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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, nor hIP C308S,C309S,C311S coupled to G q . Taken together, these data confirm that the hIP is isoprenylated and palmitoylated, and collectively these modifications modulate its G protein coupling and effector signaling. We propose that through lipid modification followed by membrane insertion, the C-tail domain of the IP may contain a double loop structure anchored by the dynamically regulated palmitoyl groups proximal to transmembrane domain 7 and by a distal farnesyl isoprenoid permanently attached to its carboxyl terminus.Modification of proteins through the covalent attachment of lipid groups occurs within a wide variety of cellular proteins and may be involved in mediating protein-membrane and/or protein-protein interactions (1-3). Three of the most common lipid modifications are N-myristoylation, isoprenylation and thio(S)-acylation. In contrast to myristoylation and isoprenylation, which typically occur either as co-translational or as immediate post-translational events, S-acylation through attachment of palmitate to Cys residue(s), via a labile thioester bond, occurs post-translationally (1). Moreover, whereas the former two modifications remain attached until protein degradation, palmitoylation is a reversible, dynamic modification that turns over more rapidly than the protein itself and thus has the potential to be regulated (2, 3). A diverse family of cellular proteins are established to be palmitoylated including ␣ subunits of the heterotrimeric G protein subunits, for example G␣ s and G␣ q , Ha-and N-Ras proteins, A kinase anchoring protein 15 and 18, endothelial nitric-oxide synthase, adenylyl cyclase, G protein-coupled receptor kinases 4 and 6, diverse members of the Src family of nonreceptor tyrosine kinases, in addition to several members of the G protein-coupled receptor (GPCR)

1 The prostanoid-IP receptor may be unique among G protein coupled receptors in that it is isoprenylated. In this study, we investigated the eects of the statins lovastatin and cerivastatin on signalling by the mouse (m) IP and the human (h) IP receptors, over-expressed in human embryonic kidney (HEK) 293 cells and by the hIP receptor, endogenously expressed in human erythroleukaemia cells. 2 Both statins signi®cantly reduced IP receptor-mediated cyclic AMP generation and intracellular calcium ([Ca 2+ ] i ) mobilization in a time and concentration dependent manner but had no eect on signalling by the non-isoprenylated b 2 adrenergic receptor or by the human prostanoid-TP receptor isoforms. 3 Cerivastatin (IC 50 , 50 ± 90 nM) was signi®cantly more potent than lovastatin (IC 50 , 0.80 ± 4.2 mM) in inhibiting IP receptor signalling. 4 Whereas IC 50 values indicated that the hIP receptor was signi®cantly more sensitive than the mIP receptor to the statins, the extent of inhibition of cyclic AMP generation by the mIP receptor was signi®cantly greater than that of the hIP receptor to either statin, even at the highest concentrations used. 5 Pretreatment with either statin signi®cantly reduced IP receptor mediated desensitization of signalling by the h.TPa, but not by the h.TPb, receptor isoform. 6 These data generated in whole cells point to the possibility that statin therapy may interfere with IP receptor signalling in vivo; such interference may be extenuated under conditions where circulating statin levels are elevated and may account, in part, for some of the pleiotropic aects of the statins not attributed solely to their lipid lowering properties.

During a recent review of this article, the authors realized that there may have been unspecified reordering of lanes in Fig. 11B and possible duplication of lanes 2 between Fig. 11, B and C. As the original data were no longer available, replicate data are provided.