title: Identification of a novel endoplasmic reticulum export motif within the eighth α-helical domain (α-H8) of the human prostacyclin receptor.
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.
authors.affiliation: School of Biomeolecular and Biomedical Sciences, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Donnellan
authors.forename: Cisca C
authors.initials: CC
authors.lastname: Kimbembe
authors.forename: Helen M
authors.initials: HM
authors.lastname: Reid
authors.forename: B Therese
authors.initials: BT
authors.lastname: Kinsella
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D037281:Calnexin / Q000378:metabolism; D019295:Computational Biology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011869:Radioligand Assay; D044006:Receptors, Epoprostenol / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D044006:Receptors, Epoprostenol; D037281:Calnexin; D001120:Arginine; D008239:Lysine; D002118:Calcium