanciones y privará a Rusia de acceso a mercados clave. Sin embargo, Hungría, Eslovaquia y la República Checa recibirán exenciones temporales debido a su dependencia del petróleo ruso.
Örnek - Çıkmış ÜDS & KPDS Soru Çözümlerimuhammedhoca
The document contains 19 sentences from past ÜDS & KPDS exam questions in Turkish. The questions test vocabulary in context and grammar points like tense agreement. The summaries focus on parts of speech, tense, context and grammar structures tested in the sample questions.
Los dientes son una fuente abundante de células madre. Los tratamientos con estas células han adquirido gran relevancia en los últimos años, y hoy sabemos que preservar dichas células permite a los pacientes tener un seguro biológico para tratar enfermedades y otros problemas de salud en un futuro próximo.
Estos nuevos descubrimientos están ubicando a los dentistas a la vanguardia de servicios de salud y están cambiando el futuro de la odontología.
En 2003, el Dr. Songtao Shi investigador del National Institute of Health, descubrió células madre en los dientes primarios o de leche. Éstas células madre dentales a diferencia de otro tipo de células madre, se multiplican muy rápidamente, pueden diferenciarse en varios tipos de células y desarrollar múltiples tipos de tejidos.
Las células madre son las células que dan origen a todos los tejidos y órganos del cuerpo tales como el corazón, hígado, cerebro y la piel. Dichas células, en condiciones controladas, pueden desarrollar diferentes tipos de tejidos y hasta reparar el sistema inmunológico.
En la pulpa dental se encuentran células madre multipotenciales llamadas mesenquimatosas, las cuales tienen el potencial de diferenciarse en los siguientes tipos de células que desempeñan distintas funciones en todo el cuerpo humano:
Condrocitos: Son células que tienen la habilidad de generar cartílago, tienen una importante función en el tratamiento de la artritis y lesiones en las articulaciones.
Osteoblastos: Son células que tienen la habilidad de generar hueso y reparar destrucciones óseas por cáncer o accidentes. También pueden ser utilizadas para regenerar dientes.
Adipositos: Tienen la habilidad de reparar tejido dañado después de un ataque cardiaco o infarto. Hay algunos datos preliminares que muestran que estas células podrán tratar condiciones cardiovasculares, problemas ortopédicos y de columna vertebral, insuficiencia cardiaca congestiva, la enfermedad de Crohn y utilizarlas en cirugía plástica.
Miocitos: Estas células pueden reparar lesiones musculares y regenerar tejido muscular. Las células madre mesenquimatosas también se diferencian en cardiomiocitos lo cual nos ayuda a reparar tejido cardiaco infartado o isquémico.
Células Beta: Éste tipo de células pancreáticas producen insulina y actualmente hay muchos estudios para utilizar esta potencialidad de diferenciación y tratar a pacientes con diabetes.
Células Nerviosas: Las células madre mesenquimatosas han demostrado la capacidad de diferenciarse en células nerviosas y ya que estas células pueden formar grupos neuronales, tienen el potencial de tratar enfermedades neurodegenerativas como Parkinson, Alzheimer y parálisis cerebral.
Además de todo el potencial ya visto anteriormente, las células madre mesenquimatosas han reparado satisfactoriamente lesiones en la médula espinal y han devuelto movilidad y sensibilidad a pacientes con parálisis.
The VITAL Project aims to promote regional economic growth through matching validated ideas with experienced entrepreneurs/SMEs and fast-tracking them to market. It identifies ideas, validates them, develops business cases, identifies suitable implementers, and provides customized support including capital. Four cases are reported: EazyIce formed from an ice machine idea matched to an entrepreneur; Shnuggle fast-tracked two baby product ideas to an SME; Viltra scouted and transferred technology; and Gem Plastics licensed technology. Customized support included determining markets, establishing service models, identifying distributors, and accessing capital to commercialize ideas and technologies. The VITAL Project provides a model for open innovation and new venture creation.
9137 P7 Planificacion de Emergencias en los AeropuertosMARTIN GUTIERREZ
Este documento presenta el Manual de Servicios de Aeropuertos - Parte 7: Planificación de Emergencia en los Aeropuertos de la Organización de Aviación Civil Internacional (OACI). El manual proporciona orientación para ayudar a los estados a establecer planes de emergencia en los aeropuertos de acuerdo con las especificaciones de la OACI. Incluye información sobre los tipos de emergencias que deben considerarse, las dependencias participantes y sus funciones, y las directrices para la operación del centro de emerg
Este documento presenta varias herramientas gratuitas de Microsoft para educación, incluyendo Learning Essentials 2.0 para crear recursos educativos y administrar el aprendizaje, Microsoft Mathematics 4.0 para apoyar el aprendizaje de matemáticas, y complementos para Microsoft Word y OneNote que permiten realizar cálculos matemáticos. También presenta herramientas como Math Worksheet Generator para crear hojas de trabajo matemáticas y Complemento de Química para Word para trabajar con información química.
The document discusses the importance of strong password security and Coalition Technologies' password policies. It explains that passwords act as digital security guards controlling access and should be unique, complex, and changed regularly. The policies also outline how employees and clients should handle sharing of credentials to ensure privacy and prevent unauthorized access.
The document describes the RheoSwitch Mammalian Inducible Expression System which provides precise control of gene expression in mammalian cells. It contains three plasmids - pNEBR-R1 encodes a nuclear receptor heterodimer for regulating transcription of genes cloned into pNEBR-X1. pNEBR-X1 is used to clone the gene of interest and contains response elements regulated by the receptor. pNEBR-X1GLuc is a control plasmid expressing Gaussia luciferase. The system allows inducible expression of a gene of interest in the presence of a small molecule ligand.
The document discusses signal transduction, which is the process by which extracellular signals are converted into intracellular responses. There are six main steps: 1) synthesis and release of signaling molecules, 2) transport to target cell, 3) detection by receptor, 4) change in cell function triggered by receptor-signal complex, 5) removal of signal, and 6) termination of response. Signal transduction involves cell surface receptors and intracellular receptors that bind ligands and mediate specific cellular responses. Major types of signaling include endocrine, paracrine, and autocrine signaling.
The document summarizes different types of receptors and their classification. It discusses four main types of receptors: ligand gated ion channel receptors (inotropic), G-protein coupled receptors (metabotropic), kinase linked receptors, and nuclear receptors. It provides details about their molecular structure, signaling mechanisms, examples, and comparisons between receptor types. In summary, the document provides an overview of receptor pharmacology, classification of receptors, and their role in drug action and signaling pathways.
This study investigated protein-DNA interactions within the cAMP-responsive region of the murine steroidogenic acute regulatory (StAR) protein gene. The researchers found that:
1) Steroidogenic factor 1 (SF-1) binds to an element at -95 bp (SF1-3) in the mouse StAR promoter and this site is required for full basal promoter activity.
2) SF-1 stabilizes protein-DNA interactions at the C/EBPβ/AP-1/nuclear receptor half-site (CAN) region located at -79 bp.
3) GATA-4 binds to the StAR promoter at -68 bp and contributes to approximately 20% of the cAMP-
This document reports on a study examining how carbon monoxide (CO) induces heme oxygenase-1 (HO-1) expression and inhibits endothelial cell apoptosis triggered by endoplasmic reticulum (ER) stress. The key findings are:
1) CO activates the transcription factor Nrf2 through phosphorylation of the protein kinase R-like ER kinase (PERK), leading to increased HO-1 expression.
2) CO-induced PERK activation results in phosphorylation of eukaryotic translation initiation factor 2α and expression of activating transcription factor 4.
3) CO prevents ER stress-induced expression of X-box binding protein 1 and cleavage of activating transcription factor 6.
