A Case Study of Signal Transduction: Heterotrimeric G-Protein Signaling

1. A Case Study of Signal Transduction: Heterotrimeric G-Protein Signaling MCSB Bootcamp 2009 9/16/09

2. Outline • Heterotrimeric G-protein signaling • Introduction to chemotaxis

3. Signal Transduction Pathway Input (Signal) Receptor Transducers Regulation Effectors Outputs

4. Where does the ligand come from? Where is the receptor? In Cell Biology We Are Concerned With Spatial Characteristics

5. Checklist of Signal Transduction Systems

6. Evolutionarily conserved in eukaryotes • > 50% of all clinically marketed drugs are targeted against G-protein systems. Heterotrimeric G-Protein Systems

7. Overview of G-Protein Activation heterotrimer (3 subunits: a, b, g)

8. bg GTP GDP G G bg G-Protein Cycle Active Guanine Exchange Factor GTPase Activating Protein GEF (receptor) GAP (RGS) Regulator of G-protein Signaling Exchange of GTP for GDP Hydrolysis of GTP to GDP Inactive

9. G-Protein Structure Lipid modifications of G-protein subunits farnesyl geranylgeranyl

10. G-Protein Classification(Mammalian Sequences) • In mammals: • 4 classes of a-subunits • Gs, Gi, Gq, G12 • 17 a-subunits • 4 b-subunits • 6 g-subunits

11. Evolutionary Conservation of G-Proteins in Eukaryotes • From simplest (e.g. yeast) to most sophisticated eukaryotes (e.g. humans) • “First” among eukaryotic signal transduction pathways • Conservation of sequence • Conservation of structure • Conservation of function • Human G-protein subunits can substitute for yeast proteins • Conservation of regulators and effectors

12. G-Protein Coupled Receptors (GPCRs) • Possess canonical 7 transmembrane helix structure • Bind variety of ligands • Small molecules, peptides, proteins • 5 GPCR families by sequence alignment • A) Rhodopsin (largest class) • B) Secretin (hormones) • C) Glutamate • D) Fungal pheromone • E) cAMP (Dictyostelium) • ~ 1400 human GPCRs (~ 5% of human genome) • ½ are olfactory receptors • Primary sensors for humans (as well as other eukaryotes)

13. Pharmacology: Agonists and Antagonists • G-protein systems (e.g. GPCRs) are common targets of pharmaceuticals • An agonist binds to and activates the receptor • An antagonist binds to the receptor and prevents its activation • Partial agonists partially activate the receptor • Inverse agonists lower the basal level of receptor activity

14. Response Log Concentration Dose-Response Curves • Measure the response as a function of drug dose (or input signal) • EC50 is the concentration of drug that produces 50% of maximal effect • Often the Kd (equilibrium dissociation binding constant) is close in value to the EC50. • Potency = concentration (EC50) of a drug required to produce 50% of drug’s maximal effect • Maximal Efficacy = maximal response to the drug Greater Sensitivity Greater Response

15. Some G-Protein Effectors • Effectors are activated (or inhibited) by Ga-GTP or Gbg • Adenylyl cyclase (Gs) • cAMP • Ion channels (Gbg) • Kinases • Phospholipase C (Gq and Gbg) • IP3 (activates Ca++) and diacylglycerol (activates protein kinase C) • Small G-protein exchange factors (G12/G13) • e.g. activator of Rho • Scaffold proteins

16. Cross-Talk between GPCRs and Other Receptor Systems

17. Signal Adaptation (Alberts p. 851) • Dynamic Range = cell detect changes in signal over a wide range of stimulus intensities • Broad dynamic range means “wide” dose-response curve to input signal • Requires that the target cells undergo a reversible adaptation or desensitization • Prolonged exposure to stimulus decreases cells’ response to that level of stimulus

18. (1) X (2) (3) Regulation of Signaling L k1 (1) Ligand (2) Receptor (3) RGS k2 RL R k3 Ga Ga* k4

19. Receptor Regulation: Phosphorylation

20. Receptor Regulation:Endocytosis (late endosome)

21. Experimental Tools for G-Proteins • Genetics • Loss-of-function mutants (e.g. dominant negative) • Gain-of-function mutants (e.g. constituitively active) • Pharmacology • Pertussis toxin (inactivates Gi subunit) • Receptor agonists/antagonists • Molecular biology • Reporters of signaling • Biochemistry • In vitro system • Cell Biology • GFP-labeled proteins and microscopy • Physiology and behavior

22. Biological (G-Protein) Sensors … Olfaction Vision Immune … much better than technological sensors

23. Complex Dynamic Behaviors:Neutrophil Chasing Bacterium • http://www.biochemweb.org/fenteany/research/cell_migration/neutrophil.html • Sensing (Heterotrimeric G-Protein) • Response (Small G-Protein)

24. Chemotaxing Eukaryotic Cell Leading Edge (Pseudopod) Ligand: = fMLP Formyl-methiomyl-leucine-phenylalanine Trailing Edge (Uropod)

25. Chemotaxis • Directed movement toward or away from specific chemicals • Move toward nutrients, mating partner, etc. • Move away from toxins, harsh conditions, etc. • Universal behavior in living organisms • Involves sensing and movement

26. x2 t2 Spatial Sensing x1 t1 Two Chemotactic Strategies Temporal Sensing (Differentiator) (reorient) (project)

27. C. ? Neutrophil Chemotaxis Examples B. A. Temporal Sensing: Bacterial Chemotaxis Spatial Sensing: Yeast Mating a a

28. Lecture 1: Some Questions to Ponder (Optional Homework) • What is the role of signaling adaptation? • What types of mechanisms and dynamics can produce adaptation? • What happens if adaptation is disrupted?