In this study, we examined the effects the prostacyclin receptor (IP) agonist cicaprost exhibited on U46619-mediated thromboxane A 2 receptor (TP) signaling in platelets and compared it to that which occurs in human embryonic kidney (

The human thromboxane A2 receptor (TP), a G protein-coupled receptor, exists as two isoforms, TPalpha and TPbeta, which arise by alternative mRNA splicing and differ exclusively in their carboxyl terminal cytoplasmic regions. In this study, a reverse transcriptase-polymerase chain reaction (RT-PCR)-based strategy was developed to examine the expression of the TPs in tissues of physiologic relevance to TXA2. Although most of the 17 different cell/tissue types examined expressed both TP isoforms, the liver hepatoblastoma HepG2 cell line was found to exclusively express TPalpha mRNA. In most cell types, TPalpha mRNA predominated over TPbeta mRNA. Moreover, although the levels of TPalpha mRNA expression were similar in most of the cell/tissue types examined, extensive differences in the levels of TPbeta mRNA were observed. Consequently, the relative expression of TPalpha: TPbeta mRNA varied considerably due to extensive differences in TPbeta mRNA expression. Most strikingly, primary HUVECs were found to express: (i) low levels of TPbeta and (ii) approximately 6-fold greater levels of TPalpha than TPbeta. These data were confirmed in the spontaneously transformed HUVEC derived ECV304 cell line. Expression of TP mRNAs in the various tissue/cells correlated with protein expression, as assessed by radioligand binding using the selective TP antagonist [3H]SQ29,548.

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In the current study, we have established that the human (h) prostacyclin receptor (IP) is isoprenylated in whole cells. Through site directed mutagenesis and generation of the isoprenylation defective hIP SSLC , it was established that while isoprenylation of hIP does not influence ligand binding, it is obligatory for agonist activation of adenylyl cyclase and cAMP generation. Overexpression of Ga S significantly augmented cAMP generation by the hIP but not by the hIP SSLC . Moreover, Ga S co-immunoprecipitated with hIP following agonist activation but did not co-immunoprecipitate with hIP SSLC . Whereas hIP mediated concentrationdependent activation of phospholipase C (PLC); the extent of PLC activation by hIP SSLC was impaired compared to hIP. Co-expression of Ga q significantly augmentated intracellular calcium mobilization by the hIP but not by hIP SSLC . Moreover, whereas Ga q co-immunoprecipitated with hIP, it failed to co-immunoprecipitate with hIP SSLC . While both the hIP and hIP SSLC underwent agonist-induced internalization, the kinetics and extent of hIP SSLC internalization was impaired compared to hIP. Altering the CAAX motif of the hIP from a farnesyl (-CSLC) to a geranylgeranyl (-CSLL) isoprene acceptor, to generate hIP CSLL , did not affect ligand binding and yielded a receptor that exhibited identical signalling through both G s -and G q -coupled effectors to that of hIP.Thus, whereas isoprenylation of hIP does not influence ligand binding, it is functionally imperative in regulating post-receptor events including agonist-activation of adenylyl cyclase, for efficient activation of PLC and for receptor internalization. Though the nature of the isoprenoid attached to hIP does not act as a major determinant, the presence of an isoprenoid group, for example farnesyl or geranylgeranyl, is required for functional receptor-G protein interaction and coupling and for efficient agonistinduced receptor internalization.

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