␣ 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.

The potential role of dimerization in controlling the expression and pharmacological properties of ␣ 1 -adrenoceptor subtypes was examined using coimmunoprecipitation of epitope-tagged receptors. Human ␣ 1 -adrenoceptor subtypes (␣ 1A , ␣ 1B , ␣ 1D ) were tagged at their amino-termini with Flag or hemagglutinin epitopes and transfected into human embryonic kidney 293 cells. Homodimerization of all three subtypes was observed by coimmunoprecipitation of receptors with different tags and was not altered by norepinephrine treatment. Heterodimer formation between hemagglutinin-tagged ␣ 1B -adrenoceptors and Flag-tagged ␣ 1A -or ␣ 1D -adrenoceptors was also observed. However, no ␣ 1A /␣ 1D -adrenoceptor heterodimers were observed, suggesting that dimerization is subtype-specific. The extent of heterodimerization was also unaltered by norepinephrine treatment. ␣ 1 -Adrenoceptor truncation mutants lacking carboxyl or amino-terminal sequences formed homo-and heterodimers similarly to full-length receptors, suggesting that these domains play little or no role in dimerization. Biotinylation with a membrane-impermeable agent showed that monomers and homo-and hetero-oligomers of all three subtypes are expressed on the cell surface. Radioligand binding studies showed that heterodimerization did not alter the affinity of ␣ 1 -adrenoceptors for norepinephrine, prazosin, or subtype-selective antagonists, suggesting that dimerization does not result in pharmacologically distinct subtypes. However, coexpression of ␣ 1B -adrenoceptors significantly increased both binding site density and protein expression of ␣ 1A -and ␣ 1D -adrenoceptors, and increased cell surface expression of ␣ 1D -adrenoceptors, suggesting a functional role for heterodimerization. Conversely, coexpression of ␣ 1A -with ␣ 1D -adrenoceptors, which did not heterodimerize, had no effect on receptor density or protein. These studies demonstrate subtype-selective heterodimerization of ␣ 1 -adrenoceptors, which does not change their pharmacological properties but seems to have functional consequences in regulating receptor expression and trafficking.

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The ␣ 1D -adrenergic receptor (␣ 1D -AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote ␣ 1D -AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of ␣ 1D -AR at the cell surface: ␣ 1B -AR and ␤ 2 -AR. Confocal imaging confirmed that coexpression with ␤ 2 -AR resulted in translocation of ␣ 1D -AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that ␣ 1D -AR and ␤ 2 -AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total ␣ 1D -AR binding sites upon coexpression with ␤ 2 -AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with ␤ 2 -AR significantly enhanced the coupling of ␣ 1D -AR to norepinephrine-stimulated Ca 2ϩ mobilization. Heterodimerization of ␤ 2 -AR with ␣ 1D -AR also conferred the ability of ␣ 1D -AR to cointernalize upon ␤ 2 -AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of ␣ 1D -AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between ␣ 1 -and ␤ 2 -ARs that is mediated through heterodimerization and cross-internalization.

Olfactory receptors (ORs) comprise more than half of the large class I G protein-coupled receptor (GPCR) superfamily. Although cloned over a decade ago, little is known about their properties because wild-type ORs do not efficiently reach the cell surface following heterologous expression. Receptor-receptor interactions strongly influence surface trafficking of other GPCRs, and we examined whether a similar mechanism might be involved in OR surface expression. Olfactory neurons are known to express ␤-adrenergic receptors (ARs), and we found that coexpression with ␤ 2 -ARs, but not any other AR subtypes, dramatically increased mouse 71 (M71) OR surface expression in human embryonic kidney 293 cells. A persistent physical interaction between M71 ORs and ␤2-ARs was shown by coimmunoprecipitation and by cointernalization of the two receptors in response to their specific ligands. Also, coexpression of wild-type M71 ORs with ␤2-ARs resulted in cAMP responses to the M71 ligand acetophenone. Finally, in situ hybridization studies showed extensive colocalization of M71 OR and ␤2-AR expression in mouse olfactory epithelium. These data demonstrate the successful heterologous surface expression of a functional wild-type OR and reveal that persistent physical association with other GPCRs can control OR surface expression. P erception of smell begins with stimulation of olfactory receptors (ORs) on neurons within the olfactory epithelium, leading to excitation and propagation of currents to the main olfactory bulb (1, 2). ORs are class I G protein-coupled receptors (GPCRs) that signal through stimulation of G␣ olf , which leads to activation of type III adenylyl cyclase and opening of cAMPgated cation channels (3). Since the completion of the human and mouse genome sequencing projects, Ϸ350 receptors in humans (4) and Ϸ1,000 receptors in mice (5) have been identified, presumably to aid in the selective recognition of Ͼ100,000 different odors. However, the mechanism by which the olfactory system selectively recognizes specific odors remains unclear. It was initially hypothesized that each olfactory neuron expresses a single OR and that the axons of olfactory neurons expressing the same OR then converge in the main olfactory bulb (6, 7). However, increasing evidence suggests that detection is substantially more complex than previously thought. For example, olfactory neurons are not restricted to expression of a single OR subtype (8). In addition to ORs, olfactory neurons can express many other receptors, which facilitate modulation of olfactory responses by hormones and neurotransmitters. For example, epinephrine stimulation of endogenous ␤-adrenergic receptors (ARs) modifies the signaling of coexpressed ORs within olfactory neurons (9). Furthermore, multiple OR subtypes can respond to the same ligand, a single OR can respond to multiple ligands (10-12), and structurally similar odorant ligands can act as either agonists or antagonists (13). Thus, as the complexity of the olfactory system becomes increasingly clear, the need t...