Cell Surface Expression of α1D-Adrenergic Receptors Is Controlled by Heterodimerization with α1B-Adrenergic Receptors

Abstract

␣ 1 -Adrenergic receptors (ARs) belong to the large Class I G protein-coupled receptor superfamily and comprise three subtypes (␣ 1A , ␣ 1B , and ␣ 1D ). Previous work with heterologously expressed C-terminal green fluorescent protein (GFP)-tagged ␣ 1 -ARs showed that ␣ 1A -and ␣ 1B -ARs localize to the plasma membrane, whereas ␣ 1D -ARs accumulate intracellularly. We recently showed that ␣ 1D -and ␣ 1B -ARs form heterodimers, whereas ␣ 1D -and ␣ 1A -ARs do not. Here, we examined the role of heterodimerization in regulating ␣ 1D -AR localization using both confocal imaging of GFP-or CFPtagged ␣ 1 -ARs and a luminometer-based surface expression assay in HEK293 cells. Co-expression with ␣ 1B -ARs caused ␣ 1D -ARs to quantitatively translocate to the cell surface, but co-expression with ␣ 1A -ARs did not. Truncation of the ␣ 1B -AR extracellular N terminus or intracellular C terminus had no effect on surface expression of ␣ 1D -ARs, suggesting primary involvement of the hydrophobic core. Co-transfection with an uncoupled mutant ␣ 1B -AR (⌬12␣ 1B ) increased both ␣ 1D -AR surface expression and coupling to norepinephrine-stimulated Ca 2؉ mobilization. Finally, GFP-tagged ␣ 1D -ARs were not detected on the cell surface when expressed in rat aortic smooth muscle cells that express no endogenous ARs, but were almost exclusively localized on the surface when expressed in DDT 1 MF-2 cells, which express endogenous ␣ 1B -ARs. These studies demonstrate that ␣ 1B /␣ 1D -AR heterodimerization controls surface expression and functional coupling of ␣ 1D -ARs, the N-and Cterminal domains are not involved in this interaction, and that ␣ 1B -AR G protein coupling is not required. These observations may be relevant to many other Class I G protein-coupled receptors, where the functional consequences of heterodimerization are still poorly understood.

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

“…Since we have demonstrated that inhibition of α 2B -AR cell-surface expression is mediated through dimerization of mutant and WT receptors, it is possible that the inhibitory effect of α 2B -ARm on the transport of α 2A -AR and α 2C -AR is also due to heterodimerization of α 2B -ARm with α 2A -AR or α 2C -AR. This possibility is supported by a number of observations demonstrating that closely related members of G protein-coupled receptors may heterodimerize [11,14,[37][38][39]. Furthermore, α 2A -AR and α 2C -AR indeed form heterodimers with β 1 -AR and M 3 -muscarinc receptor, respectively [40,41].…”

“…Therefore, dimers are unable to export from the ER. Dimerization has been well described for a variety of G protein-coupled receptors [9][10][11][12][13][14][15][34][35][36][37][38][39][40][41]. However, whether α 2B -AR is capable of forming homodimers has not been reported.…”

“…In contrast, AT1R-GFP was not found in the anti-HA immunoprecipitate when co-expressed with HA-α 2B -AR. These data indicate that HA-and GFP-tagged α 2B -AR or α 2B -ARm were co-immunoprecipitated as a complex and suggest that α 2B -AR as well as α 2B -ARm underwent homodimerization, similar to many other G protein-coupled receptors [9][10][11][12][13][14][15].…”

“…Recently, Bouvier's group has demonstrated that mutation of the putative dimerization motif GxxxGxxxL in the β 2 -adrenergic receptor (AR) prevents normal trafficking of the receptor to the plasma membrane [9]. Although dimerization has been demonstrated for a variety of G protein-coupled receptors [10][11][12][13][14][15], whether α 2 -AR is able to homodimerize and the role of dimerization in the trafficking of α 2 -AR remain unknown. Fourth, receptor exit from the ER may be directed by specific signals or motifs embedded in the receptors.…”

α1‐Adrenoceptor subtype substitution in knockout mice

“…However, 3-month-old KO mice had almost no hepatic a 1 -adrenoceptor binding. This would indicate that replacement of the missing a 1B adrenoceptor by the a 1A is a relatively slow process, and suggest that, in general, comparisons of receptor density between WT and KO mice should be made at multiple time points.Based on multiple literature reports, it is now clear that a 1 -adrenoceptors are not confined to the cell membrane, and that the subcellular localization can differ between subtypes (Mackenzie et al, 2000;Stanasila et al, 2003;Hague et al, 2004). Deighan et al (2004) show that the distribution of a 1A adrenoceptors in KO mice is identical to that of the a 1B in WT mice, consistent with the ability of the two subtypes to mediate the same physiological function.…”