Prostacyclin (PG I2) and thromboxane (TxA2), two major cyclooxygenase products formed in the vasculature, exert opposing biological effects. PGI2 is a potent inhibitor of platelet aggregation, vasodilator, anti-thrombotic and anti-mitogen which may be protective in settings of vascular disease. In contrast, TxA2, which induces platelet aggregation, mitogenesis and vasoconstriction, appears to play a detrimental role in the pathogenesis of cardiovascular disorders. Both PGI2 and TxA2 transduce their effects by binding to their cell surface receptors, IP and TP (two splice variants alpha and beta), respectively. IP and TP are frequently co-expressed in cardiovascular cells and elevated PGI2 and TxA2 biosynthesis is coincident in vascular disorders. Cross-regulation of PGI2 and TxA2-responsive events is evident in smooth muscle cells, platelets and other cells types. However, despite our understanding of the regulation and signal transduction pathways utilized by these two systems, little is known about the impact of each signaling cascade on the other. This proposal aims to examine whether and how human (h) IP and hTP activation alone and in concert may modulate each other and seeks to understand the molecular mechanisms attendant to these interactions. We shall address following specific hypotheses.1. That co-activation of hIP and hTP modulates their regulation and G protein coupling. We shall examine heterologous regulation of hIP and hTPalpha/beta in vascular smooth muscle cells endogenously express both receptor types, or deficient in one or the other, and in cell lines expressing the receptors alone or in combination.2. That agonist-induced homo- and hetero- dimerization of IP and TPalpha/beta occurs and has functional and regulatory consequences. We shall examine dimer formation and receptor signaling in vascular smooth muscle cells from humans or cultured from IP or TP deficient mice and in hIP/hTP expressing cell lines.3. That hIP and hIP interact with novel cellular proteins. The role of the cGMP-phosphodiesterase-6 delta subunit, a novel hIP associated factor, in hIP signaling and regulation will be investigated. We shall use yeast-two hybrid technology to screen a vascular smooth muscle cell cDNA library and a proteomic approach to investigate other uncharacterized proteins that interact with hIP or hTP.These studies will define the interaction of two vasoactive eicosanoids at the level of receptor signaling and regulation and identify novel pathways through which G protein-coupled receptors in general, and IP and TP in particular, can heterologously modulate receptor function.
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