Investigation of the role of the carboxyl-terminal tails of the α and β isoforms of the human thromboxane A2 receptor (TP) in mediating receptor:effector coupling

We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A(2) receptor (TP) to G alpha(16) and Gait members of the G(q) and G(12) families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha 10 or HEK.beta 3, stably over-expressing TP alpha and TP beta. respectively. Moreover, using HEK.TPDelta 328 cells which overexpress a variant of TP truncated at the point of divergence of TP alpha and TP beta, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TP alpha and TP beta couple similarly to G alpha(16) to affect increases in inositol 1,4,5-trisphosphate (IP3) and mobilisation of intracellular calcium ([Ca2+](i)) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca2+](i) mobilisation in cells co-transfected with G alpha(12), neither receptor generated corresponding increases in IP3, indicating that the G alpha(12)-mediated increases in [Ca2+](i) do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca2+ channels, reduced [Ca2+](i) mobilisation in TP alpha and TP beta cells cotransfected with G alpha(12) to approximately 40% of that mobilised in its absence, whereas [8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride] (TMB-8), an antagonist of intracellular Ca2+ release, had no effect on [Ca2+](i) mobilisation by either receptor isoform co-transfected with G alpha(1)2. Despite the tack of differential coupling specificity by TP alpha and TP beta, TPDelta 328 signalled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected G alpha(11), G alpha(12) or G alpha(16) Subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TP alpha but not TP beta, TPDelta 328 coupled to G alpha(s), leading to increased adenosine 3',5'-cyclic monophosphate (cAMP), rather than to G alpha(i). Whereas TPDelta 328 signalled more efficiently in the absence of co-transfected G protein compared to the wild type TP alpha, co-transfection of G alpha(s) did not augment cAMP generation by TPDelta 328. Hence, from these studies involving the wild type TP alpha, TP beta and Tp(Delta 238), we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to G alpha(11) and Ga-16 members of the G(q) family or to G alpha(12) it may play a role in determining G(s) versus G(i) coupling and may act as a determinant of coupling efficiency. (C) 2000 Elsevier Science B.V. All rights reserved.