Date of Graduation

5-2015

Document Type

Dissertation (PhD)

Program Affiliation

Cell and Regulatory Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Carmen W. Dessauer

Committee Member

Jeffrey A. Frost

Committee Member

Richard B. Clark

Committee Member

Alemayehu A. Gorfe

Committee Member

John S. McMurray

Abstract

The nine membrane-bound isoforms of adenylyl cyclase (AC), via synthesis of the signaling molecule cyclic AMP (cAMP), are involved in many isoform specific physiological functions. All nine isoforms share a similar structural organization; thus, AC isoform differences in physiological function are due to different regulatory profiles. A physiological example is Gβγ, which can conditionally enhance stimulation of ACs 2, 4, 5, 6, and 7, but inhibit ACs 1, 3, and 8. There is also pharmacological control of AC isoforms through small molecule inhibitors.

Isoform specific AC functions could be explained by regulatory differences as subtle as single amino acid changes. For both pharmacological targeting and known physiological regulators, differences between isoforms are not well understood. Two approaches were taken to explore AC5/6 isoform selectivity. The first approach was to more completely characterize allegedly AC5 selective small molecule AC inhibitors. The other approach was to examine AC isoform regulation by Gβγ.

The AC inhibitors SQ22,536 and Ara-A, rather than being AC5 selective as previously described, are AC5/6 inhibitors. Another AC inhibitor, NB001, showed AC1-dependent decreases in cAMP within cells without directly inhibiting AC1. These results highlighted the importance of AC inhibitor characterizations for AC specificity and isoform selectivity.

Gβγ regulation of AC5/6 was also explored. We showed that Gβγ binds to many AC N-termini (NT), but only AC5 NT binding did not require the Gβγ “hotspot”, a surface involved in many Gβγ regulatory events. Mutations of the hotspot showed it was necessary for AC5 stimulation but not AC5 NT binding. In contrast, the hotspot was required for both AC6 NT binding and AC6 stimulation. Peptide competition to disrupt Gβγ hotspot dependent binding events affected regulation of AC1/2/6 by Gβγ, but not that of AC5. Gβγ also interacted with both the AC5/6 C1/C2 catalytic domains, with similar hotspot dependency. This work suggested that Gβγ stimulation of AC5/6 occurs in a similar hotspot-dependent binding event, but that Gβγ/AC5NT binding is an isoform specific G protein anchoring event.

Keywords

adenylyl cylcase, cyclic AMP, small molecule inhibitors, G proteins, structure-function, regulation, isoform selectivity, protein scaffolding

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