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

Xianlong Gao,Rachna Sadana,Carmen W Dessauer,Tarun B Patel

doi:10.1074/jbc.m607522200

Abstract

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 beta2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the Gbeta(1) 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 Gbetagamma subunits enhance the activity of these enzymes provided either Galpha(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 Gbetagamma 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 Gbetagamma subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous Gbetagamma 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, Gbetagamma subunits both in vitro and in intact cells. Moreover, Gbetagamma subunits derived from a source(s) other than G(i) are necessary for the full activation of hACVI by isoproterenol in intact cells.

Highlights

Adenylyl cyclase (AC)2 catalyzes the conversion of ATP into cAMP
Because G␤1 is highly homologous to G␤2, we transformed cells with constructs to express the N-terminal region of human ACV (hACV) and G␤1
Because human ACVI (hACVI) and its close homologue hACV are regulated [2, 3], using the yeast two-hybrid assay we investigated whether the N terminus of hACVI interacted with the G␤1 subunit

Summary

Introduction

Adenylyl cyclase (AC) catalyzes the conversion of ATP into cAMP. To date at least nine isoforms of membrane-bound mammalian adenylyl cyclases have been cloned and characterized (for review, see Refs. 1–3). The N-terminal segment of the different AC isoforms is highly variable, and this region may play an important role in type-specific regulation of these enzymes. Among the AC isoforms, ACII, ACIV, and ACVII are stimulated by G␤␥ subunits provided that some G␣s is present [13], whereas ACI and ACVIII are inhibited by G␤␥ [13, 32]. Either nothing (ACV) or very little (ACVI) is known about the role of the N-terminal segments of these enzymes in regulating their activity. Pertussis toxin treatment did not affect the ability of GRK2-CT to attenuate isoproterenol-stimulated cAMP accumulation in these cells Together these results show that the G␤␥ subunits derived from sources other than Gi participate in mediating the full activation of hACVI by the ␤-adrenergic receptor agonist, isoproterenol

Methods

Results

Conclusion