Regulation of specific types of adenylyl cyclases by G proteins.
Item
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Title
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Regulation of specific types of adenylyl cyclases by G proteins.
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Identifier
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AAI9630447
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identifier
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9630447
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Creator
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Chen, Jianqiang.
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Contributor
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Adviser: Ravi Iyengar
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Date
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1996
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Language
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English
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Publisher
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City University of New York.
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Subject
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Health Sciences, Pharmacology | Biology, Molecular
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Abstract
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The hormone-stimulated adenylyl cyclase (EC 4.6.1.1) is a prototypical G protein regulated effector. With the cloning of several types of mammalian adenylyl cyclases, it is of interest to know whether various types of adenylyl cyclases are regulated by G protein {dollar}\rm\alpha\ (G\sb\alpha{dollar}) and {dollar}\beta\gamma{dollar} subunits (G{dollar}\beta\gamma{dollar}) as well as components from other signal transduction pathways. It is also important to identify sites in adenylyl cyclase that are involved in regulation by G{dollar}\alpha{dollar} and G{dollar}\beta\gamma{dollar}.;I studied whether various types of adenylyl cyclases are subject to inhibition by {dollar}\rm\alpha\sb{lcub}i{rcub}{dollar}. Cos-7 cells were cotransfected with cDNAs encoding (1) LH receptor, (2) Q205L{dollar}\rm\alpha\sb{lcub}i{rcub}{dollar} or Q205L{dollar}\rm\alpha\sb{lcub}o{rcub}{dollar}, and (3) required types of adenylyl cyclases (AC). The human chorionic gonadotropin (hCG)-stimulated cAMP levels were measured in transfected cells. AC2, AC3 and AC6 were chosen as representatives of each subfamily. My data indicated that all three types of adenylyl cyclases were subject to inhibition by Q205L {dollar}\rm\alpha\sb{lcub}i{rcub}{dollar}. The inhibition by Q205L{dollar}\rm\alpha\sb{lcub}i{rcub}{dollar} seems to be specific since Q205L{dollar}\rm\alpha\sb{lcub}i{rcub}{dollar}, but not Q205L{dollar}\rm\alpha\sb{lcub}o{rcub}{dollar}, inhibited hCG-stimulated cAMP accumulation in Cos-7 cells cotransfected with AC2. Phorbol ester pretreatment of Cos-7 cells cotransfected with AC2 resulted in complete attenuation of inhibition of AC2 by Q205L{dollar}\rm\alpha\sb{lcub}i{rcub}{dollar}, whereas similar treatment did not affect inhibition of AC3 and AC6 by Q205L{dollar}\rm\alpha\sb{lcub}i{rcub}{dollar}.;I also attempted to identify the regions in AC2 that may be involved in G{dollar}\beta\gamma{dollar} regulation. One peptide (the QEHA peptide), encoding residues 956 to 982 of AC2, was effective in blocking G{dollar}\beta\gamma{dollar}-stimulated AC2 in presence of activated {dollar}\rm\alpha\sb{lcub}s{rcub}{dollar}, but did not significantly affect basal, {dollar}\rm\alpha\sb{lcub}s{rcub}{dollar} and forskolin-stimulated adenylyl cyclase activity. The inhibition of G{dollar}\beta\gamma{dollar} stimulation by the QEHA peptide was specific since only the QEHA peptide, but not the SKEE peptide encoding the cognate region from AC3, was capable of inhibiting G{dollar}\beta\gamma{dollar} stimulation of AC2. The QEHA peptide also completely relieved G{dollar}\beta\gamma{dollar} inhibition of Ca{dollar}\sp{lcub}2+{rcub}{dollar}/calmodulin-stimulated adenylyl cyclase activity (AC 1-like) in rat brain membranes, indicating that the QEHA peptide is capable of blocking G{dollar}\beta\gamma{dollar} regulation of adenylyl cyclases, irrespective of the direction of the regulation. Two peptides QEHA{dollar}\rm\sb{lcub}s1{rcub}{dollar} and QEHA{dollar}\rm\sb{lcub}s2{rcub},{dollar} in which critical residues have been substituted by alanine, were less effective in blocking G{dollar}\beta\gamma{dollar} stimulation of AC 2, indicating that the QxxER motif may be important for AC2 interaction with G{dollar}\beta\gamma{dollar}.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
