Lecture 7 Chapter 15 Cell signaling Communication Between

Lecture #7 Chapter 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.

What are G-proteins? • G proteins bind GTP: guanosine triphosphate. Control and amplify intracellular signaling pathways Exist in two states (hormone, GF, drug) 1) bound GTP: active 2) bound GDP: inactive Examples of GTPase proteins Ras, Cdc-42 Fig. 15. 1

Jennifer studies G-proteins! 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.

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 constitutivelyactive (GTP-bound) form in approximately 20% of human cancers.

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).

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

Fig 15. 3 The G Protein Cycle

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. Minireview Translocation and Reversible Localization of Signaling Proteins: A Dynamic Future for Signal Transduction Mary N. Teruel * 1 and Tobias Meyer * 1 Department of Molecular Pharmacology, Stanford University Medical School, 269 Campus Drive, Stanford, California 94305 1

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

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

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

Ras Activation and the MAP Kinase Cascade Tyrosine Kinases, G-Protein coupled receptors See Fig. 15. 2 Raf-1 (Map kinase) MEK (MAP kinase) Nuclear regulatory proteins Cytoplasmic substrates ERK (MAP kinase)

What does Ras interact with? Raf

G proteins • G protein structure • G protein regulation

Why do we care about the structure of G proteins: including the Ga and Gbg interface? • • 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 • Fig. 15. 11 The a subunit binds and hydrolyzes GTP Ergo: contact surface between Ga and Gbg has major regulatory importance.

The regulation of G proteins. Fig. 15. 12

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

A Schematic of the Gb Propeller Structure Each propeller has 4 b-sheets 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.

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?

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.

Why do we need GAPs? • To buy kakis

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, are required to assist G proteins in hydrolyzing GTP.

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

Why do we need GEFs?

Answer The somnolescent state attained after hydrolysis 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 GDP in the cytosol. Replacement of GDP by GTP in the active site of a G protein is the turn-on signal that almost invariably requires the assistance of a guanine nucleotide exchange factor, or GEF.

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: c. AMP response element binding protein. Binds to DNA at the CRE (c. AMP Response Element) and activates transcription.

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

Can eating CREB make you smart? No But eating donuts can make you happy, and happiness is good psychological health! • Hopefully donuts do not stimulate activation of CREB repressor genes!

c. AMP is generated from ATP by an enzyme: adenylyl cyclase. AC is regulated by G proteins

c. AMP activates one or more kinases. What are phosphatases?

Activation of c. AMP and Protein Kinase A also play major roles in response of liver to glucagon or epinephrine. Figure 15. 7

Thursday, IP 3, Calcium and Receptor tyrosine kinases • END

Regulation of PIP 2 and PIP 3 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).

Plasma Membrane Functions that Require . Function Phosphoinositide Possible Mechanism Membrane Trafficking • • Endocytosis • PIP 2 • Recruitment of AP 2 to membrane initiating clathrin coating • • PIP 2 • Uncoating of clathrin-coated vesicles, through synaptojanin-1 -mediated PIP 2 hydrolysis • Regulated exocytosis • PIP 2 • Recruitment of CAP protein to sites of vesicle fusion • Membrane/Cytoskeletal Interface • Micovilli formation • PIP 2 • Activation of ERM proteins • Membrane attachment to cytoskeleton • PIP 2 • Binding to gelsolin, profilin, other actin regulator proteins • Phagocytosis • PIP 2/PIP 3 • Regulation of ARF 6, PLD and actin assembly