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J. Biol. Chem., Vol. 278, Issue 9, 6947-6958, February 28, 2003

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Palmitoylation of the Human Prostacyclin Receptor
FUNCTIONAL IMPLICATIONS OF PALMITOYLATION AND ISOPRENYLATION^*

Sinead M. Miggin, Orlaith A. Lawler, and B. Therese Kinsella

From the Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, Merville House, University College Dublin, Belfield, Dublin 4, Ireland

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 Cys³⁰⁸ and Cys³¹¹, proximal to transmembrane domain 7 within its carboxyl-terminal (C)-tail domain, whereas Cys³⁰⁹ 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 Val³⁰⁷ generated hIP³⁰⁷ that was neither palmitoylated nor isoprenylated and did not efficiently couple to G_s or to G_q, whereas hIP³¹² was palmitoylated and ably coupled to both effector systems. Conversion of Cys³⁰⁸, Cys³⁰⁹, Cys³¹¹, 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 Cys³⁰⁸ or Cys³¹¹ is sufficient to maintain functional G_s coupling. Although mutation of Cys³⁰⁹ and Cys³¹¹ did not affect hIP-mediated G_q coupling, mutation of Cys³⁰⁸ abolished signaling, indicating a specific requirement for palmitoylation of Cys³⁰⁸ 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) Z93039.

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