4) CO inhibits
This research article investigates how plant ribosome-inactivating proteins (RIPs) induce cellular stress responses in human cancer cells. The researchers found that two human cancer cell lines exposed to three RIPs - ricin, riproximin and volkensin - activated the unfolded protein response (UPR), a stress response pathway in the endoplasmic reticulum. This suggests the UPR induction better explains the cellular effects of RIPs, as apoptosis was induced even when some protein translation was still occurring due to ribosomal damage. The study provides new insights into the molecular mechanisms by which RIPs exert their toxic effects on cells.
10 nazir ahmad malla and mudasir bashir 215 plant protein kinases in signal ...Dheeraj Vasu
ABSTRACT: A protein kinase is a enzyme that modifies other proteins by adding phosphate groups to them. This results in a functional change of the target protein by changing enzyme activity, cellular location, or association with other proteins. Cells can interact to environmental fluctuations by transduction of extracellular signals, to produce intracellular responses. Membrane-impermeable signal molecules are recognized by receptors, which are localized on the plasma membrane of the cell. Binding of a ligand can result in the stimulation of an intrinsic enzymatic activity of its receptor or the modulation of a transducing protein. This review discusses the various protein kinases and their role in plants.
The document summarizes the Hippo signaling pathway. It discusses:
1) The Hippo pathway is conserved and regulates proliferation and survival. Its core is a kinase cascade with MST1/2 phosphorylating LATS1/2.
2) In Drosophila, Hippo was first discovered and controls organ size. It phosphorylates Yki which activates genes for proliferation.
3) In mammals, all core components are conserved including MST1/2, LATS1/2, and YAP/TAZ which are phosphorylated by LATS1/2 to inhibit their functions.
g protein coupled receptors, ion channels, types of receptors, wnt signalling, cell signalling, tranduction pathway, disorders regarding the signalling
Set7/9 is a lysine methyltransferase that interacts with the transcription factor Pdx1. This study found that:
1) Set7/9 methylates two lysine residues (Lys-123 and Lys-131) on Pdx1. Methylation only occurred with full-length Pdx1, indicating structural requirements.
2) Lys-131 methylation by Set7/9 augments Pdx1's transcriptional activity in luciferase reporter assays.
3) Mice with beta-cell specific deletion of Set7/9 (SetΔβ mice) showed glucose intolerance and impaired insulin secretion from islets, similar to Pdx1-deficient mice. Target genes
Beyond transcription: RNA-binding proteins as emerging regulator of plant res...BALASAHEB BIRADAR
This document provides an overview of RNA-binding proteins (RBPs) and their functions in plants, particularly in response to environmental stresses. It begins with an introduction to RBPs, describing the different types of RNA-binding domains and how RBPs bind to RNA. It then discusses the diverse functions of RBPs in processes like alternative splicing, RNA modification, polyadenylation, and mRNA localization, transport, stability, and translation. A major section explores the role of RBPs in responding to environmental stresses like temperature, osmotic stress, drought, and flooding. It presents examples of specific RBPs that are regulated under stress conditions. The document concludes with discussing future research perspectives on further characterizing plant
This document discusses receptor and signal transduction mechanisms. It begins by defining receptors as proteins that bind hormones, neurotransmitters, and other chemicals with specificity and affinity to produce cellular responses. It then describes the main functions of receptors. The document outlines the major categories of signal transduction mechanisms: ion channel linked, G protein coupled, enzyme linked, JAK-STAT binding, and nuclear receptors. For each category, it provides examples and describes the basic process of signal propagation and cellular response. In summary, it provides an overview of receptor types and the main pathways of signal transduction in cells.
G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors. Upon ligand binding, GPCRs activate intracellular G proteins that propagate signals via second messenger molecules. There are three major families of G proteins - Gs stimulates adenylate cyclase and increases cAMP, Gi inhibits adenylate cyclase and decreases cAMP, and Gq activates phospholipase C and increases intracellular calcium. Receptor tyrosine kinases (RTKs) activate intracellular signaling pathways through autophosphorylation upon ligand binding, which recruits signaling proteins containing SH2 domains. Major RTK pathways include Ras-MAPK, which regulates cell growth and proliferation.
The PI3-Kinase pathway is a complex signaling pathway that regulates many important cellular processes like growth, translation, and apoptosis. Studies in simpler organisms helped illuminate the basic pathway but it is more complex in mammals. At its core, extracellular signals activate PI3K which catalyzes the formation of PIP3 from PIP2 to transmit signals by recruiting proteins to the membrane like AKT. AKT then phosphorylates many substrates to influence processes like cell growth, survival and metabolism. Deregulation of this pathway through mutations in genes like PI3K, PTEN and AKT is strongly implicated in cancer development.
Vitamin A and its derivatives (retinoids) play an important role in cell growth, differentiation and apoptosis. They are obtained through diet as retinol, retinyl esters or beta-carotene and stored in the liver. Retinol is converted through a series of oxidation reactions to produce retinoic acid, which functions as a ligand for nuclear retinoid receptors and regulates gene expression. Retinoic acid also has non-genomic functions and can regulate pathways such as PI3K independently of nuclear receptors. Rheumatoid arthritis is associated with environmental and biological factors such as smoking, elevated tumor necrosis factor-alpha levels and abnormal B cell activity. TNF-alpha promotes inflammation and is a
This document discusses various protein modification and signaling pathways in plants. It mentions that protein kinases can phosphorylate enzymes, affecting their activity. Mitogen-activated protein kinase (MAPK) cascades and 14-3-3 proteins are involved in phosphorylation signaling. O-linked N-acetylglucosamine and ubiquitination are other types of post-translational modifications. Lipoxygenase pathways produce oxylipins like jasmonates that regulate defenses against pathogens. Sphingolipids also play a role in resistance responses.
1) RAGE is a receptor that binds various ligands, including AGEs, HMGB1, S100 proteins, and amyloid beta.
2) These ligands bind to the positively charged V1 domain of RAGE and induce intracellular signaling pathways.
3) Binding of ligands to RAGE activates cell signaling pathways involved in processes like cell migration and transcription. This signaling has roles in both normal physiology and disease pathology where inflammation is involved.
The document summarizes several important signaling pathways involved in regulating gene expression, including the TGF-β, JAK-STAT, and Ras-MAPK pathways. The TGF-β pathway involves TGF-β ligands binding to serine/threonine kinase receptors, activating R-Smad transcription factors. The JAK-STAT pathway involves cytokine receptors associating with JAK kinases; ligand binding activates JAKs to phosphorylate and activate STAT transcription factors. The Ras-MAPK pathway involves receptor tyrosine kinases activating the Ras GTPase, which activates a kinase cascade culminating in MAPK activation of gene transcription.
2. RIN proteins have been proposed as regulators of early Rab5a-
dependent endocytosis due to their binding properties for Rab5a and
their Rab5a-specific GEF activity. RIN1 stimulates Rab5a-dependent
endosome fusion and ligand-mediated endocytosis of the EGF-receptor
(EGFR), the insulin receptor and the EphA4 receptor [12–14]. In
addition, RIN3 has been shown to interact with amphiphysin II, which
is involved in clathrin- and dynamin-dependent endocytosis [15]. These
observations are of special interest since increasing evidence supports a
close interplay between signaling and endocytosis of receptor tyrosine
kinases (RTK). In particular, it was shown that receptors remain active
within endosomes [16]. This led to the hypothesis that membrane
trafficking could control signal transduction. Studies on different RTKs
have now demonstrated that there are two main types of functions that
receptor trafficking has in regulating receptor signaling: it controls the
magnitude of the response or it controls the specificity of the response
[17].
