1. You are studying the control of cAMP levels in brain slices. You have confirm
ID: 209727 • Letter: 1
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 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. Doparrine Receptor++ Adenylyl Cydae + Always active a-s Pertussis Tcxin A. (10 points) Explain the effects of pertussis toxin in your experiments. B. (10 points) What do you 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 C. (10 points) Predict the effects of expressing the cDNA for transducin (an unrelated G protein alpha subunit in the GDHP form) which does not bind to adenylyl cyclase but does bind tightly to free beta-gamma subunitsExplanation / Answer
A) In the central nervous system Go, the most generous G protein, there in mammalian brains it consist about 1% of membrane protein. It perform as couple receptors of cell surface to intercellular effectors, that is a main process for cells to get, depict as well as respond to extracellular signals. Go protein integrated to the G proteins pertussis toxin-sensitive Gi/Go subfamily. A number of GPCR impart signal to intercellular effectors through Go. Go maintain different cellular effectors, involving enzymes, ion channels, also small GTPases to mediate cellular function.
B) Bordetellapertussis toxin (PTX), found as the prtein activating islet, maintains the ADP ribosylation of Gi proteins that reduces activity of adenylyl cyclase. Cells Treated with PTX causes the lack of adenylyl cyclase inhibition. Go was labelled as the ‘another’ GTP-binding protein after Gs, Gi, as well as transducin. PTX block proteins Gi and Go and innhibit stimulus of the D2S receptor. D2S receptor signaling was reconstructed by transfecting individual with pertussis toxin-resistant Gi/o subunit mutants and observing D2-stimulated responses in cells treated with pertussis toxin.
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