Regulation of anterograde transport of adrenergic and angiotensin II receptors by Rab2 and Rab6 GTPases

Abstract

Three Rab GTPases, Rab1, Rab2 and Rab6, are involved in protein transport between the endoplasmic reticulum (ER) and the Golgi. Whereas Rab1 regulates the anterograde ER-to-Golgi transport, Rab2 and Rab6 coordinate the retrograde Golgi-to-ER transport. We have previously demonstrated that Rab1 differentially modulates the export trafficking of distinct G protein-coupled receptors (GPCRs). In this report, we determined the role of Rab2 and Rab6 in the cell surface expression and signaling of α 2B -adrenergic (α 2B -AR), β 2 -AR and angiotensin II type 1 receptors (AT1R). Expression of the GTP-bound mutant Rab2Q65L significantly attenuated the cell-surface expression of both α 2B -AR and β 2 -AR, whereas the GTP-bound mutant Rab6Q72L selectively inhibited the transport of β 2 -AR, but not α 2B -AR. Similar results were obtained by siRNA-mediated selective knockdown of endogenous Rab2 and Rab6. Consistently, Rab2Q65L and Rab2 siRNA inhibited α 2B -AR and β 2 -AR signaling measured as ERK1/2 activation and cAMP production, respectively, whereas Rab6Q72L and Rab6 siRNA reduced signaling of β 2 -AR, but not α 2B -AR. Similar to the β 2 -AR, AT1R expression at the cell surface and AT1R-promoted inositol phosphate accumulation were inhibited by Rab6Q72L. Furthermore, the nucleotide-free mutant Rab6N126I selectively attenuated the cell-surface expression of β 2 -AR and AT1R, but not α 2B -AR. These data demonstrate that Rab2 and Rab6 differentially influence anterograde transport and signaling of GPCRs. These data also provide the first evidence indicating that Rab6-coordinated retrograde transport selectively modulates intracellular trafficking and signaling of GPCRs.

Rab8 Interacts with Distinct Motifs in α2B- and β2-Adrenergic Receptors and Differentially Modulates Their Transport

“…In contrast, much less is known about the involvement of Rab GTPases in GPCR export from the Golgi to the plasma membrane. Our previous studies have demonstrated the roles of several Rab GTPases in regulating GPCR transport along the early secretory pathway (25)(26)(27)(28)(29). In this article, we expand these studies to determine the role of Rab8 GTPase in the cell surface targeting of ␣ 2B -AR and ␤ 2 -AR and the possible mechanism involved.…”

“…The PCR product was digested with XhoI and HindIII, purified, and ligated to 3HA-tagged pcDNA3.1(Ϫ) vector, which was digested with XhoI and HindIII. The GFP and HA epitopes have been used to label GPCRs, including ␣ 2B -AR and ␤ 2 -AR, resulting in receptors with similar characteristics to the wildtype receptors (12,29,38,39).…”

“…Rab8 and its GDP-bound mutant T22N tagged with the FLAG epitope at their N termini were kindly provided by Dr. Terrance E. Hebert (Department of Pharmacology, McGill University, Canada). Rab8 was also conjugated with GFP at its N terminus as described previously for Rab2 (29). The ␣ 2B -AR and ␤ 2 -AR gene segments encoding the C termini of the receptors were amplified by PCR and subcloned into the BamHI and XhoI restriction sites of pGEX-4T-1 as described previously (7).…”

“…[ 3 H]CGP12177 (specific activity ϭ 51 Ci/mmol) and [ 3 H]RX821002 (50 Ci/mmol) were purchased from Amersham Biosciences. All other materials were obtained as described elsewhere (25,29).…”

An escort for GPCRs: implications for regulation of receptor density at the cell surface

“…Retrograde transport to the ER is likely involved in re-targeting misfolded proteins to the ERAD 28 . Other defective proteins are targeted to lysosomal degradation after sorting to endosomes 28 Perturbing the function of the Rab6 GTPAse, which regulates vesicular transport in the Golgi 30 , inhibited the anterograde transport of Drosophila rhodopsin 31 and of mammalian GPCRs 25 . There is no evidence for GPCR sorting from the Golgi to endosomes or ER and it remains unknown whether GPCR targeting to the cell surface from the Golgi is regulated.…”