Here we report the identification of a novel RIN family member,
Rin-like (Rinl), which was isolated as interaction partner of the
muscle-specific RTK MuSK. Signal transduction events induced by
MuSK are crucially linked to the formation of the neuromuscular
synapse (NMS) [18]. MuSK is activated by the motor neuron-derived
heparansulfate proteoglycan agrin [19]. Agrin does not bind MuSK
directly but interacts with Lrp4, a member of the LDL receptor family
[20,21]. Upon binding, the Lrp4/MuSK complex presumably under-
goes a structural rearrangement that results in a dimerization and
subsequent autophosphorylation of MuSK. The subsequent activation
of the MuSK kinase induces a signaling cascade leading to the
formation of the NMS including postsynaptic differentiation charac-
terized by the accumulation of acetylcholine receptors (AChRs) at
synaptic sites and presynaptic differentiation as depicted by the
development of active zones. Consistent with this model, agrin, MuSK
and lrp4 mutant mice fail to form NMSs, lacking all features of pre- and
postsynaptic specializations [22–24].
Rinl interacts with MuSK independent of its phosphorylation and
localizes to NMSs. Further, we show that Rinl acts as GEF for Rab5a.
Moreover, Rinl interacts with inactive Rab22 and catalyzes the GDP/GTP
exchange on Rab22. Co-localization of Rinl with Rab5a or Rab22 in actin-
rich membrane ruffles implicates a novel mechanism for the recruit-
ment of Rab5a and Rab22 to the cytoskeleton via binding to Rinl. In
addition, increased fluid-phase uptake and EGFR endocytosis upon Rinl
overexpression implicates Rinl as regulator of early endocytotic
processes.
2. Materials and methods
2.1. Antibodies and reagents
The following antibodies were purchased from commercial
sources: anti-phosphotyrosine PY99 (Santa Cruz) and PY-100 (Cell
Signaling), anti-myc 9E10 (Sigma-Aldrich), anti-GFP (Santa Cruz).
Antibodies against the C-terminal sequence of MuSK were described
previously [25]. Alexa 594-conjugated α-bungarotoxin (BGT), Alexa
555-conjugated EGF and Alexa 488-conjugated secondary antibodies
were obtained from Invitrogen. Horseradish-peroxidase-coupled and
fluorophore-conjugated secondary antibodies were purchased from
Jackson ImmunoResearch. TRITC-conjugated phalloidin and cytocha-
lasin D were purchased from Sigma-Aldrich.
2.2. Generation of antibodies against Rinl
Rinl (aa 243–307, Mus musculus) was amplified by PCR (5′-
TATGAATTCCACTTTTCCTGTCCT-3′ and 5′-TATAAGCTTTGGGCTG-
TAGGGATTT-3′) and cloned into modified pMALc and pGEX-4T-3
vectors, which encode for seven C-terminal histidines in addition to
MBP or GST respectively [26]. Recombinant proteins were expressed
in the Escherichia coli strain XL1 blue in LB medium supplemented
with 0.4% glucose by induction with 0.2 mM IPTG and purified via HIS-
affinity chromatography. The MBP fusion protein was used to raise
polyclonal antisera in rabbits as described previously [26]. Reactive
serum was affinity purified on an Affi-Gel 10 (Bio-Rad) column
coupled with the GST fusion protein mentioned above. The column
was sequentially washed with phosphate buffered saline (PBS), 0.1 M
NaHCO3 and 0.1 M NaHCO3/0.5 M NaCl. The bound antibodies were
eluted with 7 ml acidic elution buffer (0.1 M glycine, pH 2.5) and
fractions of 1 ml were collected. Purified antibodies were tested for
immunoblotting and immunoprecipitation as shown in Fig. S4.
2.3. Plasmids
The MuSK cytoplasmic domain carrying the mutations Y553F or
Y750, 754, 755F (KD)wassubcloned into theyeast bait vector pBTM116.
It was previously demonstrated that MuSK Y750, Y754, 755F, which
affects the activation loop of the kinase domain, behaves as kinase-
deficient mutant similar to a K608A mutant, which blocks ATP binding
within the autocatalytic loop [27,28]. The MMT bait construct contains
the TrkA cytoplasmic domain including the MuSK juxtamembrane
region in pBTM116 [27]. To generate BTM/tpr-met-JM, the juxtamem-
brane region of MuSK (Rattus norvegicus, aa 528–570) was subcloned
into BTM/tpr-met using the primers 5′-AATGAATTCAGAGAGTCGG-
CAGC-3′ and 5′-TAGTCGACTACGGATACTCCAGGCTGAG-3′. Kinase-dead
(K608A) and kinase-active MuSK (LS745,746MT) carrying a myc-tag
were provided by Dr. Burden (NYU School of Medicine, New York, [20]).
The Rinl 5′-end was amplified from mouse muscle cDNA using the
following primers: 5′-CTGAGCAGCCTTGAGTCTTGTCTT-3′ (located up-
stream of the predicted start codon) and 5′-CCTGGCCAGAGCACG-
GATGT-3′. Full length Rinl was then cloned into pcDNA3 (Invitrogen)
with or without an N-terminal myc-tag and into pEGFP-C3 (Clontech).
Truncations ΔSH2 (aa 200–563) and ΔVps9 (aa 1–394) with an N-
terminal myc-tag were subcloned into pcDNA3. For yeast two-hybrid
and GST-pulldown experiments Rinl and truncation mutants were
cloned into pACT2 (Clontech) and pGEX (GE Healthcare) vectors
respectively: GST-Rinl (aa 1–563 in pGEX-5X-2), GST-Y2H (aa 200–563
in pGEX-3), GST-RH/ΔVps9 (aa 200–479 in pGEX-2), GST-RH/Vps9 (aa
248–563 in pGEX-2).
pLexA-Rab5a (wt, S34N, Q79L) plasmids were a gift from Dr. Zerial
(Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden), for GFP-tagged constructs Rab5a wt and mutants were
cloned into pEGFP-C2 (Clontech) using EcoRI. pEGFP-Rab22 (wt,
S19N, Q64L) was provided by Dr. Donaldson (NIH, Bethesda). Rab22
inserts were amplified by PCR (Rab22 MunI fw 5′-AAACAATT-
GATGGCGCTGAGGGAGCTCAAA-3′ and Rab22 SalI rev 5′-AAAGTC-
GACGCAGCAGCTTCGCTGCGGCTC-3′) and cloned into pLexA
linearized with EcoRI and SalI. GFP-Rab7a (wt, T22N, Q67L) constructs
were a gift from Dr. Bucci (University of Salento, Lecce).
All constructs used in this study were checked by sequencing.
2.4. Cell culture
HEK 293T cells, COS7 and HeLa cells were maintained in DMEM
supplemented with glutamine, 4.5 mg/ml glucose, 10% fetal bovine
serum (FBS) and 100 μg/ml penicillin/streptomycin. HEK 293T cells
were transfected using the protocol described by Chen and Okayama
[29]. COS7 and HeLa cells were transfected using TurboFect
(Fermentas). Briefly, COS7 and HeLa cells were plated on glass
coverslips and transfected the next day with 1 μg DNA and 1.5 μl
TurboFect in 2 ml DMEM.
2.5. Yeast two-hybrid screen
The MuSK cytoplasmic domain (Rattus norvegicus, aa 517–868) was
subcloned into the modified pBTM116 vector, BTM/tpr-met (a gift from
Dr. Birchmeier, Max-Delbrueck Center for Molecular Medicine, Berlin),
1199B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
3. which contains an active c-Met kinase lacking the multiple docking site
[30]. Insertion of MuSK produces a fusion between c-Met and MuSK. The
MuSK bait was screened against a mouse muscle cDNA library (a gift
from Dr. Chamberlain, University of Michigan Medical School, Ann
Arbor) cloned into the pACT2 prey vector [31]. The BTM/tpr-met-MuSK
bait plasmid was introduced into the L40 yeast strain. Large scale
transformations with the prey library were performed according to
Vojtek and Hollenberg [32]. A total of 7.2×106
transformants were
screened for growth on –HIS. Forty-six positive clones were recovered.
