Abstract
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 (Arg(310)-Arg(311)) 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 Arg(310)-Arg(311) 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.
Highlights
The role of membrane-proximal basic residues (MPBRs) in protein trafficking is not well understood
In this work we addressed the question whether the Arg310Arg311 dibasic signal at the membrane-proximal C terminus would play roles in cell surface trafficking
We found that the specific mutation of these MPBRs to Ala abolished cell surface expression and O-glycosylation of GPR15 (Fig. 1)
Summary
The role of membrane-proximal basic residues (MPBRs) in protein trafficking is not well understood. 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. GPCRs2 represent the largest class of cell surface receptors and play a central role in regulating a plethora of biological pathways. The positive role of MPBRs in anterograde trafficking was reported for the Golgi-resident glycosyltransferases that employ type II membrane protein topology with a short N-terminal tail in the cytoplasmic side [13] In this case, specific mutations of these MPBRs abolished the interaction of the enzyme with Sar GTPase and blocked the export of enzymes from the ER. Our results suggest that C-terminal MPBRs play an important role in mediating the anterograde trafficking of a broad range of membrane proteins, including GPCRs
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