Abstract 18304: Dimerization with the Prostacyclin Receptor Triggers Thromboxane Receptor Relocation to Lipid Rafts

Abstract

Membrane rafts are liquid-ordered microdomains characterized by high levels of sphingolipids, cholesterol and saturated phospholipids. Prostacyclin (PGI2) effects its anti-thrombotic, anti-proliferative and vasodilator actions via a Gs coupled receptor, IP, opposing those of thromboxane A2 (TxA2) via TP, a Gq coupled receptor. IP is raft localized but the relevance is ill-defined. We reported a TP signaling shift to IP-like function through IP-TP heterodimerzation. In the present study, we examined IP and TP membrane localization and explored the importance of raft integrity to receptor dimerization and function. COS-7 cells were co-transfected with IP and/or TP. Gs activation was examined by measuring cAMP stimulation with cicaprost (PGI2 analog) or U46619 (TxA2 analog). Membrane localization was examined by measuring bioluminesence resonance energy transfer (BRET) from IP or TP fused to renilla luciferase (rLuc) to a raft-associated fluorophore, DiIC16. IP was localized in rafts, identified as saturable BRET from rLucIP to DiIC16. By comparison, rLucTP transfer was shallow and inefficient, similar to the non-raft associated ß2-adrenoreceptor, consistent with raft exclusion. Interestingly, the rLucTP BRET curve was strongly left-shifted, readily saturable and indistinguishable from rLucIP alone, when rLucTP-cells were cotransfected with untagged IP. This indicated IP-triggered relocation of TP to lipid rafts. Disruption of rafts by cholesterol depletion (2-hydroxypropyl-β-cyclodextrin; 20mM, 1hr) reduced cicaprost-stimulated cAMP in IP transfected cells (48.49±9.18 % inhibition) as well as cicaprost- and U46619-stimulated cAMP in IP-TP co-transfected cells (51.18+10.47 and 52.78+6.83 % inhibition) consistent with IPTP heterodimer function in rafts. Our data confirm IP localization to, and TP exclusion from, membrane rafts. IP function alone and as part of the IPTP heterodimer was at least partially dependent on raft integrity. Further, the shift in TP signaling to the cAMP pathway likely follows its relocation to rafts as the IPTP heterodimer. These data highlight the intricate cellular mechanisms through which IP can limit the cardiovascular effects of TP activation contributing to cardiovascular homeostasis and disease.