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Lecture

Lecture 31

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Department
Biology (Sci)
Course
BIOL 300
Professor
Siegfried Hekimi
Semester
Fall

Description
st BIOL 300 November 21 2012 Lecture 31 Dr. Shock Enzyme-linked receptors are typically linked to slow transcriptional responses: growth, proliferation, differentiation and survival • The enzyme involved with these receptors are usually kinases, and involve mainly paracrine signaling • In contrast to GPCRs, they have only one TM domain (GPCRs have 7), and their cytosolic domain is an enzyme, or is associated with one • While GPCRs typically bind a small molecule as a ligand, kinase receptors usually bind protein ligands GPRC mutations cause mainly mental diseases (they’re involved mainly in neural transmission) while enzyme-linked receptor mutation can lead to cancer Receptor tyrosine kinases are a class of kinase receptors who have cytosolic kinase domains which phosphorylate tyrosines (nothing else) • Ligand binding on the extracellular side causes activation of the intracellular kinase domain • The receptors are usually named by the ligand they bind to: • E.g. the EGF receptor binds to EGF (epidermal growth factor); we will be talking about this one quite a bit • EGF mediates growth and proliferation of many epithelial tissues, including the skin; the skin is one of the one tissues which is in constant growth (even in adulthood) • EGF also mediates growth of the breasts during pregnancy • This is why skin or breast cancer is often associated with a mutation involving a constitutively active EFG pathway • The insulin receptor is another important receptor involved in general growth and determination of organ size • NGF (neural growth factor) regulates growth of neural tissue and VEGF for blood cells How is the extracellular ligand being transduced to the inside? GPCRs shift the orientation of their TM helices (as we saw before), but how does this work for kinase receptors, which only have one TM helix. All receptors with only 1 TM domain must dimerize in order to activate; they are inactive in their monomeric state, and binding of the ligand causes activation through dimerization. • There are different methods of dimerization The PDGF receptor binds PDGF as its ligand; each adjacent receptor binds 1 ligand, and the ligand itself acts as a bridge to form the dimer For the FGF receptor, the ligand binding to the receptor allows binding to a large membrane component known as a proteoglycan 1 st BIOL 300 November 21 2012 Lecture 31 Dr. Shock • A proteoglycan is part protein (which is embedded in the membrane) and part sugars (which stick out extracellularly) • The FGF ligand is able to bind to these sugar side chains, forming clusters of receptors which induces dimerization • Proteoglycans are important for signaling, and also help reduce the range of signaling (slows down diffusion of ligands in paracrine signaling by binding transiently to them) The ephrin receptor binds ephrin, which itself is a membrane bound ligand • The ephrin proteins themselves are clustered on their membrane, causing clustering of their receptors on the target cell’s membrane, which promotes dimerization The EGF receptor binds the ligand as a monomer, which induces a conformational change which allows for the dimerization of two adjacent ligand-bound monomers. • This all takes place in the extracellular domain There are two important steps for dimerization involving phosphorylation : • First, the kinase domains of the two subunits phosphorylate each other; these kinases have weak intrinsic activity which allows them to phosphorylate one another when they come into close proximity of one another • This target is at a special location known as the activation lip, an area, which, when phosphorylated, makes the kinase more active • Second, it can phosphorylate any tyrosine residues located anywhere also on the intracellular domain (not on the 2 st BIOL 300 November 21 2012 Lecture 31 Dr. Shock kinase itself) • Again, this is a cross-phosphorylation, i.e. each monomer phosphorylates the other, and not itself (i.e. no autophosphorylation) You can analyze the function of these receptors through dominant negative mutations on their kinase domain • It’s a dominant negative mutation because if a mutant monomer binds a wild-type monomer, the resulting dimer will be non-functional because the there will be no cross-phosphorylation of the wild- type monomer • Remember, to activate the receptor, you need phosphorylation of both monomers, i.e. both monomers have to be working • The wild-type tyrosine on the activation lip will never be activated, and will not be able to phosphorylate all the tyrosines on the mutant monomer’s intracellular domain without being activated • This method relies on transfecting enough of the mutant receptor into the cell such that you have enough mutant monomers to eliminate the possibility of wild-type – wild-type interactions Downstream, you will bind some kind of receptor protein which is able to recognize phosphorylated tyrosines; there are two types of these proteins: • Ones that contain an SH2 domain • Ones that contain a PTB (phospho- tyrosine binding domains) These proteins can be signaling or adaptor proteins which can then then link the receptor to a proteins which binds a GTPase and further transduces the signal. SH2 domains are not able to bind any phosphorylated tyrosine on the intracellular domain of the receptor; in addition to the tyrosine binding domain, they also have a specificity factor • Therefore the phosphorylated tyrosine has to be surrounded by a specific set of amino acids to bind to a given SH2 domain 3 st BIOL 300 November 21 2012 Lecture 31 Dr. Shock
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