Abstract
We have previously established that isoprenylation of the prostacyclin receptor (IP) is required for its efficient G protein coupling and effector signaling (Hayes, J. S., Lawler, O. A., Walsh, M. T., and Kinsella, B. T. (1999) J. Biol. Chem. 274, 23707-23718). In the present study, we sought to investigate whether the IP may actually be subject to palmitoylation in addition to isoprenylation and to establish the functional significance thereof. The human (h) IP was efficiently palmitoylated at Cys(308) and Cys(311), proximal to transmembrane domain 7 within its carboxyl-terminal (C)-tail domain, whereas Cys(309) was not palmitoylated. The isoprenylation-defective hIP(SSLC) underwent palmitoylation but did not efficiently couple to G(s) or G(q), confirming that isoprenylation is required for G protein coupling. Deletion of C-tail sequences distal to Val(307) generated hIP(Delta307) that was neither palmitoylated nor isoprenylated and did not efficiently couple to G(s) or to G(q), whereas hIP(Delta312) was palmitoylated and ably coupled to both effector systems. Conversion of Cys(308), Cys(309), Cys(311), Cys(308,309), or Cys(309,311) to corresponding Ser residues, while leaving the isoprenylation CAAX motif intact, did not affect hIP coupling to G(s) signaling, whereas mutation of Cys(308,311) and Cys(308,309,311) abolished signaling, indicating that palmitoylation of either Cys(308) or Cys(311) is sufficient to maintain functional G(s) coupling. Although mutation of Cys(309) and Cys(311) did not affect hIP-mediated G(q) coupling, mutation of Cys(308) abolished signaling, indicating a specific requirement for palmitoylation of Cys(308) for G(q) coupling. Consistent with this, neither hIP(C308S,C309S), hIP(C308S,C311S), 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.
Highlights
We propose that through lipid modification followed by membrane insertion, the C9yAs3d09etewrmithininedthtehiraitntmraacnelylufleaar tcuarrbeosxyl-terminal tail (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
Mutation of the CAAX motif of the hIP from -C383SLC to -S383SLC abolished isoprenylation and generated hIPSSLC that exhibited identical ligand binding properties to that of the wild type hIP but failed to show significant coupling to G␣s/adenylyl cyclase or efficient coupling to G␣q/PLC activation [25]. In contrast to these findings, another study demonstrated that hIP⌬312, a variant of hIP lacking a substantial portion of the C-tail including the isoprenylation CAAX motif, exhibited both G␣s coupling at levels comparable with hIP and G␣q coupling, albeit at somewhat reduced efficacy [19]
Through a combination of deletion and site-directed mutagenesis, in vivo metabolic labeling studies, and the use of pharmacologic inhibitors of protein isoprenylation, we investigated the requirement for palmitoylation and isoprenylation for IP function and intracellular signaling
Summary
Consistent with this, neither hIPC308S,C309S, the hydrophobic palmitate moiety becomes integrated into the hIPC308S,C311S, nor hIPC308S,C309S,C311S coupled to Gq. lipid membrane bilayer, resulting in the formation of a putative Taken together, these data confirm that the hIP is iso- fourth intracellular loop. 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. This is the case [8]. The 2-adrenergic receptor (AR) is palmitoylated at Cys341, and palmitoylation at this site is required for 2-AR coupling to adenylyl cyclase [9]
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