After retransformation of the recovered cDNA plasmids six clones grew
on –HIS and produced a positive signal in a β-gal assay with MuSK but
were negative for control plasmids (BTM/tpr-met, BTM/cMet and BTM/
lamin). To analyze the interaction between MuSK and Rinl or Rinl and
Rab5a/Rab22 respectively, the bait and prey plasmids were transformed
into the yeast strain L40. Single colonies were restreaked on –LEU/TRP or
–LEU/TRP/HIS. A β-gal assay screening for lacZ expression was
performed as described previously [32].
2.6. GST-pulldown experiments
All GST fusion proteins were expressed in the bacterial strain
Rosetta by induction with 0.5 mM IPTG. The harvested bacteria were
resuspended in ice-cold buffer R (PBS, 1% Triton X-100, 1 mg/ml
lysozyme, 0.2 mM phenylmethylsulfonyl fluoride (PMSF)). Cells were
lysed on ice for 1 hour, 0.25% sarkosyl was added and samples were
sonicated. Cleared lysates were incubated with glutathione agarose
beads (Sigma) for 1 hour under constant rotation at 4 °C, followed by
three washing steps with buffer R (without lysozyme). Protein
concentration and quality were assayed by SDS-PAGE and subsequent
Coomassie staining. Recombinant proteins were stored at −80 °C
until use. Cell lysates were incubated with immobilized GST fusion
proteins for 4–18 hours at 4 °C. Beads were washed three times with
NP-40 lysis buffer (1% Nonidet P-40, 5 mM EGTA, 50 mM NaCl, 30 mM
triethanolamine [pH 7.5], 50 mM NaF). Proteins bound to the beads
were subjected to SDS-PAGE and immunoblotting.
2.7. Lysate preparation, immunoprecipitation and immunoblotting
Lysates were prepared from cultured cells or adult mouse tissues
using NP-40 lysis buffer supplemented with fresh proteinase inhibitors
(1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, 0.2 mM PMSF,
1 mM sodium orthovanadate). Protein concentrations were determined
using Roti-Nanoquant (Roth) protein assay reagent.
For co-immunoprecipitation experiments, HEK 293T cells were
co-transfected with indicated constructs. Cleared lysates were
precipitated with polyclonal affinity-purified antibodies directed to
either MuSK or Rinl overnight. The next day protein A agarose
(Roche) was added for 1–3 hours, the beads were washed three times
with NP-40 lysis buffer with increased (150 mM) NaCl concentration
and precipitated protein complexes were analyzed by SDS-PAGE and
immunoblotting.
2.8. RT-PCR and quantitative PCR
RNA was isolated from C57BL/6 mice using TRI reagent (Sigma)
according to the manufacturer's protocol and reverse transcribed to
cDNA. The following primers were used for subsequent PCR reactions:
Rinl-1154fw (5′-GAAGATCTTGGCCCCGCTGT-3′), Rinl-1645rev (5′-
CTGGTAGTGAGCAATGTGGT-3′), actin1 (5′-TTCTACAAT-
GAGCTGCGTGTGG-3′), actin2 (5′-CTCGGTCAGGATCTTCATGAGG-3′),
Gapdh/fw (5′-TGCATCCTGCACCACCAACT-3′), Gapdh/rev (5′-
ATGCCTGCTTCACCACCTTC-3′). Quantitative real-time PCR (qPCR)
was performed with IQTM
SYBR Green Supermix (Bio-Rad) using a
Bio-Rad iCycler. Rinl levels were normalized to Gapdh using the
formula: Rinl/Gapdh=2(CTx −CTrel)/2(CTx −CTrel). Spleen was used
as reference tissue (rel) with a value set to 1. PCR reactions were
performed in duplicates or triplicates.
2.9. Immunofluorescence
Eighteen hours after transfection, COS7 cells were fixed with 4%
paraformaldehyde (PFA)/PBS for 10 minutes at room temperature (RT),
permeabilized with 0.1% Triton/PBS for 5 minutes at RT and incubated
with blocking solution (PBS containing 10% FBS) followed by incubation
with appropriate primary antibodies for 1 hour at RT. Cells were washed
with PBS and incubated with fluorophore-conjugated secondary
antibodies with or without TRITC-labeled phalloidin. Cells were
mounted in Mowiol and visualized using a Leica TCS SP5 spectral
confocal microscope with a HCX PL APO CS 63×/1.4 oil objective. For the
disruption of the actin cytoskeleton, transfected cells were treated with
1 μM cytochalasin D or the solvent DMSO in DMEM for 30 minutes
before the cells were fixed and stained with antibodies. The distribution
of Rab5a or Rab22 in Rinl positive cells was categorized into four
different classes and plotted as the average±s.e.m. of three indepen-
dent experiments (sum of all subcategories is 100%).
Mouse muscle cryosections were stained as previously described
[33]. Stained muscle sections were viewed on a confocal microscope
as described above.
2.10. EGF uptake
Twenty to 24 hours post transfection cells were starved for 3 hours
in DMEM, labeled with Alexa 555-conjugated EGF for 30 minutes at
4 °C and washed four times with ice-cold DMEM supplemented with
1 mg/ml BSA. Then the cells were put back at 37 °C for endocytosis to
occur. At indicated time points cells were fixed with 1% PFA in PBS and
embedded in Mowiol. Cells were analyzed by confocal microscopy as
described above. Representative images are shown.
2.11. HRP uptake
HEK 293T cells were transfected with the following constructs:
pEGFP, pEGFP-Rab5a wt, pEGFP-Rinl as stated. About 20 hours after
transfection cells were sorted for GFP expression by FACS. GFP-positive
cells were seeded on polyornithine coated 12-well plates overnight. The
next day HRP uptake was analyzed as previously described [34]. Briefly,
cells were incubated in internalization medium (IM; DMEM, 1% FBS,
24 mM HEPES [pH 7.5]) containing 4 mg/ml HRP for 1 hour at 37 °C.
After internalization, cells were washed once with warm IM followed by
three washes with ice-cold PBS supplemented with 1 mM CaCl2, 1 mM
MgCl2, 2 mg/ml BSA. Finally, cells were washed once with cold PBS and
extracted for 15 minutes on ice with lysis buffer (1% w/v Triton X-100,
20 mM HEPES [pH 7.5], 0.2 mM PMSF). Total HRP activity of the lysates
was determined in duplicates of triplicate samples using the substrate
3,3′,5,5′-tetramethylbenzidine (Sigma). HRP activity was normalized to
the protein concentration of individual lysates.
2.12. GEF activity assay
Rab5a (Cane lupus, aa 15–185), Rab7a (Cane lupus, aa 1–185),
Rab22 (Cane lupus, aa 1–173) and Rinl (Mus musculus, aa 260–563)
were cloned into pGEX-4T-3 and expressed in the bacterial strain
CK600K in Standard I medium (Merck) by induction with 100 μM
IPTG at an OD600 of 0.8 at 25 °C overnight. Bacteria were harvested
by centrifugation and washed with 0.9% NaCl. Rab proteins were
purified, cleaved from GST-tag and loaded with mGDP (2′-/3′-O-(N′-
Methylanthraniloyl)guanosine-5′-O-diphosphate) essentially as de-
scribed for Rap [35]. Rinl was purified the same way except that the
buffer was MgCl2-free and contained 5 mM EDTA, and the GST-tag was
not cleaved. Fluorescence measurements were performed as described
[35]. In brief, 200 nM of the G-protein loaded with mGDP were
1200 B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
4. incubated in buffer G (50 mM Tris–HCl, pH 7.5, 50 mM NaCl, 5 mM
MgCl2, 5 mM DTT, 5% glycerol) in the presence of 20 μM GDP and
increasing amounts of Rinl. For kinetic analysis the obtained curves
were fitted as single exponential decay to obtain the rate constant kobs.
kobs were plotted against the concentration of Rinl and analyzed by
linear fitting, whereby the slope was defined as the catalytic efficiency
of the GEF reaction.
