ABSTRACT
The human prostacyclin receptor (hIP) undergoes agonist-dependent trafficking involving a direct interaction with Rab11a GTPase. The region of interaction was localised to a 14 residue Rab11a binding domain (RBD) within the proximal carboxyl-terminal (C)-tail domain of the hIP, consisting of Val(299)-Val(307) within the eighth helical domain (α-H8) adjacent to the palmitoylated residues at Cys(308)-Cys(311). However, the factors determining the anterograde transport of the newly synthesised hIP from the endoplasmic reticulum (ER) to the plasma membrane (PM) have not been identified. The aim of the current study was to identify the major ER export motif(s) within the hIP initially by investigating the role of Lys residues in its maturation and processing. Through site-directed and Ala-scanning mutational studies in combination with analyses of protein expression and maturation, functional analyses of ligand binding, agonist-induced intracellular signalling and confocal image analyses, it was determined that Lys(297), Arg(302) and Lys(304) located within α-H8 represent the critical determinants of a novel ER export motif of the hIP. Furthermore, while substitution of those critical residues significantly impaired maturation and processing of the hIP, replacement of the positively charged Lys with Arg residues, and vice versa, was functionally permissible. Hence, this study has identified a novel 8 residue ER export motif within the functionally important α-H8 of the hIP. This ER export motif, defined by "K/R(X)(4)K/R(X)K/R," has a strict requirement for positively charged, basic Lys/Arg residues at the 1st, 6th and 8th positions and appears to be evolutionarily conserved within IP sequences from mouse to man.
Subject(s)
Amino Acid Motifs , Endoplasmic Reticulum/metabolism , Receptors, Epoprostenol/chemistry , Receptors, Epoprostenol/metabolism , Amino Acid Sequence , Arginine/chemistry , Arginine/genetics , Arginine/metabolism , Binding Sites , Blotting, Western , Calcium/metabolism , Calnexin/metabolism , Computational Biology , HEK293 Cells , Humans , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Microscopy, Confocal , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , Radioligand Assay , Receptors, Epoprostenol/genetics , Sequence Homology, Amino AcidABSTRACT
The human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood. Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl-terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaprost stimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time-dependent manner following cicaprost stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11(S25N) impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val(299)-Gln(320)) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a.
