Lecture #7 Chapter 15 Cell signaling: Communication Between Cells and Their Environment

1. Lecture #7Chapter 15 Cell signaling: Communication Between Cells and Their Environment Overview of intracellular signaling: Initiation and responses Axiom #7 Study hard and stay focused on your educational goals. But always have a backup plan! Remember: while chance favors the prepared mind, you can’t always be where the lighting strikes.

2. What are G-proteins? • G proteins bind GTP: guanosine triphosphate. Control and amplify intracellular signaling pathways Exist in two states 1) bound GTP: active 2) bound GDP: inactive (hormone, GF, drug) Examples of GTPase proteins Ras, Cdc-42 Fig. 15.1

3. Jennifer studies G-proteins! The J Lo motto: A detailed understanding of the fundamental processes that govern this GTPase cycle will provide a basis for discerning how cells relay signals. G-PROTEINS

4. GTPases and disease. • Damage to these small GTPase switches can have catastrophic consequences for the cell and the organism. • Several small GTPases of the Rac/Rho subfamily are direct targets for clostridial cytotoxins. • Further, Ras proteins are mutated to a constitutively-active (GTP-bound) form in approximately 20% of human cancers.

5. G-proteins are tightly regulated 3 types of accessory proteins that modulate cycling of G-proteins between GTP/GDP 1. GAPs: GTPase-activating proteins. Stimulate GTP hydrolysis. Inactivate G-protein. Example of a GAP: PLCb. 2. GEFs: Guanine nucleotide-exchange factors: G-protein-coupled receptors (GPCR). Stimulate dissociation of GDP (inactive) from G-protein so GTP can bind (active). 3. GDIs: Guanine nucleotide-dissociation inhibitors. Inhibit release of bound GDP (maintain G-protein in inactive state).

6. The heterotrimeric G proteins transmit signals from a variety of cell surfacereceptors to enzymes and channels • Stimulated by receptors • Act on effectors • Regulated by nucleotide exchange and hydrolysis

7. Fig 15.3 The G Protein Cycle

8. MinireviewTranslocation and Reversible Localization of Signaling Proteins: A Dynamic Future for Signal Transduction Mary N. Teruel *1 and Tobias Meyer *1 1 Department of Molecular Pharmacology, Stanford University Medical School, 269 Campus Drive, Stanford, California 94305 GTP is very small and can diffuse rapidly throughout the cell • Diffusion-Mediated Random Walk of Signaling Proteins • Schematic representation of a 4 s long random walk of (left) a cytosolic protein, (middle) a membrane-bound protein, and (right) a receptor. Simulated with MATLAB.

9. GTP binds to GTPases. Hold on! • Motors on vesicle with G-proteins. • Interesting thought: Some motors has GAP domains (Myosin-IX) • G-proteins may act as motor attachment factors. (Rabs)

10. Rabs are members of the small G-protein family • Rab 6: a GTPase for Kinesin • Rab 27a: A GTPase for myosin-Va • Defects in Rab27a cause Griscelli syndrome

11. Other G Proteins • Rho Family of GTPases (convergent pathways) Cdc 42: actin-dependent membrane ruffling Rac: actin-dependent membrane ruffling Rho: actin-dependent focal contacts (FAK), stress fibers • Ras: proliferation

12. Ras Activation and the MAP Kinase Cascade Tyrosine Kinases, G-Protein coupled receptors See Fig. 15.2 Raf-1 (Map kinase kinase kinase) MEK (MAP kinase kinase) ERK (MAP kinase) Nuclear regulatory proteins Cytoplasmic substrates

13. What does Ras interact with? Raf

14. G proteins • G protein structure • G protein regulation

15. Why do we care about the structure of the structure of G proteins: including the Ga and Gbg interface? • The a subunit binds and hydrolyzes GTP • GTP-a: dissociates from Gbg (tightly associated) • Both subunits (a and bg, then activate their respective effectors). • Following hydrolysis of GTP to GDP, subunits reassemble and become inactive • Ergo: contact surface between Ga and Gbg has major regulatory importance. Fig. 15.11

16. The regulation of G proteins. Fig. 15.12

17. A Ribbon Diagram of the G a,b,g • The heterotrimer consistsof an a subunit that binds and hydrolyzes GTP and a pairof proteins, b and g, that are tightly associated with each other. • The G a subunit isdisplayed in light blue, the Gb in green, and the Gg in dark blue

18. A Schematic of the Gb Propeller Structure The schematic shows the relative placement of the four sequential strands in each of the seven blades. Also shown are the key WD repeat amino acids (see Figure 4). The seven symmetrically placed surface Asps in the tight two to three residue turn between strands b and c are indicated by green cirles on the top surface of G . These are not the D of WD. The highly conserved aromatics at the lower ends of strands a and c are shown by blue circles. The Asp of the defining WD, potentially exposed on the propeller's wider bottom surface, is indicated by a red circle. Each propeller has 4 b-sheets

19. Take Home Message #1.Cells maintain their signaling outputs by establishing a balance between the nucleotide exchange rate and the hydrolysis rate • What is the favored bound nucleotide in the resting cell? G-GTP or G-GDP?

