Lecture 16 Signal Transduction Chapter 13

Biosignaling – Key Concepts Signal Transduction in Bacteria General features of eukaryotic signal transduction G-protein coupled receptors Receptor Tyrosine Kinase Phosphoinositol Pathways

Signaling in Bacteria – 2 Component Systems

General Features of Signal Transduction Specificity is achieved by precise molecular complementarity between the signal and receptor molecules What types of IM forces do you think guide the specificity? Extra specificity built into expression profile of certain cell types Thyrotropin-releasing hormone triggers response in pituitary cells but not hepatocytes Epinephrine alters glycogen metabolism in hepatocytes but not adipocytes Affinity of receptors for signal molecules Often sub-nanomolar Cooperativity in the interaction Small changes in ligand concentration results in large changes in receptor activation 3. Amplification of the signal Receptor is activated, which catalyzes the activation of many equivalents of a 2nd enzyme….

What amino acids are likely involved? Enzyme Cascades Signal is very commonly propagated by a series of phosphate transfer reactions What amino acids are likely involved?

Receiving the signal G-protein coupled receptors Receptor: A membrane-bound or soluble protein or protein complex, which exerts a physiological effect (intrinsic effect) after binding its natural ligand. G-protein coupled receptors Epinephrine receptor Enzyme-linked receptors Insulin receptor Ligand-gated ion channels Nicotinic acetylcholine receptor Other membrane receptors Integrin receptors Nuclear receptors Steroid receptors Typical ligands are: Small ions ferric ion: bacterial ferric receptor Organic molecules Adrenalin: epinephrine receptor Polysaccharides Heparin: fibroblast growth factor Peptides Insulin: insulin receptor Proteins Vascular endothelial growth factor: VEGF receptor

General Types of Signal Transducers

GPCR – Mechanism Overview

Epinepherine Receptor Action

Heterotrimeric G-proteins Composed of 3 distinct subunits Alpha  GTPase Beta Gamma Helical Domain GTPase Domain

The structure of GTP-bound Ga is modestly different than GDP-bound Ga Subunit Switch 3 The structure of GTP-bound Ga is modestly different than GDP-bound Helical Domain Switch 2 Switch 1 GTPase Domain

Ga Subunit P-loop Common ATP/GTP binding motif Gly-rich region followed by Lysine Backbone NH of glycine in Switch 2 makes H-bond with g-phosphate Thr-OH in Switch 1 makes H-bond with g-phosphate Why is S-substitution @ g phosphate a non-hydrolyzable GTP adduct?

G Protein Activating Proteins (GAP) Ga subunits hydrolyze GTP very slowly (2-3 min-1) - this enables the Ga to activate AC for a reasonable amount of time Some are even slower  Ras = 0.02 min-1 This is NOT physiologically reasonable! GAP (Yellow) interacts with the GTP site of Ras (blue) AlF3 corresponds with the transition state structure of g phosphate during hydrolysis Finger loop region inserts between switch I and switch II R789 positioned between b and g phosphate which stabilized the hydrolysis transition state

Adenylate Cyclase

Protein Kinase A Substrate binding cleft interacts with part of regulatory subunit in the absence of cAMP cAMP causes a dissociation of the catalytic subunit g-phosphate oriented toward substrate cleft

cAMP/PKA Regulated Genes

Epinepherine Receptor Action

Inositol Phosphate as a Secondary Messenger

Receptor Tyrosine Kinases (RTK) In most cases, a signal is propagated through the membrane by receptor dimerization (Insulin receptor is the exception here) This brings two PTK domains into close proximity and induces autophosphorylation on the OTHER monomer

Insulin and the Insulin Receptor

Insulin and the Insulin Receptor IRS-1 = Insulin receptor substrate Point of contact with phosphorylated IR Plasma Membrane Localization

Insulin and the Insulin Receptor Grb2  adapter protein SH3 domain = binds proline rich sequences

Phosphotyrosine Recognition (SH2 Domains)

Insulin and the Insulin Receptor Sos = GAP for Ras