cell signalling secondary messengers and GPCRs 10-10-23.docx

G protein coupled receptors (GPCRs) – another type of receptor Largest class of human cell surface receptors e.g., involved in vision, taste smell etc. More than half of the drugs available target GPCRs receptors in Binds to huge array of molecules. Span the membrane 7 times, has 7 transmembrane...

G protein coupled receptors (GPCRs) – another type of receptor Largest class of human cell surface receptors e.g., involved in vision, taste smell etc. More than half of the drugs available target GPCRs receptors in Binds to huge array of molecules. Span the membrane 7 times, has 7 transmembrane domains. Contains extracellular part where ligand binds, and cytosol part which interacts with downstream molecules. As the name suggests, these receptors are coupled with G proteins. How G proteins are activated by GPCR’s Ligand binds to GPCR. Changes confirmation of the receptor. Activates the GPCR Results in GDP removed from alpha subunit of the G protein as its affinity decreases and replaced by GTP. Causes activation of G protein. Once the G protein is activated, often alpha subunits dissociate from beta and gamma subunits and initiate different signalling pathways. Activates downstream signalling pathways. An active GPCR can activate many other G proteins. Trimeric G proteins Acts as molecular switches. Trimeric G-proteins- contains alpha, beta and gamma subunits. Alpha subunit can bind either to GDP or GTP The alpha and gamma subunits are anchored by lipid modifications into leaflet of lipid bilayer. Can activate adenylyl cyclase. Are transmembrane proteins. Adenylyl cyclase catalyses the formation of cyclic AMP from ATP. cAMP is a secondary messenger. How is cAMP formed by adenylyl-cyclase’s which are activated by G protein. ATP molecule is hydrolysed, and 2 phosphates are removed. The adenyl cyclase forms a cyclic bond which produced cAMP which is a short-lived molecule. cAMP is further hydrolysed into monomeric AMP by cyclic AMP phosphodiesterase. Accessory proteins are needed for G-proteins to activate or de-activate them. For GDP to be removed from GTPase and replaced by GTP, an activator is needed which is Guanine nucleotide exchange factor (GEF) When GTP is hydrolysed back to GDP, it is with the help of GTPase activating protein (GAP)- this then inactivates the monomeric G proteins. Second messengers- alter metabolism or effect effector enzymes that modulate target proteins and eventually cell behaviour. E.g., cAMP, cGMP Ca2+, DAG and IP3 G-protein families- 4 major groups Gs – stimulates adenylate cyclase. Gi/o – inhibits adenylate cyclase. Gq/11 – stimulates PLCß (enzyme called phospholipase beta) G12/13 – activates RhoGEFs (don’t need to know). Stimulatory and inhibitory G proteins Different G protein coupled receptors can act through inhibitory G proteins or stimulatory G proteins therefore the amount of adenylyl cyclase produced varies therefore production of cAMP varies. Cellular effects of cAMP cAMP activates cAMP dependent protein kinase A (PKA)- involved in signalling pathway. PKA is formed of 4 subunits, 2 are regulatory subunits and 2 catalytic subunits. 4 molecules of cAMP are needed to bind to regulatory subunits. The regulatory subunits can dissociate from catalytic subunits. Catalytic subunits become active and phosphorylates their targets. PKA can influence transcription therefore gene expression can change etc. Gq can activate phospholipase C beta (PLCb)- another class of G proteins Signal molecule binds to receptor of G protein coupled receptor(GPCR). Recruits trimeric G protein and activates it. Activation of G protein activates phospholipase c-beta- enzyme that breaks down lipids Only works on phosphate inositol 4,5-bisphosphate- type of phospholipid. Glycerol stays in the membrane and inositol 1,4,5-triphosphate (IP3) is given off. Both diacylglycerol (DAG) and inositol 1,4,5-triophospahte (IP3) are important second messengers DAG activates protein kinase C IP3 releases Ca2+ from ER Both DAG and Ca2+ activate protein kinase C Cellular effects of Ca2+ Activates calmodulin. (links to the contraction of smooth muscle) Modulates activity of different proteins Activates calmodulin dependent kinases. G proteins can control channel opening Can do it indirectly via secondary messengers e.g. Ca2+ ,cAMP etc Can also directly bind to channel and module activity. G proteins themselves can act as ligands that can open or close ion channels. How is signal transmitted downstream? Opening of channel leads to influx of ions across membrane. As a result, membrane potential changes Intracellular ion concentration rises. (importantly of Ca2+ ). Example of how G-protein coupled receptors allow us to see. In dark Guanine cyclase (GC) is an enzyme that converts GTP to cGMP (secondary messenger) cGMP is the ligand for sodium channels so it binds to the receptors and keeps it open therefore constant influx of sodium ion which depolarises the membrane. In light When a photon of light hits, it activates the receptor Recruits G protein which is bound to GDP GDP is replaced by GTP. Alpha subunit in the trimeric G protein dissociates away from beta and gamma subunits. Activated alpha subunit activates phosphodiesterase (PDE). PDE counter reacts and converts the cGMP to GMP Less cGMP therefore less binding to sodium channel and sodium channel close Changes membrane potential and generate action potential therefore we can then see.