GPCRs - Lecture Note

GPCR’S Side notes: Phosphorylation of an enzyme always turns it on. • True • False G Protein Coupled Receptors Receptor activates G protein G-protein activates a target protein (enzyme or ion channel) also located at the cell membrane Enzymes can transducer signal further There are more than 700 GPCRs in humans Responsible for our sense of smell, light, taste Other GPCRs respond to hormones and neurotransmitters The family members include Photoreceptors in the eye Olfactory (smell) receptors in the nose Half of the known drugs work through GPCRs The structure of trimeric G protein Three subunits of G proteins: alpha, beta, gamma Alpha subunit is a GTPase Beta-gamma complex interacts with target proteins Activation of alpha induces a conformational change, which exposes the surface between alpha and beta-gamma This surface allows them to interact with target proteins Two main kinds of molecular switches Two mechanisms of turning protein activity “on” and “off” Which of the following is true? • GPCR is a GEF for G protein • GPCR is a GAP for G protein • G protein is a GEF for GPCR GTPase function of a G protein _____ • Allows it to bind to other proteins and activate them • Switches a G protein to a conformation when it can phosphorylate other proteins • Switches a G-protein into an inactive GDPbound form GTP binding switches an enzyme into the active state GTP-bound state is active GDP-bound state is inactive Enzymes shut themselves off by hydrolyzing GTP to GDP Two kinds: G-proteins and small monomeric GTPases Two activities of a G protein (1) Main activity is to activate other proteins in a signal transduction chain by binding to them and inducing a conformational change. They can do it only in GTP-bound form (2) GTPase activity that leads to hydrolysis of GTP, bound inside of the G protein. It results in inactive, GDP-bound form of the G protein G protein functions as a molecular switch GEF, guanine nucleotide exchange factor, turns activity “on”. GPCR functions as GEF GAPs, GTPase-activating proteins, drive the proteins into “off” state. For G protein, RGS (regulators of G protein signaling) is a GAP Other types of molecular switches Switch on and off can depend on binding of cAMP or Ca2+ It can also depend on protein modifications other than phosphorylation, such as ubiquitylation Some G proteins regulate the production of cAMP Adneylyl cyclase Activated alpha subunit of G protein activates adenylyl cyclase —> GPCR’s Side notes: Which of the following is always attached to a membrane? produces cAMP from ATP The same as adenylate cyclase Large transmembrane protein Catalytic domain is in the cytosolic side cAMP concentration can increase very fast • • • • G protein GPCR Adenylyl cyclase All of the above Which of the following is not a second messenger? • • • • cAMP NO Diacylglycerol Epinephrine What can be a function of CBP if you know that it activates transcription? • Histone acetlytransferase (HAT) • Histone deacetylase (HDAC) • Histone methyltransferase Second messengers Second messengers are intracellular signaling molecules released by the cell in response to the first messengers The cell releases second messenger molecules in response or exposure to extracellular signaling molecules - the first messengers Second messengers can be used to greatly amplify the signal For example, one activated adenylyl cyclase can generate many cAMP molecules Second messengers are small molecules (not proteins) Some are water soluble (cAMP, Ca2+, IP3) Some are hydrophobic and diffuse throughout the membranes only (diacylglycerol) Gases NO and CO are also considered second messengers cAMP mostly functions by activating protein kinase A (PKA) Binding of cAMP to regulatory subunit causes them to dissociate. The released catalytic subunits phosphorylate target proteins Regulatory subunits help to localize kinase A inside the cell: they bind to A-kinase anchoring proteins; AKAPs. AKAPs bind both to regulatory subunits and cell components (membrane, cytoskeleton, organelle) PKA has multiple roles including regulation of glycogen, sugar, and lipids Sharper responses occur when a target molecule requires simultaneous binding of many effector molecules Protein kinase A phosphorylates transcription factor CREB protein PKA can directly phosphorylate target proteins Other mechanism: it phosphorylates transcription factor CREB protein (CRE binding) CREB binds to CRE Phosphorylated CREB recruits CBP (CREB binding protein) CBP activates transcription of target genes CREB has a role in development, in neurons (formation of long-term memory, survival), and many other organs A change in cAMP concentration can alter gene transcription GPCR activates G protein G protein activates adenylyl cyclase Increase in cAMP concentration Activation of PKA Phosphorylation and activation of CREB CREB binds to CRE and recruits CBP to promoter Transcription activation Turning on the pathway immediately turns it off: PKA activates cAMP GPCR’s Side notes: phosphodiesterase which lowers cAMP G Proteins exist in many flavors Gi = inhibitory G-protein, inhibits adenylyl cyclase Gs = stimulatory G-protein, stimulates adenylyl cyclase Gq = atypical G-protein, couples to other effector molecules (for example, opens or closes ion channels)