3. Results
3.1. Rinl: a novel interaction partner of MuSK
To identify proteins that bind to MuSK, a mouse muscle cDNA
library was screened with the MuSK cytoplasmic domain using the
yeast two-hybrid system. Screening of 7.2×106
transformants
yielded six positive clones that interacted strongly with MuSK. One
of these clones, termed CL-6, was identified independently three
times. It consisted of a 2204 bp cDNA encoding 363 amino acids, an
in-frame stop codon, the 3′ UTR and the poly(A) tail. The isolated
cDNA was identical to ENSMUST00000059857 lacking however the
first 199 amino acids. We therefore isolated the missing 5′ sequence
by RT-PCR. Analysis of the total cDNA revealed an open reading frame
that encoded 563 amino acids in which the original CL-6 sequence
corresponded to amino acids 200–563 (Fig. S1A).
Homology search revealed a sequence similarity and structural
homology between the novel MuSK interacting protein and the family
of RIN proteins, which currently consists of RIN1, 2 and 3 (Fig. S1B).
Therefore, this protein was designated Rin-like (Rinl) (gene locus:
Rinl). Similar to the so-far known family members Rinl contains an
SH2 domain, an RH domain and a Vps9 domain. In contrast, the RA
domain and proline-rich regions found in RIN1, 2 and 3 are not
present in Rinl.
To determine the region(s) within Rinl necessary for interaction
with MuSK, we generated several Rinl deletion mutants fused to the
Gal4 activation domain (Fig. 1A). Using the reporter genes HIS and
lacZ, interactions with the full-length MuSK cytoplasmic domain were
assayed in yeast. As shown in Fig. 1B, deletion mutants lacking either
the SH2 domain (Y2H and RH/Vps9) or the Vps9 domain (RH/ΔVps9)
were able to interact with MuSK. In contrast, no interaction was
detected with a deletion construct that encodes the Vps9 domain only
(Vps9). We confirmed these findings by GST pulldowns of MuSK
expressed in HEK 293T cells using the same Rinl deletion constructs
fused to GST (Fig. 1C). These results suggest that the Rinl/MuSK
interaction depends on the RH domain of Rinl.
The MuSK cytoplasmic domain consists of a short juxtamembrane
region, a tyrosine kinase domain and an eight amino acid C-terminal tail.
Upon agrin stimulation MuSK becomes tyrosine-phosphorylated, which
induces recruitment of downstream factors and consequently down-
stream signaling [25,27,36]. To test whether the interaction between
MuSK and Rinl is phosphorylation dependent, we expressed MuSK wild-
type, MuSK kinase-active or MuSK kinase-dead together with Rinl in
HEK 293T cells. Co-immunoprecipitation of Rinl with MuSK was
detected independent of MuSK phosphorylation (Fig. 1D and Fig. S2).
Tonarrow down thesite of interaction within MuSK we generated MuSK
deletion and point mutants fused to the lexA binding domain. Binding of
Rinl to these MuSK mutant proteins was assayed in yeast using the
reporter genes HIS and lacZ (Fig. 1E). Mutation of tyrosines in the
autoactivation loop of the kinase domain (MuSK KD) do not affect Rinl/
MuSK interaction. Similarly, the juxtamembrane tyrosine Y553 is
dispensable for Rinl binding to MuSK. We did not include any
Fig. 1. Rinl is a specific binding partner of MuSK. (A) A scheme of Rinl and corresponding truncation mutants. (B) Yeast was transformed with bait and prey constructs as indicated.
Interaction was assayed by growth of yeast clones on –HIS. (C) Rinl GST-deletion constructs were purified from bacteria and used for a pulldown of MuSK-myc expressed in HEK
293T cells. Proteins were detected by immunoblotting (IB) using anti-myc antibodies. GST protein input was assayed by Ponceau staining. 5% of the total MuSK-myc input is shown
in the right panel. (D) MuSK-myc and Rinl were expressed in HEK 293T cells and co-immunoprecipitated either with anti-MuSK antibodies or anti-Rinl antibodies.
Immunoprecipitated proteins were assayed by immunoblotting using anti-myc and anti-Rinl antibodies, respectively. Phosphorylated MuSK was detected with anti-
phosphotyrosine (PY). wt, wild-type; KD, kinase-dead; KA, kinase-active. IP, immunoprecipitation. (E) Yeast was transformed with the indicated MuSK bait constructs and the
Rinl (Y2H) prey construct. Interaction was followed by growth of yeast clones on –HIS.
1201B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
5. truncations into the kinase domain since deletions of C-terminal parts
will destroy the correct folding of the protein and consequently
negatively influence our binding studies. We used however constructs
carrying only the MuSK juxtamembrane region (JM) or the MuSK
juxtamembrane NPXY motif inserted in the TrkA cytoplasmic domain
(MMT). These fusion proteins are unable to interact with Rinl. Taken
together, we conclude that Rinl binds to the MuSK kinase domain and
that this binding is independent of MuSK phosphorylation.
3.2. Rinl is ubiquitously expressed with highest expression in lymphoid
organs and specifically accumulated at NMSs
We performed RT-PCR using RNA isolated from adult mouse tissue.
Rinl is expressed in all tested tissues with the highest expression in
lung, spleen and thymus (Fig. 2A). These data were confirmed by qPCR
showing that Rinl is up to 40 times more expressed in thymus than
muscle (Fig. 2B). We also studied the expression of Rinl during
development and found a slight increase in expression in spleen and
thymus within the first weeks after birth but no profound develop-
mental regulation of Rinl gene expression in brain and muscle (Fig.
S3A and B). To further extend these expression studies we
investigated Rinl protein expression in thymus, spleen and muscle.
Analysis by immunoblotting revealed a similar expression profile as
detected by RT-PCR. High expression was found in thymus and spleen
compared to a weak expression in muscle (Fig. S3C).
Proteins important for NMS development are usually enriched at
synaptic sites. We therefore tested whetherRinl is localized atNMSs. We
stained muscle sections with antibodies against Rinl and Alexa-594
conjugated α-BGT (Fig. 2C and S4). As shown in Fig. 2C, a weak but
specific enrichment of Rinl at NMSs is detectable.
3.3. Rinl acts as GEF for Rab5a
The family of RIN proteins is characterized by its Vps9 domain and
its binding affinity for Rab5a. All so far known RIN proteins are able to
catalyze the GDP to GTP exchange on Rab5a [3,15,37]. First
experiments were performed using the yeast two-hybrid system to
determine whether Rinl binds to Rab5a. The dominant-negative form
Rab5a S34N showed a strong interaction with Rinl. In contrast, Rinl
did not interact with Rab5a wild-type and the constitutively-active
Rab5a Q79L variant (Fig. 3A). Dominant-negative mutations are
known to reduce the nucleotide affinity of the G-protein, resulting in
increased concentrations of nucleotide free G-protein which binds
with high affinity to its GEFs. The observed interaction profile thus
suggests that Rinl might be a Rab5a GEF. To map the site of interaction
we used the Rinl mutant constructs described in Fig. 1A and found
that the N-terminal region is dispensable for Rinl/Rab5a interaction.
However, deletion of the Vps9 domain or the RH domain abolished
the binding of Rinl to Rab5a S34N suggesting that both the RH and
Vps9 domain are required for interaction (Fig. 3A and B). These
findings were confirmed by GST pulldowns of Rab5a expressed in
COS7 cells using the same Rinl deletion constructs fused to GST
(Fig. 3C). We further tested the interaction between Rinl and Rab5a by
co-immunoprecipitation from HEK 293T cells transfected with Rinl
and Rab5a wild-type, Rab5a S34N or Rab5a Q79L, respectively. Rab5a
S34N efficiently co-immunoprecipitates with Rinl, whereas Rab5a
wild-type and Rab5a Q79L show a weak or no interaction (Fig. 3D). In
contrast, Rinl does not bind to the late endosomal marker Rab7a,
independent of its activation status.
