Hague Chris, Uberti Michelle A, Chen Zhongjian, Hall Randy A, Minneman Kenneth P
Department of Pharmacology, Emory University, Atlanta, Georgia 30322, USA.
J Biol Chem. 2004 Apr 9;279(15):15541-9. doi: 10.1074/jbc.M314014200. Epub 2004 Jan 21.
alpha(1)-Adrenergic receptors (ARs) belong to the large Class I G protein-coupled receptor superfamily and comprise three subtypes (alpha(1A), alpha(1B), and alpha(1D)). Previous work with heterologously expressed C-terminal green fluorescent protein (GFP)-tagged alpha(1)-ARs showed that alpha(1A)- and alpha(1B)-ARs localize to the plasma membrane, whereas alpha(1D)-ARs accumulate intracellularly. We recently showed that alpha(1D)- and alpha(1B)-ARs form heterodimers, whereas alpha(1D)- and alpha(1A)-ARs do not. Here, we examined the role of heterodimerization in regulating alpha(1D)-AR localization using both confocal imaging of GFP- or CFP-tagged alpha(1)-ARs and a luminometer-based surface expression assay in HEK293 cells. Co-expression with alpha(1B)-ARs caused alpha(1D)-ARs to quantitatively translocate to the cell surface, but co-expression with alpha(1A)-ARs did not. Truncation of the alpha(1B)-AR extracellular N terminus or intracellular C terminus had no effect on surface expression of alpha(1D)-ARs, suggesting primary involvement of the hydrophobic core. Co-transfection with an uncoupled mutant alpha(1B)-AR (Delta12alpha(1B)) increased both alpha(1D)-AR surface expression and coupling to norepinephrine-stimulated Ca(2+) mobilization. Finally, GFP-tagged alpha(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 alpha(1B)-ARs. These studies demonstrate that alpha(1B)/alpha(1D)-AR heterodimerization controls surface expression and functional coupling of alpha(1D)-ARs, the N- and C-terminal domains are not involved in this interaction, and that alpha(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.
α1-肾上腺素能受体（ARs）属于庞大的I类G蛋白偶联受体超家族，包括三种亚型（α1A、α1B和α1D）。先前对异源表达的C端绿色荧光蛋白（GFP）标记的α1-ARs的研究表明，α1A-ARs和α1B-ARs定位于质膜，而α1D-ARs在细胞内积累。我们最近发现α1D-ARs和α1B-ARs形成异源二聚体，而α1D-ARs和α1A-ARs则不形成。在此，我们使用GFP或CFP标记的α1-ARs的共聚焦成像以及基于光度计的表面表达分析，在HEK293细胞中研究了异源二聚化在调节α1D-AR定位中的作用。与α1B-ARs共表达导致α1D-ARs定量转运到细胞表面，但与α1A-ARs共表达则没有这种作用。α1B-AR细胞外N端或细胞内C端的截断对α1D-ARs的表面表达没有影响，这表明疏水核心起主要作用。与未偶联的突变型α1B-AR（Δ12α1B）共转染增加了α1D-ARs的表面表达以及与去甲肾上腺素刺激的Ca2+动员的偶联。最后，当在不表达内源性ARs的大鼠主动脉平滑肌细胞中表达时，在细胞表面未检测到GFP标记的α1D-ARs，但当在表达内源性α1B-ARs的DDT1MF-2细胞中表达时，它们几乎完全定位于表面。这些研究表明，α1B/α1D-AR异源二聚化控制α1D-ARs的表面表达和功能偶联，N端和C端结构域不参与这种相互作用，并且α1B-AR G蛋白偶联不是必需的。这些观察结果可能与许多其他I类G蛋白偶联受体相关，在这些受体中，异源二聚化的功能后果仍知之甚少。
