Role of C-terminal Membrane-proximal Basic Residues in Cell Surface Trafficking of HIV Coreceptor GPR15 Protein

• Published in: The Journal of biological chemistry 2013-03, Vol.288 (13), p.9189-9199
• Main Authors: Okamoto, Yukari, Bernstein, Joshua David, Shikano, Sojin
• Format: Article
• Language: English
• Subjects: Alanine - metabolism; Amino Acid Sequence; Basic; C-terminal; Cell Biology; Cell Membrane - metabolism; Endoplasmic Reticulum (ER); Endoplasmic Reticulum - metabolism; Flow Cytometry - methods; G Protein-coupled Receptor (GPCR); Golgi Apparatus - metabolism; GPR15; HEK293 Cells; HIV; HIV - metabolism; Humans; Hydrogen-Ion Concentration; Membrane Proteins; Membrane Trafficking; Molecular Sequence Data; Monomeric GTP-Binding Proteins - metabolism; Mutation; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; Proximal; Receptors, G-Protein-Coupled - metabolism; Receptors, Peptide - metabolism; Residue; Sequence Homology, Amino Acid; Vesicular Transport Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.445817

Cell surface density of G protein-coupled receptors (GPCRs) is controlled by dynamic molecular interactions that often involve recognition of the distinct sequence signals on the cargo receptors. We reported previously that the RXR-type dibasic motif in the distal C-terminal tail of an HIV coreceptor GPR15 negatively regulates the cell surface expression by mediating the coatomer protein I complex-dependent retrograde transport to the endoplasmic reticulum (ER). Here we demonstrate that another pair of basic residues (Arg310-Arg311) in the membrane-proximal region of the C-terminal tail plays a pivotal role in mediating the anterograde trafficking of GPR15. The Ala mutation of the C-terminal membrane-proximal basic residues (MPBRs) (R310/311A) abolished the O-glycosylation and cell surface expression of GPR15. The subcellular fractionation and immunocytochemistry assays indicated that the R310/311A mutant was more localized in the ER but much less in the trans-Golgi when compared with the wild-type GPR15, suggesting the positive role of Arg310-Arg311 in the ER-to-Golgi transport of GPR15. Sequence analysis on human GPCRs showed that the basic residues are frequent in the membrane-proximal region of the C-terminal tail. Similar to GPR15, mutation of the C-terminal MPBRs resulted in a marked reduction of the cell surface expression in multiple different GPCRs. Our results suggest that the C-terminal MPBRs are critically involved in mediating the anterograde trafficking of a broad range of membrane proteins, including GPCRs. Background: The role of membrane-proximal basic residues (MPBRs) in protein trafficking is not well understood. Results: Mutation of C-terminal MPBRs reduced cell surface expression of GPR15 and other GPCRs. Conclusion: C-terminal MPBRs are necessary for cell surface trafficking of GPCRs, including GPR15. Significance: C-terminal MPBRs play a pivotal role in cell surface trafficking of GPCRs.