The production of cAMP is controlled at many levels, particularly at the level of its synthesis by regulation of adenylyl cyclase (AC), the enzyme catalyzing the conversion of ATP to cAMP. Hormonal regulation of AC occurs by the receptor-catalyzed activation of heterotrimeric G proteins that in turn regulate AC activity. The alpha subunit of the heterotrimeric G protein Gs stimulates all nine isoforms of AC. Many other regulatory influences are also brought to bear on these enzymes. For example, the cardiac isoforms of AC are stimulated by PKC and are inhibited by calcium, PKA, and Gi-alpha. ACs thus serve critical roles as integrators of diverse inputs. AC activity can also be regulated by a newly appreciated family of proteins, designated RGS (regulators of G protein signaling). Family members are characterized by their ability to dampen the activity of G protein alpha subunits. No known RGS family member regulates the activity of Gs-alpha, although RGS proteins can inhibit the production of cAMP when expressed in vivo. The two cytoplasmic domains of AC create a beautifully symmetrical enzyme, forming a catalytic site at the interface of these domains. The applicant has developed a system whereby these two cytoplasmic domains are each expressed independently in E. coli. Simple mixing of the soluble proteins reconstitutes full AC activity. Initial data examining the inhibition of soluble AC by Gi-alpha has led to the hypothesis that the binding of Gi-alpha to AC induces a conformational change that decreases the affinity of the two domains for each other and in turn decreases catalytic activity. Many regulators of AC, including RGS proteins, may alter catalytic activity by influencing the conformation of the interface between the two domains. This proposal will test these hypotheses and determine the mechanism of several inhibitory regulators.
Specific aims are to (1) Determine the kinetic mechanism of Gi-alpha mediated inhibition of AC. (2) Examine the structural features of the Gi-alpha-AC complex. (3) Determine the mechanism of inhibition of AC by RGS proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060419-02
Application #
6387053
Study Section
Biochemistry Study Section (BIO)
Program Officer
Cole, Alison E
Project Start
2000-07-01
Project End
2005-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
2
Fiscal Year
2001
Total Cost
$239,200
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Biology
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
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