G Protein Coupled Receptors PDF

G-Protein-Linked Receptors Session Learning Outcomes (SLOs) SLO# 1 : List the pathways of signal transduction to generate different physiological responses. SLO# 2 : Describe the role of G proteins in the activation of specific effector proteins. SLO# 3 : Define primary and secondar...

G-Protein-Linked Receptors Session Learning Outcomes (SLOs) SLO# 1 : List the pathways of signal transduction to generate different physiological responses. SLO# 2 : Describe the role of G proteins in the activation of specific effector proteins. SLO# 3 : Define primary and secondary messengers and give specific examples of each. G-Protein-Linked Receptors G-protein-linked receptors form the largest family of cell-surface receptors, with hundreds of members already identified in mammalian cells. They mediate response to an enormous diversity of extracellular signal molecules, including hormones, local mediators, and neurotransmitters. These signal molecules are as varied in structure as they are in function. Tthey can be proteins, small peptides, or derivatives of amino acids or fatty acids, and for each one of them there is a different receptor. Despite the diversity of signal molecules that bind to them, all G-protein-linked receptors that have been analyzed possess a similar structure: each made of a single polypeptide chain threads back and forth cross the lipid bilayer seven times. All G-protein-linked receptors possess a similar structure  Stimulation of G-Protein-linked Receptors Activates G-Protein Subunits 1- Extracellular signal molecule binds to a seven-pass transmembrane receptor Loading… 2- The receptor protein undergoes a conformational change 3- The receptor protein activates a G protein located on the underside of the plasma membrane G-Proteins G-proteins are heterotrimeric, with 3 subunits α, β, γ. The α subunit of a G-protein (Gα) binds GTP, & can hydrolyze it to GDP + Pi. α & γ subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface.  G-Proteins All G-proteins – similar structure/activation G-protein is a switch turned on by the receptor There are several varieties of G proteins. Each is specific for a particular set of Loading… receptors and particular set of downstream target proteins. G-protein then activates an effector protein (usually an enzyme) There are TWO broad subclasses of trimeric G-protein-activated signal transduction pathways depends on their target effector enzymes: A. adenylyl cyclase B. phospholipase C  A: Some G-proteins Activate adenyl cylase The most frequent target enzymes for G proteins are adenyl cylase Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site. Adenylate Cyclase is responsible for production of the small intracellular signal molecule cyclic AMP (cAMP). Phospholipase C, the enzyme responsible for production of the small intracellular signaling molecules inositoltriphosphate and diacylglycerol. XV Signal Transduction Process via GPCR When there is no ligand, receptor is off; Gα has bound GDP, and α, β, & γ subunits are complexed together in an inactive state (GDP bound state)  The sequence of events by which a hormone activates cAMP signaling: 1-Ligand binds to the extracellular domain of a 7-helix receptor (GPCR), 2- A conformational change in the receptor 3- Activation of G-protein on the cytosolic side of inactive the membrane 4- Gα releases GDP & binds GTP (GDP-GTP exchange) and dissociates from βγ 5- Gα-GTP dissociates from the inhibitory βγ active complex. 6- α subunit changes conformation and binds to adenylate cyclase (AC). 7- Adenylate Cyclase is activated by the stimulatory Gα-GTP.  G-Protein-linked Receptors Receptors have 7 transmembrane helices Interact with heterotrimeric G-Proteins – Gα binds GDP at rest – Gβ and Gγ complete the hetero-trimer Loading… Activation of receptor leads to: – GDP release and GTP binding to Gα – Dissociation of Gβ and Gγ Slow hydrolysis of GTP to GDP returns the system to the resting state Both the activated α subunit and the free big complex can regulate target proteins.  8- An activated Ga-protein-GTP can trigger the formation of cAMP, which then acts as a second messenger in cellular pathways. 