As Rinl contains a classical Vps9 domain and binds preferentially to
nucleotide free Rab5a, we next asked whether Rinl indeed acts as GEF
for Rab5a. Recombinant proteins were expressed in bacteria and used
in an in vitro assay to measure nucleotide exchange. In brief, the Rab
GTPase was loaded with the fluorescent nucleotide analog mGDP. The
fluorescence intensity of mGDP is approximately twice as high if
bound to the hydrophobic environment of a protein as if exposed
freely to the buffer solution. The exchange of mGDP in the presence of
excess unlabeled GDP can thus be measured in real time as
fluorescence signal decay. As shown in Fig. 3E, the addition of Rinl
accelerates nucleotide exchange in a concentration dependent
Fig. 2. Rinl expression in different tissues and localization at the NMS. (A) RT-PCR was performed from different mouse tissue samples. Actin was used as quality control. (B) qPCR
from cDNAs generated from different tissue RNAs was performed. Spleen was set to 1. s.e.m. is shown. (C) Mouse muscle sections were stained with anti-Rinl antibodies and Alexa
594-conjugated α-BGT. Images were taken on a confocal microscope. Scale bar, 20 μm.
1202 B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
6. manner by several orders of magnitude. In contrast, no exchange
activity of Rinl for Rab7a could be detected. To validate proper
nucleotide loading of Rab7a, the Mg2+
chelator EDTA was added to
the control reaction at the indicated time point. This induces the
release of nucleotides from G-proteins as their binding is Mg2+
dependent. These data demonstrate that Rinl similar to the other
members of the RIN protein family acts as GEF for Rab5a.
3.4. Rinl colocalizes with dominant-negative Rab5a to cytoskeletal-rich
membrane ruffles
To examine the subcellular distribution of Rinl COS7 cells were
transfected with myc-tagged Rinl (Fig. 4A). We found Rinl localized to a
variety of different compartments. To analyze the distribution in more
detail we divided Rinl localization into four categories and quantified
the distribution within these categories. As shown in Fig. 4A, around 90%
of Rinl is localized either diffusely and/or in vesicles. The remaining 10%
of Rinl show a ruffle-like distribution. This localization pattern is the
same for GFP-tagged Rinl or untagged Rinl (Fig. S5). Rinl shows no co-
localization with early endosomes, recycling and late endosomes or the
Golgi (Fig. S6). Interestingly, upon co-expression of Rinl and Rab5a
S34N, Rinl and Rab5a are co-localized predominantly to membrane
ruffles (Fig. 4B and S7A). Similarly, a weak co-localization between Rinl
and Rab5a wt is detectable at membrane-ruffles. This co-localization is
increased when Rinl lacking the SH2 domain is co-expressed with Rab5a
wt (Fig. S7B). Most importantly, Rab5a S34N becomes redistributed
from a diffuse cytoplasmic localization to either ruffle-like or vesicular-
like structures. Membrane ruffles have been implicated in endocytotic
processes associated with cytoskeletal rearrangements. To test whether
actin is concentrated within the Rinl/Rab5a-positive ruffles we stained
cells with phalloidin. Fig. 4C shows a co-localization of actin with Rinl
and Rab5a. Moreover, disruption of the cytoskeleton using cytochalasin
Fig. 3. Rinl preferentially interacts with nucleotide free Rab5a and acts as GEF for Rab5a. (A) Yeast was transformed with the indicated Rab5a bait constructs and Rinl full-length and
deletion prey constructs. Interaction was screened by growth on –HIS. (B) Table showing the activation of the reporter genes HIS and lacZ. (C) Rinl GST-deletion constructs were
purified from bacteria and used for a pulldown of GFP-Rab5a (wt, S34N, Q79L) expressed in COS7 cells. Proteins were detected by immunoblotting (IB) using anti-GFP antibodies. 5%
of the total Rab5a lysates are shown as input. (D) Extracts of transiently transfected HEK 293T cells were used to immunoprecipitate Rinl with anti-Rinl antibodies. Co-
immunoprecipitated Rab5a was detected by immunoblotting with anti-GFP. No Rab7a co-immunoprecipitation is detected. 10% of the input is shown (total lysate). IP,
immunoprecipitation. (E) 200 nM Rab5a or Rab7a loaded with mGDP were incubated with increasing amounts of Rinl in the presence of 20 μM unlabeled GDP. The exchange of
mGDP for GDP was measured as decay in fluorescence signal. In case of Rab7a proper nucleotide loading was demonstrated by the addition of EDTA at the indicated time point, which
induces the release of nucleotides and a rapid decay in the fluorescence signal.
1203B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
7. 1204 B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
8. D blocked the formation of membrane ruffles and caused an
accumulation of Rinl and Rab5a in actin-positive aggregates.
3.5. Rinl acts as GEF for Rab22 and co-localizes with Rab22 to actin-rich
domains
Rab22 is the closest homologue of Rab5 with 52% sequence
identity [38]. Like Rab5a, Rab22 has been localized to early endosomes
but its role during endocytosis is so far not well understood. Since
Vps9 domain containing proteins have been reported to activate
Rab5a and its homologues Rab21 and Rab22, we tested the interaction
of Rinl with Rab22 [6]. Using the yeast two-hybrid system we detect a
strong interaction between Rinl and the dominant-negative form of
Rab22. Moreover, the binding of Rinl to Rab22 is dependent on the RH
and Vps9 domain but independent of the N-terminal region (Fig. 5A
and B). The Rinl/Rab22 interaction was confirmed by co-immunopre-
cipitation experiments in transfected HEK 293T cells. Dominant-
negative Rab22 S19N efficiently co-immunoprecipitates with Rinl
upon co-expression, whereas Rab22 wild-type and constitutively
active Rab22 Q64L show a weak or no interaction (Fig. 5C). To
determine whether Rinl acts indeed as GEF for Rab22, the GEF activity
of Rinl toward Rab22 was measured in vitro (Fig. 5D). Rinl strongly
accelerates nucleotide exchange of Rab22 and can therefore be
classified as a GEF of Rab22. For a more quantitative comparison of
the Rinl activity toward Rab5a and Rab22, the rates of the nucleotide
exchange reaction, kobs, were determined from measurements as
Fig. 5. Rinl preferentially interacts with GDP-bound Rab22 and acts as GEF for Rab22. (A) Yeast was transformed with Rab22 bait constructs (wt, S19N, Q64L) and Rinl full-length or
deletion prey plasmids. Growth on –HIS is shown. (B) The interaction between Rab22 and Rinl was assayed by growth on –HIS and LacZ expression (β-gal assay). (C) Extracts of
transiently transfected HEK 293T cells were used to immunoprecipitate Rinl with anti-Rinl antibodies. Co-immunoprecipitated Rab22 was detected by immunoblotting (IB) with
anti-GFP. Rab7a was used as negative control. 10% of the input is shown in the top panel (total lysate). (D) 200 nM of mGDP loaded Rab22 were incubated with increasing amounts of
Rinl in the presence of 20 μM unlabeled GDP. The exchange of mGDP for GDP was measured in real time as decay in fluorescence signal. (E) The velocity of the nucleotide exchange
reaction (kobs) toward Rab5a and Rab22 were determined and plotted against the concentration of Rinl.
Fig. 4. Subcellular localization of Rinl and co-localization with Rab5a and actin. (A) myc-tagged Rinl was transiently expressed in COS7 cells and stained with antibodies against Rinl
and myc. Representative confocal images are shown. Scale bar, 25 μm. Transfected cells were assayed for their Rinl expression patterns. Expression patterns were divided into four
categories. A quantification is shown (nN100). Error bars, s.e.m. (B) GFP-Rab5a S34N alone or together with myc-tagged Rinl were co-expressed in COS7 cells. Scale bar, 25 μm. A
quantification of the Rab5a S34N distribution was performed as described in A (n=50). Error bars, s.e.m. (C) Transiently transfected COS7 cells were treated with cytochalasin D or
DMSO for 30 minutes. Cells were stained with anti-myc antibodies to detect Rinl and with TRITC-conjugated phalloidin to label the actin cytoskeleton. Representative confocal
images are shown. Scale bar, 25 μm.
