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GPCR Functions.docx

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Biology 2382B
Jessica Kelly

Lecture 16 – GPCR Functions G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins that respond to a variety of extracellular stimuli. Each GPCR binds to and is activated by a specific ligand stimulus that ranges in size from small molecule catecholamines, lipids, or neurotransmitters to large protein hormones. When a GPCR is activated by its extracellular ligand, a conformational change is induced in the receptor that is transmitted to an attached intracellular heterotrimeric G protein complex. ThesG alpha subunit of the stimulated G protein complex exchanges GDP for GTP and is released from the complex. In a cAMP-dependent pathway, the activated G alsha subunit binds to and activates an enzyme called adenylyl cyclase, which, in turn, catalyzes the conversion of ATP into cyclic adenosine monophosphate [2] (cAMP). Increases in concentration of the [3]ond messenger cAMP may lead to the activation of  cyclic nucleotide-gated ion channels  exchange proteins activated by cAMP (EPAC) [4such as RAPGEF3 [5]  an enzyme called protein kinase A (PKA). The PKA enzyme is also known as cAMP-dependent enzyme because it gets activated only i[6]AMP is present. Once PKA is activated, it phosphorylates a number of other proteins including:  enzymes that convert glycogen into glucose  enzymes that promote muscle contraction in the heart leading to an increase in heart rate  transcription factors, which regulate gene expression Specificity of signaling between a GPCR and its ultimate molecular target through a cAMP-dependent pathway may be achieved through formation of a multiprotein complex that includes the GPCR, adenylyl [7] cyclase, and the effector protein. Regulation of Adenylyl Cyclase (effector) Adenylyl cyclase catalyzes formation of cyclic AMP (cAMP) … • GPCR/cAMP pathway is very common in many mammalian cells • GPCR can bind to both inhibitory and stimulatory ligands 1 Bound GPCR interact with G proteins whose Gsubunits may be inhibitory or stimulatory to the effector protein Gs  stimulates adenylyl cyclase to produce cAMP Gi  inhibits adenylyl cyclase hindering the production of cAMP Inappropriate Activation of Adenylyl Cyclase – Cholera Toxin • Bacteria V. cholera produce cholera toxin (a peptide) • Toxin that accumulates in the intestinal lumen binds to receptors on the apical surface of intestinal epithelial cells and is transported into the cell by retrograde transport • Once inside the cell the toxin binds to the Gs subunit which binds GTP but cannot hydrolyze it (Gs is always active) Constitutively active (toxin bound) Gs-GTP has strong association with adenylyl cyclase causing it to constitutively produce cAMP High Intracellular cAMP levels  activate CFTR to pump Cl- ions into the intestinal lumen… Na+ ions follow Cl- ions as well  H O2will follow by osmosis… 2 Thus through this mechanism cholera toxin leads to massive water loss and dehydration that can if not treated (by replenishment of water with electrolyte solution) can lead to death… **CFTR mutation that causes CF**  recessive allele thought to have propagated in white Europeans due to heterozygote advantage that came from having mutation  individuals that are heterozygous do not lose as much water when infected with cholera  thus less likely to die from infection Inappropriate Activation of Adenylyl Cyclase Whooping Cough (Pertussis Toxin)  Bordetella pertussis toxin enters ciliated epithelial cells in lungs  Pertussis toxin maintains Gi in inactive state (GDP) (i.e. inhibits Gi) Inactive state of inhibitory G subunit results in increase in adenylyl cyclase activity = higher levels of intracellular cAMP… Increased levels of cAMP = Increased CFTR activity = Increased pumping of Cl- out of lung epithelia cells (Na+ follows  so does 2 0 by osmosis)… 3 = Mucous secretion and electrolyte/ H 2 accumulation in lungs…cough results from lungs trying to clear the mucous secretion… Extracellular Signalling Molecules Can Have Variety of Effects Depending on the tissue/cell type in which a hormone is acting on, it can induce a variety of cellular responses…(i.e. epinephrine)… 4 cAMP Activates Protein Kinase A (PKA) Inactive when not bound to cAMP  tetramer composed of 2 catalytic subunits & 2 regulatory subunits (R subunits bind to catalytic site on C-subunits = inactivation) Activated by binding of cAMP  cAMP binds to regulatory subunits in a cooperative fashion causing the dissociation of the regulatory subunits from the catalytic subunits (which know of kinase activity) st st Cooperative Binding Fashion = binding of 1 ligand (i.e. 1 cAMP) lowers the K for d the binding of the 2 ligand (i.e. 2 cAMP)  PKA is an exception in that the catalytic domain separates from the regulatory domain… How cAMP Binds To R:  1st cAMP to CNB-B domain on R  Binding of 1 molecule lowers the Kd for binding of the 2nd cAMP molecule to CNB-A domain on R  5 Signal Amplification Signal Amplification allows a small amount of signal molecules to bind to a small number of complementary receptors and induce a large & rapid cellular response… Example: Epinephrine/cAMP • There is about a 10,000 fold amplification of the signal from epinephrine to cAMP • Enzymes  have the ability to repeat their function over and over again as long as they are in their active state = further amplification of signal o GPCR receptors & G-proteins can diffuse rapidly in the membrane  1 activated receptor can in turn activate up to 100 G-proteins, which will activate cAMP production to further amplify the signal… o Activated kinases can influence the activity of a number of other signaling molecules = further amplification Theoretically you could get a 100,000,0000 fold amplification of signal through this mechanism of signal transduction catalyzed by enzymes… 6 Regulation of Glycogen Metabolism By cAMP (Short-term Glycogenolysis  process that takes a stored sugar source (i.e. glycogen) and down to produce glucose that can be used by cells to produce energy (ATP)breaks it • Epinephrine (i.e. fight or flight hormone) results in the liberation of glucose molecules that are stored as glycogen allowing the glucose to used by a cell or transported to other cells and feed into glycolytic pathways to produce energy (ATP) • Epinephrine  activates adenylyl cyclase through GPCR  Increase in cAMP PKA  activated by binding of cAMP…is a protein kinase that phosphorylates serine and threonine residues  It acts to phosphorylate target enzymes involved in glycogen storage/breakdown to either activate or inactivate them depending on the enzymes function 7 PKA in Glycogen
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