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J. Biol. Chem., Vol. 282, Issue 1, 294-302, January 5, 2007

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Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein Subunits^*

Xianlong Gao, Rachna Sadana, Carmen W. Dessauer, and Tarun B. Patel¹

From the Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois 60153 and Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas 77030

In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein 2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the G₁ subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that G subunits enhance the activity of these enzymes provided either G_s or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of G subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by G subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous G subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G_i was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, G subunits both in vitro and in intact cells. Moreover, G subunits derived from a source(s) other than G_i are necessary for the full activation of hACVI by isoproterenol in intact cells.

Received for publication, August 7, 2006 , and in revised form, November 3, 2006.

^* This research was supported by National Institutes of Health Grants HL59679 and GM073181 (to T. B. P.) and GM060419 (to C. W. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1.

¹ To whom correspondence should be addressed: Dept. of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Ave., Maywood, IL 60153. Tel.: 708-216-5773; Fax: 708-216-6888; E-mail: tpatel7{at}lumc.edu.

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