Regulation of the Cell Surface Expression and Function of Angiotensin II Type 1 Receptor by Rab1-mediated Endoplasmic Reticulum-to-Golgi Transport in Cardiac Myocytes

Abstract

Rab1GTPase coordinates vesicle-mediated protein transport specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. We recently demonstrated that Rab1 is involved in the export of angiotensin II (Ang II) type 1 receptor (AT1R) to the cell surface in HEK293 cells and that transgenic mice overexpressing Rab1 in the myocardium develop cardiac hypertrophy. To expand these studies, we determined in this report whether the modification of Rab1-mediated ER-to-Golgi transport can alter the cell surface expression and function of endogenous AT1R and AT1R-mediated hypertrophic growth in primary cultures of neonatal rat ventricular myocytes. Adenovirus-mediated gene transfer of wild-type Rab1 (Rab1WT) significantly increased cell surface expression of endogenous AT1R in neonatal cardiomyocytes, whereas the dominant-negative mutant Rab1N124I had the opposite effect. Brefeldin A treatment blocked the Rab1WT-induced increase in AT1R cell surface expression. Fluorescence analysis of the subcellular localization of AT1R revealed that Rab1 regulated AT1R transport specifically from the ER to the Golgi in HL-1 cardiomyocytes. Consistent with their effects on AT1R export, Rab1WT and Rab1N124I differentially modified the AT1R-mediated activation of ERK1/2 and its upstream kinase MEK1. More importantly, adenovirus-mediated expression of Rab1N124I markedly attenuated the Ang II-stimulated hypertrophic growth as measured by protein synthesis, cell size, and sarcomeric organization in neonatal cardiomyocytes. In contrast, Rab1WT expression augmented the Ang II-mediated hypertrophic response. These data strongly indicate that AT1R function in cardiomyocytes can be modulated through manipulating AT1R traffic from the ER to the Golgi and provide the first evidence implicating the ERto-Golgi transport as a regulatory site for control of cardiomyocyte growth.

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

“…To address these issues, we previously determined the role of Rab1, a Ras-like GTPase that regulates vesicle-mediated protein transport in the early secretory pathway, specifically from the ER to the Golgi, in the export trafficking of α 2B -AR, β 2 -AR and AT1R [23,24]. We have demonstrated that, whereas the transport from the ER to the cell surface of AT1R and β 2 -AR is dependent on Rab1, α 2B -AR export is independent of Rab1, indicating that different G protein-coupled receptors may use distinct pathways for their transport from the ER to the cell surface [23].…”

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

“…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).…”

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

“…Whereas Rab4 and Rab11 are mainly involved in the recycling of internalized receptor from the endosome back to the plasma membrane, Rab5 regulates the internalization of GPCRs from the plasma membrane to the endosome, and Rab7 participates in the process targeting receptors to the lysosome for degradation [22][23][24][25][26][27][28][29][30][31][32]. Our laboratory has focused efforts on understanding the role of Rab1 GTPase in the export trafficking of GPCRs [33][34][35]. Rab1 is localized in the ER and the Golgi apparatus and regulates anterograde protein transport specifically from the ER to the Golgi and between the Golgi compartments [36][37][38].…”

“…We have previously demonstrated that Rab1 differentially regulates the anterograde trafficking of distinct GPCRs. Whereas the transport from the ER to the cell surface of α 1 -AR, β-AR and AT1R is dependent on Rab1, the transport of α 2B -AR to the cell surface utilizes a non-classic, Rab1-independent pathway, suggesting multiple pathways mediating the cell-surface targeting of GPCRs [33][34][35].…”