Palmitoylation of the human prostacyclin receptor. Functional implications of palmitoylation and isoprenylation.
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.
Miggin
Sinead M
SM
Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, Merville House, University College Dublin, Belfield, Dublin 4, Ireland.
Lawler
Orlaith A
OA
Kinsella
B Therese
BT
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D006801:Humans; D008024:Ligands; D008055:Lipids; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019308:Palmitic Acid / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017368:Protein Prenylation; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D004791:Enzyme Inhibitors; D008024:Ligands; D008055:Lipids; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D019308:Palmitic Acid; D012694:Serine; D000242:Cyclic AMP; D011493:Protein Kinase C; D003545:Cysteine; D002118:Calcium