PROBLEM SET 10 QUESTIONS PDF

Questions and problems #10 G-protein-coupled receptors (GPCRs) GPCRs and arrestins Agonist-activated GPCRs (agonist/ligand is shown as a green ball) bind heterotrimeric G proteins and serve as GEFs: they facilitate the release of GDP bound to the Gα subunit of inactive heterotrime...

Questions and problems #10 G-protein-coupled receptors (GPCRs) GPCRs and arrestins Agonist-activated GPCRs (agonist/ligand is shown as a green ball) bind heterotrimeric G proteins and serve as GEFs: they facilitate the release of GDP bound to the Gα subunit of inactive heterotrimer. This allows for the subsequent binding of GTP to the Gα subunit. Then Gα subunit dissociates from the GPCR and Gβγ dimer, and both GTP-Gα and Gβγ regulate signaling pathways (shown as three long arrows). GRKs (G protein-coupled receptor kinases) bind agonist/ligand-activated GPCRs and phosphorylate them. This reduces the G protein coupling of active GPCR (three shorter arrows). Complete blockade of G protein-mediated signaling takes place when arrestin binds to the phosphorylated GPCR. The binding of arrestins to GPCRs results in these receptors’ uptake (endocytosis) and desensitization. NOT included in our lecture modules: The arrestin-receptor complex could act as a scaffold facilitating different types of signaling (e.g., Raf-MEK-ERK cascade is shown). Free arrestins in the cytoplasm also function as scaffolds, facilitating signaling independently of GPCRs (e.g., ASK-MKK4/7-JNK cascade). Image from https://www.frontiersin.org/articles/10.3389/fphar.2019.00125/full. 1. Which is a cell-surface receptor that associates with an intracellular G protein upon activation by an extracellular ligand? A. Trimeric GTP-binding protein B. GPCR kinase (GRK) C. Acetylcholine receptor D. G-protein-coupled receptor (GPCR) 2. What type of regulator activity is exhibited by the Regulators of G protein Signaling (RGS) proteins, and the effectors of G alpha subunits of heterotrimeric G proteins? A. GEF-type regulators to inhibit G protein activity. B. GEF-type regulators to activate G protein activity. C. GAP-type regulators to activate G protein activity. D. GAP-type regulators to inhibit G protein activity. 3. IP3 increases the intracellular Ca2+ concentration by binding ____. A. specific plasma membrane receptors B. specific receptors on the endoplasmic reticulum membrane C. ryanodine receptors on the endoplasmic reticulum membrane D. transcription factors that increase the gene expression of Ca2+ channels 4. True or false? In a signaling pathway that utilizes a heterotrimeric GTP-binding protein, Gβγ could activate a downstream pathway, but Gαβγ could not. 5. Cholera toxin blocks the GTP hydrolysis at the α subunit of stimulatory G protein (G alpha-s). What is the effect of the toxin on intracellular cAMP? A. Cholera toxin increases cAMP by increasing the activation of adenylyl cyclase by the alpha subunit of Gs. B. Cholera toxin decreases cAMP by preventing the activation of adenylyl cyclase. C. Cholera toxin increases cAMP by increasing the activation of adenylyl cyclase by the beta-gamma (βγ) subunit of Gs. D. Cholera toxin increases cAMP by increasing the activation of cAMP phosphodiesterase. 6. The rods in our retina are specialized for night non-color vision and respond to light with changes in membrane potential. In the dark, the rods’ cGMP- gated Na+ channels are open (due to the presence of cGMP) and sodium ions enter the cells. Light detected by rhodopsin, the light-sensitive GPCR in the cells, leads to the closing of the Na+ channels. These and other events can be studied in a laboratory because the outer segments of rod photoreceptor cells can be broken off, isolated, and analyzed. How would the visual response be affected by: an inhibitor of cyclic GMP phosphodiesterase an inhibitor of the rhodopsin-specific kinase (remember the role of GRKs, the GPCR- specific kinases). 7. _____ is an enzyme bound to the cytoplasmic surface of the plasma membrane that converts membrane PI (4,5) P2 to diacylglycerol and IP3. A. Cyclic-AMP-dependent protein kinase (PKA) B. Phospholipase C C. Cyclic AMP phosphodiesterase D. GPCR kinase 8. True or false? Epinephrine (adrenaline) is a hormone that signals via cAMP as the second messenger: adrenaline binds to the adrenergic receptors, activates G proteins, and initiates the breakdown of glycogen in the liver, muscle, and adipose cells. The cells are exposed to (1) an inhibitor of phosphodiesterase or (2) a non-hydrolyzed analog of GTP (it cannot be converted into GDP) in the presence of adrenaline. Therefore, (1) glycogen breakdown will decrease, and (2) glycogen breakdown will not be affected. 9. _____ is a second messenger that is released from a phospholipid in the plasma membrane and diffuses to the endoplasmic reticulum, where it opens Ca2+- release channels. A. Cyclic AMP B. Diacylglycerol C. Inositol 1,4,5-trisphosphate (IP3) D. Calmodulin 10. True or false? Both trimeric and small G-proteins have intrinsic GTPase activity. In trimeric G- proteins, it is localized on the Gα subunit. 