1. Lecture 16
Signal Transduction
Chapter 13

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

3. Signaling in Bacteria – 2 Component Systems

4. 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….

5. 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?

6. 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

7. General Types of Signal Transducers

8. GPCR – Mechanism Overview

9. Epinepherine Receptor Action

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

11. 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

12. 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 g phosphate a non-hydrolyzable GTP adduct?

13. 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

14. Adenylate Cyclase

15. 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

16. cAMP/PKA Regulated Genes

17. Epinepherine Receptor Action

18. Inositol Phosphate as a Secondary Messenger

19. 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

20. Insulin and the Insulin Receptor

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

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

23. Phosphotyrosine Recognition (SH2 Domains)

24. Insulin and the Insulin Receptor
Sos = GAP for Ras