20. Answer: G-GDP In the basal state, G alpha releases GDP at a slow rate (0.002 s-1) compared to its rate of GTP hydrolysis (0.05 s-1 for G). This kinetic balance ensures a very low population of activated G protein molecules, and maintains the cell in a resting state. Upon binding to agonist, G protein coupled receptors accelerate G alpha subunit GDP/GTP exchange.  Receptors work as catalysts, achieving rate enhancements of up to 104-fold. As receptor-driven nucleotide exchange becomes fast relative to hydrolysis, the balance of rates in the GTPase cycle changes. The new balance increases the population of GTP-bound species, thus shifting the cell to an activated state. 

21. Why do we need GAPs? • To buy kakis

22. Answer • The kinetic barrier to GTP hydrolysis is substantial, allowing G proteins to maintain the active signaling state for seconds, potentially hours. Hence, GTPase-activating proteins, or GAPs, arerequired to assist G proteins in hydrolyzing GTP.

23. Take home message #2 Hydrolysis is the turn-off signalthat induces heterotrimeric G protein a subunits (Ga ) to disengage their effectors. Note change in structure of GTP versus GDP bound G protein

24. Why do we need GEFs?

25. Answer The somnolescent state attained afterhydrolysis should be similarly protracted without intervention; again, the kinetic barrier to product(GDP) release is high, even though GTP is in 10-fold molar excess to GDPin the cytosol. Replacement of GDP by GTP in the active site ofa G protein is the turn-on signal that almost invariably requires theassistance of a guanine nucleotide exchange factor, or GEF.

26. Do you remember everything from yesterday’s lecture?If you say yes, you get a donut! If not, then you need to pay attention to the CREB story. Fact: CREB: cAMP response element binding protein. Binds to DNA at the CRE (cAMP Response Element) and activates transcription.

27. What does CREB do? • Landmark papers in 1995 demonstrated that cAMP-dependent transcription via CREB enhance the formation of long-term memory (LTM)

28. Can eating CREB make you smart? NoBut eating donuts can make you happy, and happiness is good psychological health! • Hopefully donuts do not stimulate activation of CREB repressor genes!

29. cAMP is generated from ATP by an enzyme: adenylyl cyclase. AC is regulated by G proteins

30. cAMP activates one or more kinases. What are phosphatases?

31. Activation of cAMP and Protein Kinase A also play major roles in response of liver to glucagon or epinephrine. Figure 15.7

32. Thursday, IP3, Calcium and Receptor tyrosine kinases • END

33. Regulation of PIP2 and PIP3 Synthesis Green arrows denote stimulatory effects; blue arrows denote synthetic pathways; red denotes inhibitory effect. Feedback inhibitory loop (1). Cross-talk between receptor signaling pathways (2). Feed-forward loop (3).

34. Plasma Membrane Functions that Require • . Function    Phosphoinositide     Possible Mechanism     • Membrane Trafficking     • Endocytosis     • PIP2     • Recruitment of AP2 to membrane initiating clathrin coating     • PIP2     • Uncoating of clathrin-coated vesicles, through synaptojanin-1-mediated PIP2 hydrolysis     • Regulated exocytosis     • PIP2     • Recruitment of CAP protein to sites of vesicle fusion     • Membrane/Cytoskeletal Interface     • Micovilli formation     • PIP2     • Activation of ERM proteins     • Membrane attachment to cytoskeleton     • PIP2     • Binding to gelsolin, profilin, other actin regulator proteins     • Phagocytosis     • PIP2/PIP3     • Regulation of ARF6, PLD and actin assembly     • Cell Signaling     • Protein kinase localization and activation     • PIP2/PIP3     • Localization of PDK1 and PKB/Akt     • PtdIns(3,4)P2/PIP3     • Regulated localization of Btk tyrosine kinase     • Regulation of ARF GTPases     • PIP2/PIP3     • Localization of ARF6 exchange factors     • PIP3     • ARFGAP localization     • PIP2     • Activation of ARF GAP     • EGFR regulation of membrane ruffling     • PIP2     • Actin reorganization