Presented By- Manish Kumar Department of Pharmacology Mangalayatan university
<ul><li>What is signal transduction? </li></ul><ul><li>Any process by which a cell converts one kind of signal or stimulus into another </li></ul><ul><li>Signal transduction often involves </li></ul><ul><li>a sequence of biochemical reactions </li></ul><ul><li>inside the cell, which are carried </li></ul><ul><li>out by enzymes and linked through </li></ul><ul><li>second messengers. </li></ul>
<ul><li>Types of Signals: </li></ul><ul><li>EXTRACELLULAR: </li></ul><ul><li>Signal transduction usually involves the binding of extracellular signaling molecules to receptors that face outwards from the membrane and trigger events inside. </li></ul><ul><li>This takes place via a change in the shape or conformation of the receptor which occurs when the signal molecule "docks" or binds . </li></ul><ul><li>INTRACELLULAR : </li></ul><ul><li>Intracellular signaling molecules in eukaryotic cells include heterotrimeric G protein, small GTPases, cyclic nucleotides, such as cyclic AMP (cAMP) and cyclic GMP (cGMP), calcium ion, phophoinositide derivatives, such as Phosphatidylinositol-triphosphate (PIP3), Diacylglycerol (DAG) and Inositol-triphosphate (IP3), and various protein kinases and phosphatases. Some of these are also called second messengers . </li></ul>
<ul><li>Within endocrinology, which is the study of intercellular signaling in animals, intercellular signaling is subdivided into the following types: </li></ul><ul><li>1) Endocrine </li></ul><ul><li>2) Paracrine </li></ul><ul><li>3) Autocrine </li></ul><ul><li>4) Juxtracrine </li></ul><ul><li>5) Direct contact </li></ul><ul><li>signalling </li></ul>
Direct Contact Signaling a) Gap junctions They provide for metabolic cooperation between adjacent cells, and may help maintain homeostasis in connected cells for ion balance. Some signal molecules may move through gap junctions
b) Cell-cell recognition : Many signal molecules remain bound to surface of signaling cell & influence only cell that contact it. Important during development & immune responses.
<ul><li>Hormones: </li></ul><ul><li>They enable signaling between the cells or tissues within an individual, animal or plant. </li></ul><ul><li>Hormone-initiated signal transduction takes the following steps: </li></ul><ul><li>Biosynthesis of a hormone. </li></ul><ul><li>Storage and secretion of the hormone. </li></ul><ul><li>Transport of the hormone to the target cell. </li></ul><ul><li>Recognition of the hormone by the hormone receptor protein, leading to a conformational change. </li></ul><ul><li>Relay and amplification of the signal that leads to defined biochemical reactions within the target cell. The reactions of the target cells can, in turn, cause a signal to the hormone-producing cell that leads to the down-regulation of hormone production. </li></ul><ul><li>Removal of the hormone. </li></ul>
Signal Pathways <ul><li>Receptor cells on the surface of the plasma membrane or </li></ul><ul><li>within the cytoplasm of the cell induce changes in the cell that elicit appropriate responses, generally some type of chemical reaction or series of metabolic reactions. </li></ul><ul><li>The three stages of cell signaling are, therefore, reception , transduction and response . </li></ul><ul><li>The series of steps involved is referred to as a signal transduction pathway </li></ul><ul><li>and there are a variety of methods for signaling </li></ul>
The stages of chemical cell signaling 1. Reception The target cell must be able to detect that a signal is "arriving". This requires a chemical binding to a receptor molecule (protein), specialized for different functions. Most receptor molecules are found on the cell surface, but there area also intracellular receptors.
<ul><li>2. Transduction - Initiating the Intracellular Signal </li></ul><ul><li>The receptor molecule binds to the signal molecule in a method that brings about a change in the receptor molecule (often a conformational change). This change effectively translates (or transduces ) the signal into a form that the target cell can respond to. </li></ul><ul><li>Responder – Intracellular Secondary Transduction </li></ul><ul><li>Transduction may be a single step or a relay pathway of chemical reactions within the cell. </li></ul><ul><li>The initial receptor molecule's conformational change may serve to activate a second molecule, transducing the second molecule that resides within the target cell to initiate the response. </li></ul><ul><li>A responder will often serve to amplify the signal for a greater response. Secondary messengers are important in the signal transduction. </li></ul>
3. Response The cell makes an appropriate response to the signal. A signal can activate enzymatic activity, genetic transcription, movement of cytoskeletal components, or other cell activities. Cell signals ensure that the right kind of activity occurs in the cell at the right time and in the proper cell conditions.
