Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Emer M. Smyth

Abstract

Thromboxane A2 (TXA2) contributes to cardiovascular disease (CVD) by activating platelets and vascular smooth muscle cell constriction and proliferation. Despite their preclinical efficacy, pharmacological antagonists of the TXA2 receptor (the TP), a G protein-coupled receptor (GPCR), have not been clinically successful, raising interest in novel approaches to modifying TP function. We sought to examine molecular mechanisms underlying auto-upregulation of the TP in response to agonist activation. We first determined a lack of agonist-induced TP mRNA modulation, focusing our attention on post-translational TP regulation. GPCR dimerization contributes to post-translational regulation of receptor expression and function, therefore we characterized how TP forms dimers with itself (homodimerization) or other related receptors (heterodimerization) and defined the relative affinities. To determine how disruption of TP dimerization impacts its regulation and function, we targeted a GxxxGxxxL helical interaction motif, reportedly involved in transmembrane protein-protein interactions between other membrane proteins and GPCRs, that is located in the human TP's (α isoform) 5th transmembrane domain. We determined that disruption of this motif suppressed TP agonist-induced Gq signaling and TPα homodimerization, but not its cell surface expression, ligand affinity or Gq association. Heterodimerization of TPα with the functionally opposing receptor for prostacyclin (the IP) shifts TPα to signal via the IP-Gs cascade contributing to prostacyclin's restraint of TXA2 function. Interestingly, and in contrast to the TPα homodimer, disruption of the TPα-TM5 GxxxGxxxL motif did not modify either TPα-IP heterodimerization or its Gs-cAMP signaling. Our study indicates that distinct regions of the TPα receptor direct its homo- and hetero- dimerization and normal homodimerization appears necessary for efficient TPα-Gq activation. Targeting the TPα-TM5 GxxxGxxxL domain may allow development of biased TPα- homodimer antagonists that avoid suppression of TPα-IP heterodimer's predicted beneficial "IP-like" effects. Such novel therapeutics may prove superior in CVD compared to non-selective suppression of all TP functions with TXA2 biosynthesis inhibitors or traditional TP antagonists.

Included in

Pharmacology Commons

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