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

Abstract

We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A(2) receptor (TP) to Galpha(16) and Galpha(12) members of the G(q) and G(12) families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP(Delta328) cells which over-express a variant of TP truncated at the point of divergence of TPalpha and TPbeta, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha(16) to affect increases in inositol 1,4,5-trisphosphate (IP(3)) and mobilisation of intracellular calcium ([Ca(2+)](i)) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca(2+)](i) mobilisation in cells co-transfected with Galpha(12), neither receptor generated corresponding increases in IP(3), indicating that the Galpha(12)-mediated increases in [Ca(2+)](i) do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca(2+) channels, reduced [Ca(2+)](i) mobilisation in TPalpha and TPbeta cells co-transfected with Galpha(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 Ca(2+) release, had no effect on [Ca(2+)](i) mobilisation by either receptor isoform co-transfected with Galpha(12). Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP(Delta328) 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 Galpha(11), Galpha(12) or Galpha(16) subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP(Delta328) coupled to Galpha(s), leading to increased adenosine 3',5'-cyclic monophosphate (cAMP), rather than to Galpha(i). Whereas TP(Delta328) signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha, co-transfection of Galpha(s) did not augment cAMP generation by TP(Delta328). Hence, from these studies involving the wild type TPalpha, TPbeta and TP(Delta328), we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha(11) and Galpha(16) members of the G(q) family or to Galpha(12); it may play a role in determining G(s) versus G(i) coupling and may act as a determinant of coupling efficiency.

Thromboxane A2 Signalling in Humans: a ‘Tail’ of Two Receptors

“…However, in the absence of cotransfection of Gα s , cAMP generation by TP ∆328 was significantly greater than that of the wild-type TPα. Over-expression of Gα S , significantly augmented cAMP generation by TPα but had no effect on cAMP generation by TP ∆328 in response to U46619 [61]. Taken together, these data indicate that whilst the C-tail per se may not determine G-protein specificity to members of G q or G 12 family, it may play a role in determining G s versus G i coupling and it may be necessary for controlled, efficient G protein coupling and intracellular signalling, acting as a determinant of G protein coupling efficiency.…”

“…Whereas many of the latter reported studies have implicated various G protein α subunits in mediating TP activation [45][46][47][48][49][50][51][52][53][54][55] and the latter studies [33,60] mobilization by TPα and TPβ indicating that the TP isoforms may mediate opening of L-type channels in a Gα 12 dependent signalling mechanism [61]. Taken together, these studies investigating the signalling by the TP isoforms indicate that both TPα and TPβ couple to both the G q and G 12 family with no apparent isoform specific differences in their signalling behaviour.…”

The effects of the statins lovastatin and cerivastatin on signalling by the prostanoid IP‐receptor

“…HEK.hIP cell lines stably overexpressing the wild type hIP receptor in HEK 293 cells were established, using pHM6:hIP, and characterized essentially as previously described (Hayes et al, 1999). HEK.TPa10 cells and HEK.TPb3 cells over-expressing the human prostanoid thromboxane (TX) A 2 receptor (TP) a and b isoforms, respectively, have been previously described (Walsh et al, 1998;2000a). Cells were transfected with pCMV:Ga q as previously described (Kinsella et al, 1997).…”

Transcriptional Regulation of the Human Thromboxane A2 Receptor Gene by Wilms’ Tumour (WT)

“…Most notably, signalling by TPα, but not by TPβ, is completely desensitized/inhibited by the counter-regulatory anti-platelet and vasodilatory agents prostacyclin/prostaglandin I 2 and nitric oxide, which is mediated by direct protein kinase (PK) A and PKG phosphorylation of TPα at Ser 329 and Ser 331 , respectively, the very first residues within its unique carboxyl-terminal tail domain of TPα and divergent from those of TPβ (13,18). The conclusion from those studies is that TPα is the TP isoform essential for haemostasis/ thrombosis, while the role of TPβ in this pathophysiologic process remains unclear (13,18). Hence, TPα and TPβ have both shared and unique patterns of expression and function to mediate the (patho)physiologic actions of the potent autocrine/paracrine mediator TXA 2 in human health and disease.…”

“…While TPα and TPβ are encoded by the same TP gene, the TBXA2R (Figure 1), they are differentially expressed in several cell/tissue types being transcriptionally regulated by two different promoters referred to as Prm1 and Prm3, respectively, within the TBXA2R (7)(8)(9)(10). Functionally, as members of the G-protein-coupled receptor (GPCR) superfamily, TPα and TPβ both primarily couple to Gαq-mediated phospholipase Cβ activation, raising intracellular calcium levels in response to inositol phosphate turnover, but also readily couple to Gα12-mediated RhoA and to extracellular signal-regulated protein kinase (ERK) activation ( Figure 2) (6,(11)(12)(13)(14). In contrast, TPα and TPβ undergo distinct mechanisms of agonist-induced Structurally, TPα and TPβ are identical for their N-terminal 328 aa residues but differ exclusively in their intracellular C-terminal domains, where TPα (343 aa) and TPβ (407 aa) have 15 and 79 unique aa residues, respectively.…”