Cell Surface Receptors:- <ul><li>There are three main families of cell-surface receptors, each of which transduces extracellular signals in a different way. </li></ul><ul><li>They are as follows: </li></ul>
<ul><li>G-Protein-Linked Receptors:- </li></ul><ul><li>Largest family of cell-surface receptors. </li></ul><ul><li>G-Proteins are Guanine-nucleotide binding proteins. </li></ul>Structure of Transmembrane α -Helices Despite the chemical and functional diversity of the signaling molecules that bind to them, all of the G-Protein-Linked receptors whose amino acid sequences are known from DNA sequencing studies have a similar structure and are almost certainly evolutionarily related.
Signal transduction:- Trimeric G-Protein disassemble to Relay signals from G-Protein-Linked Receptors:-
<ul><li>The time during which the α-subunit and βγ complex remain apart and active is usually short, and it depends on how quickly the α-subunit hydrolyzes its bound GTP. </li></ul><ul><li>An isolated α-subunit is an inefficient GTPase, and, left to its own devices, the subunit would inactivate only after several minutes. </li></ul><ul><li>Its activation is usually reversed much faster than this, however, because the GTPase activity of the α subunit is greatly enhanced by the binding of a second protein, which can be either its target protein or a specific modulator known as a regulator of G protein signaling (RGS) . </li></ul>
<ul><li>G Proteins Signal By Regulating the Production of Cyclic AMP:- </li></ul><ul><li>Many extracellular signal molecules work by increasing cyclic AMP content, and they do so by increasing the activity of adenylyl cyclase rather than decreasing the activity of phosphodiesterase. </li></ul><ul><li>All receptors that act via cyclic AMP are coupled to a stimulatory G protein (Gs) , which activates adenylyl cyclase and thereby increases cyclic AMP concentration. </li></ul><ul><li>Another G protein, called inhibitory G protein (Gi) , inhibits adenylyl cyclase, but it mainly acts by directly regulating ion channels rather than by decreasing cyclic AMP content. </li></ul><ul><ul><li>Both G s and G i are targets for some medically important bacterial toxins. </li></ul></ul>
<ul><ul><li>Cholera toxin, which is produced by the bacterium that causes cholera, is an enzyme that catalyzes the transfer of ADP ribose from intracellular NAD+ to the α subunit of G s . This ADP ribosylation alters the α-subunit so that it can no longer hydrolyze its bound GTP, causing it to remain in an active state that stimulates adenylyl cyclase indefinitely. The resulting prolonged elevation in cyclic AMP levels within intestinal epithelial cells causes a large efflux of Cl - and water into the gut, thereby causing the severe diarrhea that characterizes cholera. </li></ul></ul><ul><ul><li>Pertussis toxin, which is made by the bacterium that causes pertussis (whooping cough), catalyzes the ADP ribosylation of the α subunit of G i , preventing the subunit from interacting with receptors; as a result, this α subunit retains its bound GDP and is unable to regulate its target proteins. </li></ul></ul><ul><ul><li>These two toxins are widely used as tools to determine whether a cell's response to a signal is mediated by G s or by G i . </li></ul></ul>
Cyclic-AMP-dependent Protein Kinase (PKA) Mediates Most of the Effects of Cyclic AMP:-
How gene transcription is activated by a rise in cyclic AMP concentration?
<ul><li>G Proteins Activate the Inositol Phospholipid Signaling Pathway by Activating Phospholipase C-β:- </li></ul><ul><li>Many G-Protein-Linked receptors exert their effects mainly via G proteins that activate the plasma-membrane-bound enzyme phospholipase C-β . </li></ul><ul><li>The polyphosphoinositides—PI(4)P and PI(4,5)P2—are produced by the phosphorylation of phosphatidylinositol (PI) and PI(4)P, respectively. </li></ul><ul><li>Receptors that operate through this inositol phospholipid signaling pathway mainly activate a G protein called Gq , which in turn activates phospholipase C-β, in much the same way that Gs activates adenylyl cyclase. </li></ul><ul><li>The activated phospholipase cleaves PI(4,5)P2 to generate two products: Inositol 1,4,5-trisphosphate and Diacylglycerol . At this step, the signaling pathway splits into two branches. </li></ul>
<ul><li>Inositol 1,4,5-trisphosphate (IP3) diffuses through the cytosol and releases Ca2+ from the endoplasmic reticulum by binding to and opening IP3-gated Ca2+-release channels in the endoplasmic reticulum membrane. </li></ul><ul><li>The large electrochemical gradient for Ca2+ across this membrane causes Ca2+ to escape into the cytosol. </li></ul><ul><li>Diacylglycerol remains embedded in the membrane, where it has two potential signaling roles. </li></ul><ul><li>First , it can be further cleaved to release arachidonic acid, which can either act as a messenger in its own right or be used in the synthesis of other small lipid messengers called eicosanoids. </li></ul>
