When opened, also allow Ca2+ to come into cytoplasm
ATPase in the ER uses hydrolyzed ATP to bring in calcium into the smooth ER, endergonic
Calcium pumps to go into mitochondria (doesn’t really need ATP)
Calcium exchangers to keep cytosolic concentration low (Na+ in/out Ca2+)
Phospholipase c beta
Linked to Heterotrimeric G protein linked receptors
Activates PLCB and cleaves PIP2 in the membrane
Phospholipase c gamma
links up with enzyme linked receptors aka receptor tyrosine kinases (RTKs)
transmembrane receptors that bind ligands on extracellular side of cell and dimerize to phosphorylate the two transmembrane components of the receptor, and there are lots of protein that recognize the phosphorylated sites on the receptor, one of which is PLCG. PLCG binds to the phosphorylated site on receptor and cleaves PIP2, increasing IP3 and DAG signaling (IP3 to increase Ca2+)
The Structure and Activation of a Receptor Tyrosine Kinase
Extracellular part of receptor binds ligand (usually growth factors or cytokines)
EGF – epithelial growth factor
FGF – fibroblast growth factor
NGF – Nerve growth factor
All bind receptor tyrosine kinases (RTKs)
When ligands bind to binding site, the binding sites initiates a conformational change that exposes amino acids in the PM that brings two components of receptor together.
Transmembrane region has attracting amino acids
Brings two monomers together to form a dimer
Juxtamembrane region helps hold receptors together and help attract the monomers to form the dimer (along with transmembrane region)
Cytoplasmic domain has the functional domain where tyrosines are phosphorylated by the neighboring cytosolic tail, so it [cytosolic region] has both the ability to phosphorylate and the sequence to be phosphorylated by its neighbor.
When brought close by amino acids that attract, the phosphorylating domains of cytosolic tail can reach across and phosphorylate the tyrosines on neighboring side of the receptors
Called Autophosphorylation, or cross phosphorylation, because they phosphorylate a similar protein, fundamentally exactly the same.
The pattern of phosphorylation on these receptors determines what types of downstream signaling happens after the receptor is activated.
Each tyrosine can be a binding site for binding of different types of messengers in the cell, which will lead to different pathways being activated.
The specificity in terms of activating specific pathways is determined by what amino acids are on either side of the tyrosine and how they are recognized by downstream signaling proteins
Parts of the RTKs
Extracellular – ligand binding
Trans membrane– used to attract monomers to form the dimers
Juxtamembrane – used to attract monomers to form the dimers
Cytosolic – phosphorylation function
We can call it autophosphorylation because they phosphorylate a similar protein. Both proteins are fundamentally the same. Cross phosphorylate neighboring tyrosines.
The amino acids on either side of the tyrosines can regulate what type of signaling occurs.
A dimer-like signal can also bind to the binding site
All of these intracellular signaling proteins have a domain called an SH2 domain.
Proteins use this SH2 domain to recognize this phosphorylated tyrosine and its amino acids for binding sites for the domain as well as turning on different intracellular signaling pathways
Src Homology domain = SH2.
Src = Sarcomere
Terminology came from tumorigenic cells. People had noticed that there were tumors formed in this tissue and wanted to figure out what was regulating this proliferation of cells. Well, the mutation was in the src homology domain. The mutation resulted in constant RTK signaling, like it was always on. Mutation in SH2 domain always allowed it to activate signaling pathway. Allowed it to activate signaling pathway so the cells continued to divide. SH2 turns on signaling and pathways can regulate cell mitosis.
Proteins with SH2 domains are usually called Protooncogenes – have a mutated form that has been identified as oncogenic, or associated with specific tumors. They are genes that make proteins that when mutated, have an oncogenic form that can induce tumors/cancer.
All of these signaling proteins have an SH2 domain which means they can recognize cerain phosphoryalted tyrosines and turn on their specific intracellular signaling pathway.
RTK signaling continued
Any mutation that knocks out or removes function of that protein
Tyrosine kinase domain has been removed and when signaling molecule binds, it cannot autophosphorylate so receptor doesn’t work and there is no signaling beneath it.
Determined to figure out which pathways are activated by the tyrosines
Tyrosine kinase mutation is removed and when signal molecule binds, there is no signaling.
Constitutively activating mutation
Generate receptors that are always on
Receptors that are found in tumorigenic cells and usually responsible for these mutations
Protooncogene -Per2 – major component of some types of breast cancer - Has valine amino acid in transmembrane region in normal form, and within region we have amino acids that help dimerize receptors that are only exposed upon conformationl change.
In the oncogneic mutation, valine is mutated to glutamine (and attract one another and leads to activation of receptor), which causes spontaneous dimerization of the receptor, and act as if growth factor is bound. Recepteors dimierizae and act as if growth factors were bound.
People study these to determine what is the effect of activating these types of receptors.
In the EXS, EGF receptors normally would have to bind EGF. There are certain mutations that remove extracellular region and the absence of extracellular part leads to dimerization and leads to active receptor at all times.
Activation of Receptor Tyrosine Kinase
Linked to activation of small g proteins
MAP Kinase Pathway
Begins with the activation of a small g protein called Ras
Ras links up with RTK Activation and itself is then activated. These small g proteins are kinases themselves and will Phosphoporylate targets on serines and threonine. They are intimately linked to regulation of cell mitiosis and cell differentiation. Remember small g proteins are active when bound to GTP. Guanine Nucleotide Exchange Factors remove GDP and allow GTP to bind, and GAPs enhance andogenous hydrolysis of GTP to leave a GDP for inactivity
Linking to RTKs requires linkers proteins (all of this occurs at plasma membrane)
Ras is linked to inner leaflet of PM and Phosphporylation of the RTK leads to Ras activation indirectly through SH2 protein that can bind the RTK and another protein that acts as a Ras-GEF
An SH2 domain protein links a GEF to Ras at the membrane
Growth factors dimerizes receptor
Receptor undergoes autophosphorylation
A specific phosphorylated tyrosine is recognized by the SH2 domain on the adapter protein (Grb-2 for Ras pathway)
Binding of Grb-2 to Phosphorylated tyrosine releases or activates a region on protein that can be recognized by the GEF
The Ras GEF is called SOS (sauce) – ‘son of sevenless’ because the protein was indentified in drosophila.
Drosophila have eyes that have 8 omatidia, but when mutated, 7, a mutation on SOS protein because the eye cells don’t differentiate correctly.
SOS exchanges GDP for GTP to activate Ras
Once Ras is activated
Sets up a series of phosphorylation events that can regulate gene transcription, also cytoskeletal dynamic, or transport of proteins in cells. Ras is a highly diverse pathway.
Downstream targets are also phosphorylated etc etc