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Lecture 10

Lecture 10 - GCPR functions.docx

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Biology 2382B
Cumming/ Damjanovski

Lecture 10: Functions of G protein-coupled receptors in cells Learning objectives • GPCRs fast acting effectors K+ channels • GPCRs that regulate adenylyl cyclase - – cAMP, epithelial cell Cl channels, PKA, CREB – all of these are 2 messengers • GPCRs that 2+tivate phospholipase C – Ca , IP 3 DAG, PKC, NO, cGMP, PKG In different contexts different effectors and secondary messengers can be produced Activation of muscarinic acetylcholine receptor and K+ channels in the heart Regulation of the speed of the heart • Acetylcholine: produced to slow down the heart • Muscarinic acetylcholine receptor: A GCPR that when activated leads to the efflux of K + which slows down muscle contractions • When acetylcholine bind to its receptor, leads to opening of K+ channel and cellular hyperpolarisation • Slows the rate of heart muscle contraction • Gbγ subunit activates the effector protein (K+ channel) 1. Binding of acetylcholine to muscarinic acetylcholine receptor promotes dissociation of the trimeric G protein complex coupled to it. 2. GDP bound to alpha subunit is displaced and lets GTP bind 3. Promotes dissociation of the alpha subunit from the trimeric complex and the GPCR 4. Exception: Beta-gamma subunit is the one that is freed and goes on to activate the effector K+ channel protein. 5. Potassium channel opens and the K+ ions leave the cells to slow the heart rate Page 1 of8 Regulation of Adenylyl Cyclase • Adenylyl cyclase: effector enzyme associated with trimeric G protein complex. It catalyzes formation of cyclic AMP (cAMP): Major second messenger • The only ligand we need to worry about is epinephrine. • Depending on the ligand-receptor complex you can have either increased cAMP or decreased cAMP. • GPCR/cAMP pathway is very common in many mammalian cells. For increased cAMP 1. Stimulatory ligand I.e. epinephrine can bind to stimulatory hormone receptor (GCPR) 2. Activation of the trimeric G protein complex (stimulatory) 3. Dissociation of Ga subunit to activate effector Adenylyl Cyclase 4. Production of cAMP For decreased cAMP 1. Another inhibitory ligand can bind to an inhibitory hormone receptor (GCPR) 2. Activation of the trimeric G protein complex (inhibitory) 3. Dissociation of the Ga subunit to deactivate the effector Adenylyl Cyclase 4. Production of cAMP is stopped Inappropriate activation of Adenylyl Cylase- Cholera Toxin • Cholera - 200-500,000 cases/yr with 20-50% mortality associated with dirty water • Cholera bacteria will enter the intestine and if it is in high numbers starts producing cholera toxin • toxin enters enterocytes (intestinal epithelial cell) of small intestine using a GM1 ganglioside receptor – the toxin is internalized and binds to G-alpha subunit and activates it • toxin maintains Ga sstimulatory Ga) subunit in active state (GTP) • mass activation of Adenylyl Cyclase • massive increase in cAMP levels – cAMP levels binds an effector protein CFTR Page 2 of 8 • increase leads to hyperactivity CFTR ion channel – high level of cAMP leads to high level of Cl- pumped - + • Cl pumped out & Na follows & H O los2 to intestine(passive and osmotic) • Massive diarrhea and dehydration Whooping Cough – Pertussis Toxin • Bordetella pertussis toxin enters ciliated epithelial cells in lungs • pertussis toxin maintains Ga (iihibitory Ga) subunit in inactive state (GDP) – inhibiting the inhibitor • mass activation of Adenylyl Cyclase – there is always a low level of activation of adenylyl cyclases by Ga subsnit but that is normally held in check by Ga i – Removal of that check causes a gradual accumulation of cAMP • mass increase in cAMP levels • increase activity in ion pumps – same scenario occurs as in cholera • Mucous secretion and electrolyte/ H O ac2umulation in lungs – Cough happens to try and clear the water from the lungs – Opposite scenario than that of Cystic Fibrosis where not enough water is outside of the cells because not enough or any Cl- is pumped out it produces dry mucous as opposed to the watery mucous Fight or flight response Epinephrine is a hormone used in a flight or flight response to increase the levels of cAMP in various tissues Page 3 of 8 • i.e. need to make sugar or glucose to fuel your muscles to run and your heart to pump faster and increase blood flow. • Epinephrine – helps break down tryglycerides, breaks down glycogen and releases it into glucose, increase contraction rate and redirect blood flow away from gastrointestinal tract to the muscles and heart. • Epinephrine works through G Protein Coupled Receptors which turn on Adenylyl Cyclase • Adenylyl Cyclase catalyze the production of cAMP cAMP activates protein kinase A • cAMP accumulates in the cytosol and binds to different proteins including PKA. • Protein Kinase A (PKA) is a tetrameric complex: – 2 catalytic subunits (C) – 2 regulatory subunits (R) • PKA has two cAMP binding sites on a single R – cAMP binds to R in a cooperative fashion: the binding of the 1st cAMP to CNB-B causes the conformational change that lowers the Kd for binding of the 2nd cAMP molecule to CNB-A (both on the same R) • cAMP biniding activates PKA an exception in that the catalytic domain separates from the regulatory domain. • PKA’s catalytic subunits start to phosphorylate things. Four common intracellular second messengers Need to know where they are produced and where they are going to act. • cAMP: activates Protein Kinase A (PKA) • cGMP: activates Protein Kinase G (PKG) • 1,2 Diacylglycerol (DAG): activates Protein Kinase C (PKC) – Lipid-like group • Inositol 1,4,5 triphosphate (3P ): Opens Ca2+ channels in the endoplasmic reticulum – Sugar-like group DAG and IP3 are a class of precursors second messengers Signal Amplification – Amplification increases the response time. – Epinephrine binds GPCR in very low levels. – The activated effector protein Adenyl cyclase can create high levels of second messenger – cAMP can activate Protein kinase A can phosphorylate lots of things which is another step in amplification. – PKA can phosphorylate other enzymes that can create the final produce which is another step in amplification. Page 4 of 8 Regulation of Glycogen by cAMP For increased cAMP  epinephrine binding which means that we want to make as much glucose as possible 1. PKA is activated by cAMP and phosphorylates GPK (glycogen phosphorylase kinase). 2. Activated GPK phosphorylates GP (glycogen phosphorylase) 3. Activated GP transfers phosphate group from glycogen onto glucose to create G1P. This makes glucose available to the cell. PKA activated by cAMP also starts a pathway to inhibit
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