Distinct Pathways for the Trafficking of Angiotensin II and Adrenergic Receptors from the Endoplasmic Reticulum to the Cell Surface

Abstract

The molecular mechanism underlying the transport of G protein-coupled receptors from the endoplasmic reticulum (ER) to the cell surface is poorly understood. This issue was addressed by determining the role of Rab1, a Ras-related small GTPase that coordinates vesicular protein transport in the early secretory pathway, in the subcellular distribution and function of the angiotensin II type 1A receptor (AT1R), ␤ 2 -adrenergic receptor (AR), and ␣ 2B -AR in HEK293T cells. Inhibition of endogenous Rab1 function by transient expression of dominant-negative Rab1 mutants or Rab1 small interfering RNA (siRNA) induced a marked perinuclear accumulation and a significant reduction in cell-surface expression of AT1R and ␤ 2 -AR. The accumulated receptors were colocalized with calregulin (an ER marker) and GM130 (a Golgi marker), consistent with Rab1 function in regulating protein transport from the ER to the Golgi. In contrast, dominant-negative Rab1 mutants and siRNA had no effect on the subcellular distribution of ␣ 2B -AR. Similarly, expression of dominant-negative Rab1 mutants and siRNA depletion of Rab1 significantly attenuated AT1R-mediated inositol phosphate accumulation and ERK1/2 activation and ␤ 2 -AR-mediated ERK1/2 activation, but not ␣ 2B -AR-stimulated ERK1/2 activation. These data indicate that Rab1 GTPase selectively regulates intracellular trafficking and signaling of G protein-coupled receptors and suggest a novel, as yet undefined pathway for movement of G protein-coupled receptors from the ER to the cell surface.

Cell-surface targeting of α2-adrenergic receptors — Inhibition by a transport deficient mutant through dimerization

“…Penicillin-streptomycin, L-glutamine and trypsin-EDTA were from Invitrogen. All other materials were obtained as described elsewhere [23][24][25][26].…”

“…α 2B -AR and AT1R tagged with green fluorescent protein (GFP) at their carboxyl termini (α 2B -AR-GFP and AT1R-GFP) and α 2B -AR tagged with HA epitope at its amino terminus (HA-α 2B -AR) were generated as previously described [22,23]. The GFP and HA epitopes have been used to label G protein-coupled receptors, including α 2B -AR and AT1R, resulting in receptors with similar characteristics to the WT receptors [22][23][24]27] were mutated to alanines, were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).…”

“…The GFP and HA epitopes have been used to label G protein-coupled receptors, including α 2B -AR and AT1R, resulting in receptors with similar characteristics to the WT receptors [22][23][24]27] were mutated to alanines, were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The sequence of each construct used in this study was verified by restriction mapping and nucleotide sequence analysis (Louisiana State University Health Sciences Center DNA Sequence Core).…”

“…NG108-15 neuroblastoma-glioma cells expressing endogenous α 2B -AR subtype were cultured in DMEM containing 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ ml streptomycin, 100 µM hypoxanthine, 0.4 µM aminopterin, and 16 µM thymidine. Transient transfection of the cells was carried out using Lipofect-AMINE 2000 reagent (Invitrogen) as described previously [22,23]. Based on the GFP fluorescence, 70-85% of the cells were transfected.…”

“…The coverslips were mounted, and fluorescence was detected with a Leica DMRA2 epifluorescent microscope. Images were deconvolved using SlideBook software and the nearest neighbors deconvolution algorithm (Intelligent Imaging Innovations, Denver, CO) as previously described [22,23].…”