Question: 1. You Are Studying The Control Of CAMP Levels In Brain Slices. You Have Confirmed That Drugs Such As Isoproteronol That Act Through Beta-adrenergic Receptors Cause A Modest Increase In CAMP As Expected From G-protein Mediated Coupling Between The Receptor And Adenylyl Cyclase. You Find A Puzzling Synergy, However, Between Isoproteronol And A Number …

1. You are studying the control of cAMP levels in brain slices. You have confirmed that drugs such as isoproteronol that act through beta-adrenergic receptors cause a modest increase in CAMP as expected from G-protein mediated coupling between the receptor and adenylyl cyclase. You find a puzzling synergy, however, between isoproteronol and a number of pharmacological agents that be themselves have no effect of cAMP levels. What is the basis for this paradoxical augmentation of cAMP levels? A biochemist friend of yours has suggested a possible explanation: she has found in in-vitro experiments that beta-gamma subunits from inhibitory trimeric G proteins stimulate type II adenylyl cyclase, which is expressed in the brain. To test this idea in cells you plan to express the cDNAs encoding the component proteins in human kidney cells, which lack the receptors found in the brain. In this way you hope to reconstruct the effects you observed in the brain slices. You transfect the kidney cells with various combinations of cDNAs encoding type II adenylyl cyclase, the dopamine receptor (which interacts with an inhibitory G protein), and a mutant (constitutively active) alpha-s subunit. You measure the levels of CAMP in the resulting cell lines in the presence or absence of quinprole (Which activates the dopamine receptor). You also measure the effects of pertussis toxin, which blocks the signal from Gi coupled receptors by modifying the alpha-i subunit in such a way that it can no longer bind GTP and dissociate from its beta-gamma subunit. Orice o + inpedia CAMPM 2 + + + + Doporne Rotor + Ad Cydon Awon active Pation A. (5 points) Describe the molecular effects of pertussis toxin in your experiments (Describe why the addition of pertussis toxin B. (10 points) What do your experiments indicate is required for maximal activation of type II adenylyl cyclase? Propose a molecular explanation for the augmented activation of type II adenylyl cyclase when quinprole and the always active alpha-S are present. Explain why quinprole by itself has no effect. C. (5 points) Predict the effects of expressing the cDNA for transducin (an unrelated protein alpha subunit in the GDP form) which does not bind to adenylyl cyclase but does bind tightly to free beta-gamma subunits.