9- cAMP activates the enzyme cyclic-AMP- dependent protein kinase (PKA).  The Cyclic AMP Pathway Can Activate Enzymes and Turn On Genes The activated G-protein alpha subunit switches on the adneylyl cyclase, causing a dramatic and sudden increase in the synthesis of cAMP from ATP (Gs). To help eliminate the signal, a second enzyme called cAMP phosphodiesterase, rapidly converts cAMP to ordinary AMP. cAMP-dependent protein kinase (PKA) mediate most of the effects of cyclic AMP Role of cAMP, PKA in glycogen metabolism  10- PKA catalyzes transfer of phosphate from ATP to serine or threonine residues of various cellular proteins, altering their activity. 11- PKA phosphorylates. stimulaspecific gene regulatory proteins te the transcription of a whole set of target genes. This type of signaling pathway controls many processes in cells, ranging from hormone synthesis in endocrine cells to the production of proteins involved in long-term memory in the brain.  The b -adrenergic pathway Activated PKA can also phosphorylate and thereby regulate other proteins and enzymes in the cytosol ACTH: Adrenocorticotropic hormone  B: Some G-Proteins activates phospholipase C Some extracellular signal molecules exert their effects via a type of G- protein that activates the membrane-bound enzyme phospholipase C instead of adenylyl cyclase. The Inositol Phospholipids Pathway Triggers a Rise in Intracellular Ca2+  target effector enzyme is Phospholipase C PLC cleaves a membrane phospholipid (Phoshatidyl inositol) Two 2nd Messengers Inositol-1,4,5-Trisphosphate Diacylglycerol (InsP3) (DAG) Release of Ca++ ions Activation of PKC  Response: Release of Ca++ ions Activated Protein Kinase C Binds & activates Phosphorylates Calmodulin-binding proteins Activates kinases & phosphatases Target proteins (ser & thr) Cell growth Regulation of ion channels cytoskeleton increases cell pH Protein secretion  A Ca2+ Triggers Many Biological Processes Ca 2+ has such an important and widespread role as an intracellular messenger. The effects of Ca 2+ in the cytosol are largely indirect; they are mediated through the interaction of Ca2+ with various transducer proteins known as Ca2+ - binding proteins. The most widespread and common of these is the Ca2+ responsive protein called calmodulin. G-protein-linked signaling : Inositol phospholipid pathway: A Ca2+ signal triggers many biological process Example: the fertilization of an egg by a sperm triggers an increase cytosolic Ca2+ in the egg. C: Some G Proteins Regulate Ion Channels The target proteins for G-protein subunits are either ion channels or membrane bound enzymes. Different targets are affected by different types of G proteins, and these various G-proteins are themselves activated by different classes of cell surface receptors. In this way, binding of an extracellular signal molecule to a G-protein- linked receptor leads to effects on a particular subset of the possible target proteins, eliciting a response that is appropriate for that signal and that type of cell. G proteins couple receptor activation to the opening of K+ channels in the plasma membrane of heart muscle cells. 1. Binding of the neurotransmitter acetylcholine to its G- protein–linked receptor on heart muscle cells results in the dissociation of the G protein into an activated βγ complex and an activated α subunit. G proteins couple receptor activation to the opening of K+ channels in the plasma membrane of heart muscle cells. 2- The activated γβ complex binds to and opens a K+ channel in the heart cell plasma membrane. G proteins couple receptor activation to the opening of K+ channels in the plasma membrane of heart muscle cells. 3- Inactivation of the α subunit by hydrolysis of bound GTP causes it to re-associate with the βγ complex to form an inactive G protein, allowing the K+ channel to close  Fine-Tuning of the Response There are four aspects of fine-tuning to consider 1- Amplifying the signal (and thus the response). 2- Specificity of the response. 3- Overall efficiency of response, enhanced by scaffolding proteins. 