<ul><li>The second , and more important, function of diacylglycerol is to activate a crucial serine/threonine protein kinase called protein kinase C (PKC) , so named because it is Ca2+-dependent . </li></ul><ul><li>The initial rise in cytosolic Ca2+ induced by IP3 alters the PKC so that it translocates from the cytosol to the cytoplasmic face of the plasma membrane. </li></ul><ul><li>There it is activated by the combination of Ca2+, diacylglycerol, and the negatively charged membrane phospholipid phosphatidylserine. </li></ul><ul><li>Once activated, PKC phosphorylate target proteins that vary depending on the cell type. </li></ul>
<ul><li>Enzyme-Linked Receptors:- </li></ul><ul><li>Like G-protein-linked receptors, enzyme-linked receptors are transmembrane proteins with their ligand-binding domain on the outer surface of the plasma membrane. </li></ul><ul><li>Instead of having a cytosolic domain that associates with a trimeric G protein, however, their cytosolic domain either has an intrinsic enzyme activity or associates directly with an enzyme. </li></ul><ul><li>Each subunit of an enzyme-linked receptor usually has only one. </li></ul><ul><li>They were recognized initially through their role in responses to extracellular signal proteins that promote the growth, proliferation, differentiation, or survival of cells in animal tissues. </li></ul><ul><li>These signal proteins are often collectively called growth factors , and they usually act as local mediators at very low concentrations . </li></ul>
<ul><li>The responses to them are typically slow (on the order of hours) and usually require many intracellular signaling steps that eventually lead to changes in gene expression. </li></ul><ul><li>Enzyme-linked receptors have since been found also to mediate direct, rapid effects on the cytoskeleton, controlling the way a cell moves and changes its shape. </li></ul><ul><li>The extracellular signals that induce these rapid responses are often not diffusible but are instead attached to surfaces over which the cell is crawling. </li></ul><ul><li>Disorders of cell proliferation, differentiation, survival, and migration are fundamental events that can give rise to cancer, and abnormalities of signaling through enzyme-linked receptors have major roles in this class of disease. </li></ul>
<ul><li>The largest group of receptors with intrinsic enzymatic activity consists of cell surface protein kinases, which exert their regulatory effects by phosphorylating diverse effector proteins at the inner face of the plasma membrane. </li></ul><ul><li>Protein phosphorylation can alter the biochemical activities of an effector or its interactions with other proteins. </li></ul><ul><li>Indeed, of all the possible reversible covalent modification of proteins that regulate their function, phosphorylation is the most common. </li></ul><ul><li>Most receptors that are protein kinases phosphorylate tyrosine residues in their substrates; these include receptors for insulin, and diverse polypeptides that direct growth or differentiation, such as Epidermal Growth Factor (EGF) and Nerve Growth Factor (NGF). </li></ul><ul><li>. </li></ul>
<ul><li>The most structurally simple receptor protein kinases are composed of an agonist-binding domain on the extracellular surface of the plasma membrane, a single membrane-spanning element, and a protein kinase domain on the inner membrane face. </li></ul>Seven subfamilies of receptor tyrosine kinases
<ul><li>Protein phosphorylation and kinase cascade mechanism: </li></ul><ul><ul><li>The two important pathways are: </li></ul></ul><ul><li>The Ras/Raf/MAP kinase pathway , which is important in cell division, growth and differentiation & </li></ul><ul><li>The Jak/Stat pathway , which is activated by many cytokines and which controls the synthesis and release of many inflammatory mediators. </li></ul>
<ul><ul><li>Ras/Raf/MAP Pathway: </li></ul></ul><ul><ul><li>The Ras/Raf/MAP pathway mediates the effect of many growth factors and mitogens. </li></ul></ul><ul><ul><li>Ras, which is a proto-oncogene product, functions like a G-Protein and conveys the signal (by GDP/GTP exchange) from the SH2-domain protein Grb, which is phosphorylated by the receptor tyrosine kinase. </li></ul></ul><ul><ul><li>Activation of Ras, in turn, activates Raf, which is the first of a sequence of serine/threonine kinases, each of which phosphorylates, and activates, the next in line. </li></ul></ul><ul><ul><li>The last of these, MAP (Mitogen-Activated Protein) kinase, phosphorylates one or more transcription factors that initiate gene expression, resulting in a variety of cellular responses, including cell division. </li></ul></ul>
