Rab26 Modulates the Cell Surface Transport of α2-Adrenergic Receptors from the Golgi

Abstract

The molecular mechanisms underlying the transport from the Golgi to the cell surface of G protein‐coupled receptors (GPCRs) remain poorly elucidated. Here we determined the role of Rab26, a Ras‐like small GTPase involved in vesicle‐mediated secretion, in the cell‐surface export of α2‐adrenergic receptors (α2‐ARs). We found that transient expression of Rab26 mutants and siRNA‐mediated depletion of Rab26 significantly attenuated the cell‐surface numbers of α2A‐AR and α2B‐AR as well as ERK1/2 activation by α2B‐AR. Furthermore, the receptors were extensively arrested in the Golgi by Rab26 mutants and siRNA. Moreover, Rab26 directly and activation‐dependently interacted with α2BAR, specifically the third intracellular loop. These data demonstrate that the small GTPase Rab26 regulates the Golgi‐to‐cell surface traffic of α2‐ARs, likely through a physical interaction. These data also provide the first evidence implicating an important function of Rab26 in coordinating plasma membrane protein transport (R01GM076167, R01GM096762 and R01GM078319).

GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

“…All three ␣ 2 -ARs couple to the Gi/Go family G proteins, and their activation has been well shown to stimulate the mitogen-activated protein kinases (MAPK) ERK1/2, inhibit adenylyl cyclases, and suppress voltage-gated calcium channels (24,26,(37)(38)(39). To investigate if GGA3 knockdown-induced reduction of cell surface ␣ 2B -AR expression could result in a concomitant defective signaling, the activation of ERK1/2 and the reduction of cAMP production were chosen as functional readouts.…”

“…HEK293 cells were grown on coverslips precoated with poly-L-lysine in 6-well plates and transfected with 50 ng of ␣ 2B -AR-GFP together with 400 ng of control or GGA3 shRNA. The cells were permeabilized with PBS containing 0.2% Triton X-100 for 5 min and blocked with 5% normal donkey serum for 1 h. The cells were then incubated with antibodies against p230 (1:100 dilution) for 1 h. After washing with PBS (3 times for 5 min), the cells were incubated with Alexa Fluor 594-labeled secondary antibody (1:2,000 dilution) for 1 h. Images were captured using an LSM510 Meta Zeiss confocal microscope as described previously (24,33). To detect the cell surface expression of inducibly expressed HA-␣ 2B -AR, the cells were stained with anti-HA antibodies (1:500 dilution) without permeabilization.…”

“…For GST fusion protein pulldown assays, the GST fusion proteins were expressed in bacteria and purified as described previously (23,24). GST fusion proteins immobilized on the glutathione resin were either used immediately or stored at 4°C for no longer than 3 days.…”

RAB26 coordinates lysosome traffic and mitochondrial localization

“…Recent studies have found that RAB26 is associated with RAB11 recycling endosomes (Chan et al, 2011) in Drosophila neurons, and a giantin-positive Golgi sorting compartment (Li et al, 2012) when transfected into HEK293 cells. These results would seem to contradict those presented here, although we have focused here exclusively on exocrine secretory cells, trying to mimic the effects of scaling RAB26 as occurs in differentiating cells in tissue.…”

“…S1B-F). The lack of RAB26 association with the Golgi was of interest, because recent studies have demonstrated that RAB26 might occupy a recycling endosomal (Chan et al, 2011) or giantin-positive Golgi compartment (Li et al, 2012). Because of the proximity, if not substantial direct overlap, of RAB26 vesicles with our trans-Golgi marker, we next examined membrane compartments that interact with the trans-Golgi.…”