Investigation of a functional requirement for isoprenylation by the human prostacyclin receptor

Abstract

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.

Interaction of the Human Prostacyclin Receptor with Rab11

“…It has been suggested that regulated cycles of palmitoylation/depalmitoylation, such as in response to agonist, may result in the formation of a transient fourth intracellular loop (IC 4 ) to regulate receptor-G protein coupling, downregulation, and/or internalization, to list but a few processes. More specifically, in the case of the hIP, in addition to its established isoprenylation (24,25), it undergoes palmitoylation at Cys 308 and Cys 311 in response to stimulation with the selective IP agonist cicaprost (2 h), whereas Cys 309 was found not to be palmitoylated under those conditions (27). Because the proposed RBD contains Cys 308 and Cys 311 that are known to undergo palmitoylation, in addition to Cys 309 , the current study sought to investigate the role of those Cys residue(s) and the possible influence of palmitoylation on the interaction between the hIP and Rab11a and to identify the key structural determinants of the RBD.…”

“…The IP is primarily coupled to G s /adenylyl cyclase activation but may regulate other effectors in a cell-and/or species-specific manner (23,24). The IP is somewhat unusual among GPCRs in that it undergoes both isoprenylation and palmitoylation within its carboxyl-terminal tail (C-tail) domain, modifications that are critical for its signaling and function (24 -30).…”

“…The IP is somewhat unusual among GPCRs in that it undergoes both isoprenylation and palmitoylation within its carboxyl-terminal tail (C-tail) domain, modifications that are critical for its signaling and function (24 -30). More specifically, in the case of the human (hIP), it undergoes farnesylation at Cys 383 within an evolutionarily conserved -CAAX motif (24,25) and is dually palmitoylated at Cys 308 and Cys 311 , whereas an intervening Cys 309 was found not to be palmitoylated, at least under the experimental conditions used (27). Although neither lipidation affected its ligand binding properties, it is proposed that farnesylation in addition to palmitoylation of the hIP may confer a double loop structure within its C-tail domain to provide and/or orientate the critical structural domains for its G protein/effector(s) coupling and, possibly, for its interaction with components of the intracellular trafficking machinery to modulate its internalization after agonist activation (24,25,27).…”

“…More specifically, in the case of the human (hIP), it undergoes farnesylation at Cys 383 within an evolutionarily conserved -CAAX motif (24,25) and is dually palmitoylated at Cys 308 and Cys 311 , whereas an intervening Cys 309 was found not to be palmitoylated, at least under the experimental conditions used (27). Although neither lipidation affected its ligand binding properties, it is proposed that farnesylation in addition to palmitoylation of the hIP may confer a double loop structure within its C-tail domain to provide and/or orientate the critical structural domains for its G protein/effector(s) coupling and, possibly, for its interaction with components of the intracellular trafficking machinery to modulate its internalization after agonist activation (24,25,27). More specifically, although disruption of farnesylation effectively abolishes agonist-induced G s /adenylyl cyclase activation and cAMP generation by the hIP, palmitoylation at either Cys 308 or Cys 311 is sufficient to maintain functional G s coupling, whereas disruption of palmitoylation at both sites abolishes that signaling (24,25,27).…”

Prostacyclin receptors: Transcriptional regulation and novel signalling mechanisms

“…The IP is somewhat unusual among GPCRs in that it undergoes isoprenylation and palmitoylation within its carboxyl-terminal tail (C-tail) domain, modifications critical for IP signalling and function 96-100 . More specifically, the human IP undergoes farnesylation at Cys 383 within its carboxy-terminal conserved -C 383 SLC 386 , or 'CaaX', motif 99,100 and palmitoylation at Cys 308 , Cys 309 , and Cys 311 (Figure 6) 97,98 . While neither lipid modification affect the ligand binding properties of the IP, they modulate its G protein coupling/intracellular signalling and, in the case of palmitoylation, may influence its ability to directly interact with Rab11a, to regulate agonist-induced trafficking of the IP following activation [96][97][98][99] .…”