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

Abstract

We previously demonstrated that the α 2B -adrenergic receptor mutant, in which the F(x) 6 IL motif in the membrane-proximal carboxyl terminus were mutated to alanines (α 2B -ARm), is deficient in export from the endoplasmic reticulum (ER). In this report, we determined if α 2B -ARm could modulate transport from the ER to the cell surface and signaling of its wild-type counterpart. Transient expression of α 2B -ARm in HEK293T cells markedly inhibited cell-surface expression of wild-type α 2B -AR, as measured by radioligand binding. Subcellular localization demonstrated that α 2B -ARm trapped α 2B -AR in the ER. The α 2B -AR was shown to form homodimers and heterodimers with α 2B -ARm as measured by co-immunoprecipitation of the receptors tagged with green fluorescent protein and hemagglutinin epitopes. In addition to α 2B -AR, the transport of α 2A -AR and α 2C -AR to the cell surface was also inhibited by α 2B -ARm. Furthermore, transient expression of α 2B -ARm significantly reduced cell-surface expression of endogenous α 2 -AR in NG108-15 and HT29 cells. Consistent with its effect on α 2 -AR cell-surface expression, α 2B -ARm attenuated α 2A -AR-and α 2B -AR-mediated ERK1/2 activation. These data demonstrated that the ER-retained mutant α 2B -ARm conferred a dominant negative effect on the cell-surface expression of wild-type α 2 -AR, which is likely mediated through heterodimerization. These data indicate a crucial role of ER export in the regulation of cell-surface targeting and signaling of G protein-coupled receptors.

Di-acidic Motifs in the Membrane-distal C Termini Modulate the Transport of Angiotensin II Receptors from the Endoplasmic Reticulum to the Cell Surface

“…We have demonstrated that the cell surface expression of AT1R is regulated by Ras-like small GTPases, such as Rab1, Rab2, Rab6, Sar1, and ARF1 in HEK293 cells and cardiac myocytes (29,(43)(44)(45)(46). We have also demonstrated that AT1R exit from the ER is mediated through the COPII vesicles and controlled by the F(X) 6 LL motif in the membrane-proximal C terminus and a single Leu residue in the center of the first intracellular loop (23,24,29,47). These studies indicate that, similar to endocytosis, cell surface export of AT1R is a highly regulated process.…”

“…Mutagenesis studies have also identified a number of highly conserved motifs in the membrane-proximal C termini essential for GPCR export from the ER, such as the E(x) 3 LL, FN(x) 2 LL(x) 3 L, and F(x) 3 F(x) 3 F motifs (22,26,27). We have recently identified the F(x) 6 LL motif in the 8 th ␣-helix, which modulates export of ␣ 2B -AR, ␤ 2 -AR, ␣ 1B -AR, and AT1R from the ER (24,25,47). In the present study, we have demonstrated that the membrane-distal C terminus is also absolutely required for AT2R anterograde transport to the cell surface as the AT2R mutant lacking the C-terminal 7 residues was unable to transport to the cell surface, which is similar to the mutant lacking the entire C-terminal 42 residues.…”

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

“…NG108-15 neuroblastoma-glioma cells were cultured in DMEM containing 10% fetal bovine serum, 100 units/ml of penicillin, 100 g/ml of streptomycin, 100 M hypoxanthine, 0.4 M aminopterin, and 16 M thymidine as described previously (40). HL-1 cardiac myocytes were plated onto fibronectin-gelatin-coated plates or coverslips and cultured in Claycomb medium supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, 100 g/ml of streptomycin, 0.1 mM norepinephrine, and 2 mM L-glutamine as described previously (26,41).…”

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

“…It has been demonstrated that export from the ER is a rate-limiting step for maturation of the δ-opioid receptor [8], and accumulation of misfolded receptors in the ER is associated with the pathogenesis of human disease [9,10]. Second, GPCR export from the ER is directed by highly conserved motifs in the membrane-proximal termini [11][12][13][14][15]. We have recently identified the F(X) 6 LL motif in the C-termini required for α 2B -adrenergic (α 2B -AR) and angiotensin II type 1 receptor (AT1R) export from the ER [14,15] and the YS motif in the N-termini essential for α 2 -AR export from the Golgi [16].…”

“…Second, GPCR export from the ER is directed by highly conserved motifs in the membrane-proximal termini [11][12][13][14][15]. We have recently identified the F(X) 6 LL motif in the C-termini required for α 2B -adrenergic (α 2B -AR) and angiotensin II type 1 receptor (AT1R) export from the ER [14,15] and the YS motif in the N-termini essential for α 2 -AR export from the Golgi [16]. Third, dimerization of GPCRs also plays an important role in proper receptor folding to achieve a confirmation competent for export, in addition to regulating ligand binding, signal transduction and internalization [17].…”