<ul><li>2) Jak/Stat Pathway:- </li></ul><ul><ul><li>The Jak/Stat pathway is involved in responses to many cytokines. </li></ul></ul><ul><ul><li>Dimerisation of these receptors occurs when the cytokine binds, and this attracts a cytosolic tyrosine kinase unit (Jak) to associate with, and phosphorylate, the receptor dimer. </li></ul></ul><ul><ul><li>Jaks belong to a family of proteins, different members having specificity for different cytokine receptors. </li></ul></ul><ul><ul><li>Among the targets for phosphorylation by Jak are a family of transcription factors (Stats). </li></ul></ul><ul><ul><li>These are SH2-domain proteins that bind to the phosphotyrosine groups on the receptor-Jak complex, & are themselves phosphorylated. </li></ul></ul><ul><ul><li>Thus activated, Stat migrates to the nucleus and activates gene expression. </li></ul></ul>
<ul><li>Receptor Guanylyl Cyclases Generate Cyclic GMP Directly:- </li></ul><ul><li>Receptor guanylyl cyclases are single-pass transmembrane proteins with an extracellular binding site for a signal molecule and an intracellular guanylyl cyclase catalytic domain. </li></ul><ul><li>The binding of the signal molecule activates the cyclase domain to produce cyclic GMP, which in turn binds to and activates a cyclic GMP-dependent protein kinase (PKG), which phosphorylates specific proteins on serine or threonine. </li></ul><ul><li>Thus, receptor guanylyl cyclases use cyclic GMP as an intracellular mediator in the same way that some G-protein-linked receptors use cyclic AMP, except that the linkage between ligand binding and cyclase activity is a direct one. </li></ul>
<ul><li>Among the signal molecules that use receptor guanylyl cyclase receptors are the natriuretic peptides (NPs) , a family of structurally related secreted signal peptides that regulate salt and water balance and dilate blood vessels. </li></ul><ul><li>There are several types of NPs, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) . </li></ul><ul><li>Muscle cells in the atrium of the heart secrete ANP when blood pressure rises. </li></ul><ul><li>The ANP stimulates the kidneys to secrete Na+ and water and induces the smooth muscle cells in blood vessels walls to relax. Both of these effects tend to lower the blood pressure. </li></ul><ul><li>When gene targeting is used to inactivate the ANP receptor guanylyl cyclase in mice, the mice have chronically elevated blood pressure, resulting in progressive heart enlargement. </li></ul>
<ul><li>Extracellular calcium acts as a "third messenger" to regulate enzyme and alkaline secretion </li></ul><ul><li>It is generally assumed that the functional consequences of stimulation with Ca 2+ -mobilizing agonists are derived exclusively from the second messenger action of intracellular Ca 2+ , acting on targets inside the cells. However, during Ca 2+ signaling events, Ca 2+ moves in and out of the cell, causing changes not only in intracellular Ca 2+ , but also in local extracellular Ca 2+ . The fact that numerous cell types possess an extracellular Ca 2+ "sensor" raises the question of whether these dynamic changes in external [Ca 2+ ] may serve some sort of messenger function. </li></ul>
It was found that in intact gastric mucosa, the changes in extracellular [Ca 2+ ] secondary to carbachol-induced increases in intracellular [Ca 2+ ] were sufficient and necessary to elicit alkaline secretion and pepsinogen secretion, independent of intracellular [Ca 2+ ] changes. These findings suggest that extracellular Ca 2+ can act as a "third messenger" via Ca 2+ sensor(s) to regulate specific subsets of tissue function previously assumed to be under the direct control of intracellular Ca 2+ .
<ul><li>Intracellular cAMP is typically generated via adenylyl cyclases (AC) following hormonal activation of G-protein-coupled receptors (GPCRs) linked to the heterotrimeric G-protein, G s . Once formed, the second messenger can be actively transported to the extracellular space via a probencid- and sulfinpyrazone-sensitive efflux mechanism belonging to the ATP-binding cassette (ABC) transporter family. </li></ul>
Extracellular cAMP is hypothesized to have direct actions on putative receptor proteins (as of yet unidentified) that are expressed on neighboring cells. Alternatively, it is well established that extracellular cAMP can be sequentially metabolized, first by ectophosphodiesterase to adenosine monophosphate (AMP), and then by ecto-5'-nucleotidase to adenosine. Adenosine can then act as a paracrine or autocrine messenger to activate other signal transduction cascades via one of four subtypes of adenosine receptors (A 1 , A 2A , A 2B , A 3 ). In addition, since cAMP is relatively stable in blood, circulating cAMP can be converted by ectoenzymes at a distant site, effectively rendering cAMP as a prohormone for adenosine (which has a fleeting half-life in the circulation).