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Publication Date

2009-11-18

Availability

UM campus only

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2009-10-23

First Committee Member

Charles W. Luetje - Committee Chair

Second Committee Member

Stephen D. Roper - Committee Member

Third Committee Member

Kerry L. Burnstein - Committee Member

Fourth Committee Member

Vladlen Z. Slepak - Mentor

Fifth Committee Member

Alan V. Smrcka - Outside Committee Member

Abstract

Regulators of G protein signaling (RGS) are a diverse group of proteins, which play a fundamental role in modulation of G protein coupled receptor signal transduction. RGS proteins are primarily known as GTPase activating proteins (GAPs) for Gá subunits. In addition to the RGS domain, which is responsible for GAP activity, most RGS proteins also contain other structural motifs. The R7 family of RGS proteins for example, which consists of RGS-6, 7, 9 and 11 gene products, also contains DEP, DHEX and GGL domains. All R7 RGS proteins are obligatory binding partners with G protein beta subunit, G beta sub five, which binds to the GGL domain. In my dissertation work, I provide insights into significance of the multi-domain architecture of G beta sub five-RGS7. I have identified a novel intramolecular interaction within the G beta sub five-RGS7 complex; between the DEP domain of RGS7 and G beta sub five subunit. My experimental evidence supports the idea that G beta sub five-RGS7 can exist in at least two hypothetical conformations: "closed" where the DEP domain and G beta sub five subunit are bound to each other, and "open" where DEP and G beta sub five are not interacting, and as a result both these proteins can associate with other binding partners. My results indicate that in its "open" conformation, G beta sub five-RGS7 can selectively inhibit calcium mobilization elicited by stimulated muscarinic acetylcholine receptor type 3 (M3R). This inhibition is mediated by direct interaction between the third intracellular loop of M3R and the DEP domain of RGS7. In addition to the effect on M3R signaling, I observed that the G beta sub five-RGS7 complex redistributes from the cytosol to endocytic vesicles in an M3R-specific manner. These results identify a novel molecular mechanism that can impart receptor-subtype selectivity on signal transduction via G protein-coupled receptors. Lastly, I have identified a small group of compounds that inhibits the DEP-G beta sub five interaction. These compounds may serve as starting points for design of G beta sub five-RGS7 modulators in the future.

Keywords

DEP Domain; Calcium; GPCRs; Signal Transduction; RGS Proteins

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