Interaction of the Human Prostacyclin Receptor with Rab11: CHARACTERIZATION OF A NOVEL Rab11 BINDING DOMAIN WITHIN α-HELIX 8 THAT IS REGULATED BY PALMITOYLATION

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 Cys308 and Cys311, in addition to Cys309, 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 Val299 and Leu312, and proposed to be organized into an eighth α-helical domain (α-helix 8), comprising Val299–Val307, adjacent to the palmitoylated residues at Cys308–Cys311. From mutational and [3H]palmitate metabolic labeling studies, it is proposed that palmitoylation at Cys311 in addition to agonist-regulated deacylation at Cys309 > Cys308 may dynamically position α-helix 8 in proximity to Rab11a, to regulate agonist-induced intracellular trafficking of the hIP. Moreover, Ala-scanning mutagenesis identified several hydrophobic residues within α-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 α-helical 8 domain proximal to TM7 and, often, adjacent to palmitoylable cysteine(s), the identification of a functional role for α-helix 8, as exemplified as an RBD for the hIP, is likely to have broader significance for certain members of the superfamily.