title: Interaction of the human prostacyclin receptor with Rab11: characterization of a novel Rab11 binding domain within alpha-helix 8 that is regulated by palmitoylation.
abstract: The human prostacyclin receptor (hIP) undergoes agonist-induced internalization and subsequent recyclization in slowly recycling endosomes involving its direct physical interaction with Rab11a. Moreover, interaction with Rab11a localizes to a 22-residue putative Rab11 binding domain (RBD) within the carboxyl-terminal tail of the hIP, proximal to the transmembrane 7 (TM7) domain. Because the proposed RBD contains Cys(308) and Cys(311), in addition to Cys(309), that are known to undergo palmitoylation, we sought to identify the structure/function determinants of the RBD, including the influence of palmitoylation, on agonist-induced trafficking of the hIP. Through complementary approaches in yeast and mammalian cells along with computational structural studies, the RBD was localized to a 14-residue domain, between Val(299) and Leu(312), and proposed to be organized into an eighth alpha-helical domain (alpha-helix 8), comprising Val(299)-Val(307), adjacent to the palmitoylated residues at Cys(308)-Cys(311). From mutational and [(3)H]palmitate metabolic labeling studies, it is proposed that palmitoylation at Cys(311) in addition to agonist-regulated deacylation at Cys(309) > Cys(308) may dynamically position alpha-helix 8 in proximity to Rab11a, to regulate agonist-induced intracellular trafficking of the hIP. Moreover, Ala-scanning mutagenesis identified several hydrophobic residues within alpha-helix 8 as necessary for the interaction with Rab11a. Given the diverse membership of the G protein-coupled receptor superfamily, of which many members are also predicted to contain an alpha-helical 8 domain proximal to TM7 and, often, adjacent to palmitoylable cysteine(s), the identification of a functional role for alpha-helix 8, as exemplified as an RBD for the hIP, is likely to have broader significance for certain members of the superfamily.
authors.affiliation: School of Biomolecular and Biomedical Sciences, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Helen M
authors.initials: HM
authors.lastname: Reid
authors.forename: Eamon P
authors.initials: EP
authors.lastname: Mulvaney
authors.forename: Elizebeth C
authors.initials: EC
authors.lastname: Turner
authors.forename: B Therese
authors.initials: BT
authors.lastname: Kinsella
mesh_terms: D019295:Computational Biology / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D007930:Leucine / Q000737:chemistry; D019308:Palmitic Acid / Q000737:chemistry; D010169:Palmitic Acids / Q000737:chemistry*; D011453:Prostaglandins / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D044006:Receptors, Epoprostenol / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D014633:Valine / Q000737:chemistry; D020691:rab GTP-Binding Proteins / Q000737:chemistry*; D000099193:rab11 GTP-Binding Proteins
chemical_list: D003545:Cysteine; D007930:Leucine; D019308:Palmitic Acid; D010169:Palmitic Acids; D011453:Prostaglandins; D044006:Receptors, Epoprostenol; D043562:Receptors, G-Protein-Coupled; D014633:Valine; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
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