4- Termination of the signal  1- Signal Amplification Enzyme cascades amplify the cell’s response At each step, the number of activated products is much greater than in the preceding step Loading…  Reception Binding of epinephrine to G protein-coupled receptor (1 molecule) What do we have/need 2nd messengers and G proteins? Transduction Inactive G protein For amplification Active G protein (102 molecules) 1. Single hormone binds to a single Inactive adenylyl cyclase Active adenylyl cyclase (102) receptor molecule -> activates multiple G proteins (10x) -> each G protein activates a ATP Cyclic AMP (104) single adenylate cyclase (AC) -> each adenylate cyclase makes multiple cAMP Inactive protein kinase A Active protein kinase A (104) (100-1000x) -> 104 amplification with a Inactive phosphorylase kinase single hormone molecule. Active phosphorylase kinase (105) Inactive glycogen phosphorylase 2. Each cAMP binds to a single PKA -> Active glycogen phosphorylase (106) each PKA can phosphorylate multiple Response Glycogen targets. Amplifies signal from 104 to 106 Glucose 1-phosphate (108 molecules)  2- The Specificity of Cell Signaling Different kinds of cells have different collections of proteins which allow cells to detect and respond to different signals. Even the same signal can have different effects in cells with different proteins and pathways – Pathway branching and “cross-talk” further help the cell coordinate incoming signals  3- Signaling Efficiency: Scaffolding Proteins and Signaling Complexes Scaffolding proteins are large relay proteins to which other relay proteins are attached Scaffolding proteins can increase the signal transduction efficiency by grouping together different proteins involved in the same pathway Signaling Plasma molecule membrane Receptor Three different protein kinases Scaffolding protein  4- Termination of the Signal Inactivation mechanisms are an essential aspect of cell signaling When signal molecules leave the receptor, the receptor reverts to its inactive state How to shut it off? 1-Within seconds, the GTP on the a subunit is hydrolyzed to GDP by the Gα (GTPase activity). Alpha subunit dissociates from AC, turning it off –alpha binds to βγ complex to reform an inactive G protein -> signal is off. Self-Inactivation in G-protein Signaling  How to shut it off? 2-. Hydrolysis of cAMP AMP by Phosphodiesterases -> signal is off. - cAMP phosphodiesterase is inhibited by caffeine, theophylline (tea), and theobromine (chocolate) - cAMP helps improve memory cAMP- phosphodiesterase rapidly cleaves cAMP (so short lived) 0  How to shut it off? 3- In some cases the activated receptor is phosphorylated via a G-protein Receptor Kinase. The phosphorylated receptor then may bind to a protein β-arrestin, preventing interaction with G proteins -> signal is off. β-Arrestin promotes removal of the receptor from the membrane by clathrin-mediated endocytosis -> signal is off. β-Arrestin may also bind a cytosolic phosphodiesterase, bringing this enzyme close to where cAMP is being produced, converting cAMP to AMP. Therefore, contributing to signal turn off  How to shut it off? 4- cAMP activates PKA phosphorylates target proteins protein phosphatase dephosphorylates proteins target proteins converted back to original form, reversing the signal -> signal is off The G protein is now ready to couple to another receptor. kinases – phosphatases  What if you can’t turn off cascade? Vibrio cholera - causes cholera Normal gut: H20, NaCl, NaHCO3 secretion controlled by hormones via Gs/cAMP signal pathways V. cholera – secretes enterotoxin, chemically modifies Gs – no GTPase activity - stays ON The stimulatory G-protein is permanently activated. Severe watery diarrhea – dehydration, death  Summary: G-PROTEIN–COUPLED RECEPTORS Stimulation of GPCRs Activates G-Protein Subunits Some G Proteins Directly Regulate Ion Channels Some G Proteins Activate Membrane-bound Enzymes The Cyclic AMP Pathway Can Activate Enzymes and Turn On Genes The Inositol Phospholipid Pathway Triggers a Rise in Intracellular Ca2+ A Ca2+ Signal Triggers Many Biological Processes Intracellular Signaling Cascades Can Achieve Astonishing Speed, Sensitivity, and Adaptability