11. True or false? CAM-kinase II “remembers” a Ca2+ signal by binding GTP. 12. Which of the following is an immediate consequence of the activation of phospholipase C-β (PLCβ)? A. Elevation of intracellular cAMP levels, leading to the activation of protein kinase A B. Elevation of PIP3 levels in the plasma membrane, leading to the activation of protein kinase B C. Generation of diacylglycerol (DAG) and inositol trisphosphate (IP3). D. Elevation of IP3 in the plasma membrane, leading to the activation of AKT. 13. A 6-month-old male patient presents with a slight fever, rhinitis, sneezing, and forceful coughs ending with loud inspiration (whoop). Bordetella pertussis was cultured from the nasopharynx. The toxin from this microorganism prevents the normal function of the Gαi protein in cells of the respiratory tract. Which of the following disruptions in cell signaling take place in response to this infection? A. Calcium not binding to calmodulin. B. Impaired IP3-stimulated release of calcium from the endoplasmic reticulum. C. Increased phospholipase C activity and PIP2 cleavage. D. Overproduction of cAMP from uninhibited adenylyl cyclase. 14. Which is the Ca2+-release channel in the endoplasmic reticulum membrane that is activated by Ca2+ binding in the absence of IP3? A. The calmodulin protein B. The rhodopsin receptor C. The ryanodine receptor D. A specific G-protein-coupled receptor (GPCR) 15. The alteration in receptor sensitivity following repeated stimulation, reducing a cell’s response to that level of stimulus is called ______. A. sensitization B. desensitization C. contact-dependent signaling D. paracrine signaling 16. G protein that activates adenylyl cyclase, and thereby increases the concentration of cyclic AMP: A. Ryanodine receptor B. Diacylglycerol C. Calmodulin D. Stimulatory G protein (Gs) 17. Which is a ubiquitous calcium-binding protein whose interaction with other proteins is modulated by changes in the intracellular concentration of Ca2+? A. Gq B. Calmodulin C. IP3 receptor D. GPCR kinase 18. What is the name of a protein that binds to a GTP-binding protein and inactivates it by stimulating its GTPase activity (i.e., the bound GTP is hydrolyzed to GDP)? A. GTP-binding protein B. Guanine nucleotide exchange factor (GEF) C. GTPase activating protein (GAP) D. Monomeric GTPase 19. Which is the protein kinase, whose activity is regulated by the binding of Ca2+-activated calmodulin and which indirectly mediates the effects of Ca2+ by phosphorylation of other proteins? A. Calmodulin B. Ca2+/calmodulin-dependent kinase (CaM-kinase) C. Cyclic-AMP-dependent protein kinase (PKA) D. Protein kinase C (PKC) 20. Which enzyme desensitizes GPCRs by phosphorylating them after receptor-ligand binding? A. GPCR kinase (GRK) B. Protein kinase A C. Protein kinase C D. Adenylate (adenylyl) cyclase 21. You are purifying adenylyl cyclase from the brain. The assay is based upon the conversion of -32P- ATP to cAMP. You can detect activity in crude brain homogenates stimulated by isoproterenol, which binds to beta-adrenergic receptors, but the enzyme loses activity when low-molecular-weight cofactors are removed by dialysis. What single molecule do you think you could add back to restore activity? 22. You have synthesized a novel small molecule that reduces the GTP affinity of the G alpha-s subunit in a trimeric G protein and does not modify its GDP affinity. How would the treatment with this molecule change the activity of a trimeric G protein? A. The trimeric G protein exposed to this small molecule will be permanently active. B. The activity will increase. C. The activity will not change. D. The activity will decrease. 23. Phosphorylase kinase “cleaves” glucose units from glycogen. The enzyme integrates signals from the cAMP-dependent and Ca2+-dependent signaling pathways that control glycogen breakdown in liver and muscle cells. The enzyme has four subunits: one subunit is a kinase that catalyzes the addition of phosphate to the enzyme to activate it. The other three subunits are regulatory: two have sites for phosphorylation by PKA (activated by cAMP), the other subunit is calmodulin (bound by Ca2+, when its concentration rises). How does this structure allow for phosphorylase kinase to integrate multiple pathways to stimulate glycogen breakdown? 24. True or false? The inhibition of a rhodopsin-specific kinase extends signaling through the rhodopsin receptor. 25. In muscle cells, the hormone angiotensin II binds to its GPCR, and G alpha-q is activated. Which of the following will most likely happen? A. IP3 will cause the release of Ca2+ from plasma membrane-ligated ion channels. B. Protein Kinase A will cleave phosphoinositides to form DAG and IP3. C. Protein kinase C will be activated by increased diacylglycerol, calcium ions, and phosphatidylserine. D. Decreased Ca2+ release from endoplasmic reticulum stores will lead to muscle relaxation.