1205B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
9. presented in Figs. 3E and 5D. The dependency of kobs on the Rinl
concentration is presented in Fig. 5E. As can be seen, nucleotide
exchange toward Rab22 is more sensitive to the concentration of Rinl,
which thus displays a higher catalytic rate for Rab22 than for Rab5a.
Moreover, Rinl and Rab22 S19N co-localize to similar compartments
upon co-expression in COS7 cells (Fig. S7). Like for Rab5a, Rinl only
weakly co-localizes with Rab22 wt whereas Rinl lacking the SH2
domain robustly co-localizes with Rab22 wt (Fig. S7B). Rinl induces a
redistribution of diffusely localized Rab22 to ruffle-like structures
(Fig. 6A). Similar to Rab5a and Rinl, also Rab22 and Rinl co-localize to
actin-positive structures (Fig. 6B).
3.6. Rinl recruits Rab5a and Rab22 to the cytoskeleton via the Vps9 domain
Since we detected a significant redistribution of dominant-negative
Rab5a and Rab22 upon co-expression with Rinl, we next asked how this
localization is influenced by Rinl deletion mutants. For that we used
constructs that either lack the N-terminal SH2 domain or the C-terminal
Vps9 domain. These truncations were co-expressed with Rab5a S34N or
Rab22 S19N and the localization assayed by immunostaining. Rinl
lacking the SH2 domain (ΔSH2) localizes to vesicles and ruffles together
with Rab5a and Rab22 (Fig. 7). In particular, Rinl ΔSH2 has a more
pronounced localization to ruffles compared to full-length Rinl (data not
Fig. 6. Rab22 and Rinl co-localize to actin-positive membrane ruffles. (A) GFP-Rab22 S19N alone or together with myc-tagged Rinl were expressed in COS7 cells. Representative
confocal images are shown. Scale bar, 25 μm. Rab22 expression, when transfected alone or together with Rinl, was assayed by confocal microscopy. Subcellular expression patterns
were quantified as described in Fig. 5. nN50; Error bars, s.e.m. (B) Transiently transfected COS7 cells were treated with cytochalasin D or DMSO for 30 minutes. Cells were stained
with anti-myc antibodies to detect Rinl and with TRITC-conjugated phalloidin to label the actin cytoskeleton. Scale bar, 25 μm.
1206 B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
10. Fig. 7. Rinl-dependent recruitment of Rab5a and Rab22 to the cytoskeleton. (A) myc-tagged Rinl (ΔSH2 and ΔVps9) and GDP-bound Rab5a or Rab22 were co-transfected into COS7
cells. Cells were stained with anti-myc antibodies and visualized by confocal microscopy. Representative images are shown. Scale bar, 25 μm. (B) The distribution of Rab5a and Rab22
was quantified as described in Fig. 5. nN35; Error bars, s.e.m.
1207B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
11. shown). Moreover, Rinl ΔSH2 shows a higher degree of co-localization
with Rab5a or Rab22 to ruffles than full-length Rinl and Rab5a/Rab22
(compare Figs. 4B and 6A to 7B). This suggests that the SH2 domain
might have an inhibitory effect on the interaction between Rinl and Rab
proteins. In contrast, Rinl lacking the Vps9 domain (ΔVps9) does not co-
localize with Rab5a S34N and Rab22 S19N (Fig. 7). Rab5a and Rab22 are
unchanged upon co-expression with Rinl ΔVps9 and remain diffusely
distributed and/or in vesicles. Rinl ΔVps9 itself is localized mainly
throughout the cytoplasm or in vesicles but does not attach to actin-rich
membrane ruffles. Therefore, the Vps9 domain is not only responsible
for Rab5a/Rab22 activation but also mediates the localization of Rinl and
Rab proteins to the cytoskeleton.
3.7. Rinl regulates fluid-phase and receptor-mediated endocytosis
Since RIN family members have previously been implicated in
endocytotic processes we set out to study the function of Rinl during
fluid-phase endocytosis. For that we examined the HRP uptake in HEK
293T cells expressing Rinl, Rab5a wt, Rab5a wt together with Rinl, or
GFP as a control. Expression of Rinl and Rab5a induced the
internalization of HRP (Fig. 8A). The co-expression of Rab5a wt and
Rinl induced a similar degree of HRP uptake than expression of Rinl or
Rab5a wt alone.
It has previously been demonstrated that RIN1 acts as regulator of
EGFR endocytosis [12,39]. We therefore asked whether Rinl also affects
EGFR internalization. We used Alexa 555-conjugated EGF to label
endogenous EGFR in HeLa cells expressing Rinl, Rab5a wt, Rab5a S34N,
Rinl together with Rab5a wt, or GFP as a control. EGF uptake at 37 °C was
imaged at various times. As shown in Figs. 8B and S8, in control cells
EGFR endocytosis is rapidly induced within 5 minutes and perinuclear
accumulations of EGF-positive vesicles is prominent by 15 minutes.
Expression of Rab5a wt or Rinl leads to an acceleration of EGFR
endocytosis and a decrease in EGF-positive vesicles. This increase in
EGFR endocytosis is especially pronounced in cells co-expressing Rab5a
wt and Rinl. In contrast, cells expressing Rab5a S34N show a reduced
EGFR endocytosis. These results support a functional role of Rinl during
early endocytotic processes.
4. Discussion
The GTPase Rab5a is the key regulator during early endocytotic
processes. Therefore, it is of particular interest to identify mechanisms
and molecules that modulate Rab5a action. In this study we report the
identification and characterization of Rinl, a novel Rab5a GEF, which
shows a high homology to the family of RIN proteins. Rinl was isolated
via its interaction with the RTK MuSK. It specifically interacts with MuSK
through the central portion of the protein containing the RH domain.
Rinl interacts and co-localizes with Rab5a and catalyzes GDP/GTP
exchange on Rab5a. Similar biochemical and enzymatic properties were
demonstrated toward Rab22. Furthermore, we identified the Vps9
domain of Rinl as a critical molecular determinant that controls the
recruitment of Rab5a and Rab22 to cytoskeletal membrane compart-
ments. Most importantly, Rinl stimulates fluid-phase uptake and EGFR
endocytosis.
The formation of the NMS is crucially linked to signal transduction
events induced by the muscle-specific RTK MuSK [18]. MuSK
activation via agrin/Lrp4 induces a signaling cascade that leads to
post- as well as presynaptic differentiation. Several MuSK binding
proteins have been identified which include adaptor proteins like
Dok7, kinases like Abl and scaffolding proteins including Magi-1c and
Fig. 8. Rinl increases the internalization of HRP and EGFR. (A) HEK 293T cells expressing GFP-Rinl, GFP-Rab5a wt, GFP-Rinl and GFP-Rab5a wt or GFP alone were loaded with HRP.
HRP uptake was quantified from two independent experiments done in triplicates. Error bars, s.e.m. (B) HeLa cells were transfected with GFP, GFP-Rinl, GFP-Rab5a wt, GFP-Rinl and
GFP-Rab5a wt or GFP-Rab5a S34N. Cells were incubated with Alexa 555-conjugated EGF at 4 °C and EGF uptake at 37 °C was imaged after 5 and 15 minutes. Representative confocal
images are shown. Scale bar, 25 μm. The GFP signals of the equivalent cells are shown in Fig. S8.
