Mechanism of Galpha i-mediated inhibition of type V adenylyl cyclase.
The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2). The stimulatory G protein, Galpha(s), binds within a cleft in the C2 domain of adenylyl cyclase while Galpha(i) binds within the opposite cleft in the C1 domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Galpha(s) or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another. We show herein that Galpha(i) reduces C1/C2 domain interaction and thus formation of the adenylyl cyclase catalytic site. Mutants that increase the affinity of C1 for C2 decrease the ability of Galpha(i) to inhibit the enzyme. In addition, Galpha(i) can influence binding of molecules to the catalytic site, which resides at the C1/C2 interface. Adenylyl cyclase can bind substrate analogs in the presence of Galpha(i) but cannot simultaneously bind Galpha(i) and transition state analogs such as 2'd3'-AMP. Galpha(i) also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs. Thus homologous G protein alpha-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme.
Dessauer
Carmen W
CW
Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas 77030, USA. Carmen.W.Dessauer@uth.tmc.edu
Chen-Goodspeed
Misty
M
Chen
Jun
J
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D007313:Insecta; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D000199:Actins; D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; C419510:adenylyl cyclase type V