1208 B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
12. ColQ [18]. More recently the trafficking protein NSF and the E3
ubiquitin ligases PDZRN3 and PAUL were isolated as MuSK interaction
partner, which are thought to regulate MuSK endocytosis and
degradation, respectively [40–42]. Here we identified Rinl as novel
interaction partner of MuSK. Binding of Rinl to MuSK requires the
internal RH domain of Rinl but is independent of MuSK phosphory-
lation. This suggests that Rinl binding is not associated with MuSK
activation. However, a regulation of Rinl/MuSK interaction dependent
on the subcellular localization appears likely since we show a specific
localization of Rinl to vesicles and membrane ruffles. In support of this
hypothesis we detect a specific co-localization of MuSK and Rinl at
actin-positive membrane ruffles (data not shown). The identification
of Rinl as binding partner of MuSK appeared of distinct interest since it
was recently demonstrated that MuSK endocytosis regulates MuSK
signaling [42]. However so far, we have not been able to correlate Rinl
action and MuSK function. Rinl is very weakly expressed in muscle
cells questioning the importance of Rinl in cultured muscle cells.
Nevertheless, this does not exclude the possibility that Rinl plays a
role during MuSK-dependent signal transduction in vivo, either at the
NMS or in the brain, where MuSK function during memory formation
has been implicated recently [43]. A gene targeting approach in mice
will be necessary to answer these questions. Furthermore, a functional
compensation by one of the other RIN family members cannot be
ruled out at the moment.
The RIN family members have been implicated in early endocytotic
events [1]. In particular, the role of RIN1 during receptor endocytosis
has been demonstrated many-fold [12–14,39]. Here we are able to
show that Rinl overexpression accelerates fluid-phase endocytosis as
well as EGFR endocytosis. These data support our in vitro data and
provide first hints on the physiological role of Rinl. RIN1 has been
postulated as crucial regulator of EGFR endocytosis and signaling
[12,39,44]. Our findings raise the questions whether Rinl represents a
similarly important regulator of EGFR endocytosis and whether Rinl
and RIN1 play complementary roles during EGFR endocytosis due to
their differential expression patterns.
The current members of the RIN protein family share an SH2 domain,
an RH region, a Vps9 domain and an RA domain. RIN1 was isolated by its
ability to bind to H-Ras via the RA domain thereby competing with Raf1
and inhibiting Ras action [2,45]. In addition, the RA domain has been
implicated in the interaction with the EGFR via Ras, which leads to a
recruitment of RIN1 to the activated receptor linking internalized EGFR
to Rab5a-positive endosomes [3]. Rinl lacks an RA domain. Therefore it
appears unlikely that Rinl acts as an effector for Ras proteins. However, a
recruitment to activated RTKs can still occur through the N-terminal
SH2 domain, which would then bring the intracellular receptors to early
endosomes. Such an SH2-dependent interaction has been shown for
RIN1 and EGFR as well as RIN1 and EphA4 [12,13].
The RIN proteins belong to the family of Vps9 domain containing
proteins. These proteins are characterized by their ability to bind Rab5a
and to catalyze the GDP/GTP exchange on Rab5a [1]. Crystallization
studies on Rabex-5 have shown that an N-terminal helical bundle in
addition to the Vps9 domain is required for GEF activity, the so-called
HB-Vps9 tandem [6]. The helical bundle conforms to the RH domain in
the RIN proteins. Consistent with the Rabex-5 data, it was shown that a
splice-variant of RIN1 lacking part of the helical bundle is unable to
interact with dominant-negative Rab5a. Likewise, Rinl truncations
deleting the RH domain/helical bundle do not bind dominant-negative
Rab5a implicating similar biochemical properties for all Vps9 domain
containing proteins. It was also reported that full-length Vps9 domain
containing proteins including the RIN proteins have a reduced binding
activity and/or GEF activity for Rab5a [6,46]. This suggests that
autoinhibition by regulatory elements in the N- and/or C-terminus
play a role. Similarly, we find a reduced binding activity for Rab5a and
Rab22 in full-length Rinl constructs compared to Rinl constructs
carrying only the RH and Vps9 domain. Therefore, the N-terminal SH2
domain might represent such an inhibitory element. Protein interac-
tions via the SH2 domain would then relief the autoinhibition and
induce exchange activity. So far however, it remains unclear which
proteins bind to the SH2 domain of Rinl.
Analysis of the Rabex-5 HB-Vps9 tandem revealed a high specificity
toward the Rab5 subfamily but also showed a selective exchange
activity for the different Rab5 subfamily members: strong GEF activity
for Rab5 and Rab21, a weak activity for Rab22 [6]. This specificity and
selectivity is achieved on one hand by conserved exchange determi-
nants on a common surface of the Vps9 domain and on the other hand
by invariant aromatic residues in the switch regions of the Rab GTPases.
Delprato and colleagues also reported a similar catalytic activity and Rab
specificity for RIN1 [6]. In this study we show that Rinl specifically binds
Rab5a and Rab22, and there preferentially the nucleotide free forms.
Rinl acts as GEF for Rab22 and Rab5a, surprisingly however, presents a
higher efficiency for Rab22. This distinguishes Rinl from RIN1 and
suggests that different Vps9 domains have different specificity profiles
for the Rab5 subfamily. This of course also raises the question whether
this specificity is also represented at a physiological level whereby
different RIN proteins regulate different Rab5 subfamily dependent
processes. Four amino acids (D313, P317, Y354 and T357) in the Vps9
domain in Rabex-5 have been shown to be essential for exchange
activity and these residues are highly conserved among different Vps9
domains [6]. Likewise, the Rinl Vps9 domain contains these conserved
amino acids (D456, P460, Y497 and T500). Other residues, which have
been shown to lie within the binding site for the GTPase differ between
Rabex-5 and Rinl. These residues are expected to be responsible for the
differences in the specificity profile.
When studying the subcellular distribution of Rinl protein we
noticed a characteristic localization in vesicles and membrane ruffles.
Moreover, co-expression of Rinl and Rab5a or Rab22 mutants with
lowered nucleotide affinity leads to a redistribution of diffusely
expressed Rab5a and Rab22 to actin-positive membrane compartments.
The localization of Rinl to membrane ruffles as well as the recruitment of
Rab5a and Rab22 to the cytoskeleton are dependent on the Vps9
domain. It is unclear at the moment whether the Vps9 domain interacts
directly or indirectly (via a so-far unknown actin binding protein) with
the cytoskeleton. Rab mutants with lowered nucleotide affinity are
known to bind GEFs with high affinity, thereby trapping the active GEF
in an unproductive complex [47]. The ability of Rinl to recruit Rab5a
S34N and Rab22 S19N indicates that Rinl interacts with the actin
cytoskeleton in an active state. Thus it is expected that Rinl activates Rab
proteins locally at the cytoskeleton. Actin cytoskeleton remodeling has
been implicated in early endocytosis as well as recycling [48].
Furthermore, membrane ruffling is a characteristic of actin remodeling
and is closely associated with regions of active endocytosis [49]. Our
findings therefore support a model whereby Rinl-dependent activation
of Rab5a and Rab22 at the cytoskeleton regulates early endocytotic and/
or recycling processes. Future experiments will have to show how actin
remodeling, exchange activity by Rinl and Rab5a/Rab22-dependent
endocytosis are connected.
Supplementary materials related to this article can be found online
at doi:10.1016/j.bbamcr.2011.03.005.
Acknowledgments
We are grateful to Jeffrey Chamberlain for providing us with the
mouse muscle prey library. We would like to thank Walter
Birchmeier, Cecilia Bucci, Steve Burden, Judy Donaldson, Said
Hashemolhosseini and Marino Zerial for providing plasmids. Many
thanks also go to Marije Rensen-De Leeuw for technical assistance.
Wilfried Ellmeier provided useful comments on the manuscript. This
work was supported by the Austrian Science Fund (P19223-B09 to R.
H.) and by the Chemical Sciences of the Netherlands Organization for
Scientific Research (NOW-CW to H.R.). B.W. was supported by the
DOC-fFORTE Programme from the Austrian Academy of Sciences. M.P.
was supported by the TI Pharma Project T3-106.
1209B. Woller et al. / Biochimica et Biophysica Acta 1813 (2011) 1198–1210
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