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Heterologous expression of ␣ 1D -adrenergic receptors (␣ 1D -ARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with ␣ 1B -ARs promotes surface localization of ␣ 1D -ARs. Here, we report that the ␣ 1B -/␣ 1D -AR interaction has significant effects on the pharmacology and signaling of the receptors, in addition to the effects on trafficking described previously. Upon coexpression of ␣ 1B -ARs and epitope-tagged ␣ 1D -ARs in both human embryonic kidney 293 and DDT 1 MF-2 cells, ␣ 1D -AR binding sites were not detectable with the ␣ 1D -AR selective antagonist 8-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro [4,5]decane-7,9-dione (BMY 7378), despite the ability to detect ␣ 1D -AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the ␣ 1B -AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in ␣ 1B /␣ 1D -AR-expressing cells, revealing that ␣ 1D -ARs were expressed but did not bind BMY 7378 with high affinity. Studies of norepinephrine-stimulated inositol phosphate formation showed that maximal responses were greatest in ␣ 1B /␣ 1D -AR-coexpressing cells. Stable coexpression of an uncoupled mutant ␣ 1B -AR (⌬12) with ␣ 1D -ARs resulted in increased responses to norepinephrine. However, Schild plots for inhibition of norepinephrine-stimulated inositol phosphate formation showed a single low-affinity site for BMY 7378. Thus, our findings suggest that ␣ 1B /␣ 1D -AR heterodimers form a single functional entity with enhanced functional activity relative to either subtype alone and a novel pharmacological profile. These data may help to explain why ␣ 1D -ARs are often pharmacologically undetectable in native tissues when they are coexpressed with ␣ 1B -ARs.

The ␣ 2C -adrenergic receptor (␣ 2C AR) is known to be poorly trafficked to the cell surface when expressed in a variety of cell types. We tested the hypothesis that the surface expression and signaling of ␣ 2C AR might be enhanced by heterodimerization with other G protein-coupled receptors (GPCRs). Cotransfection of ␣ 2C AR with more than 25 related GPCRs revealed that only coexpression with the ␤ 2 -adrenergic receptor (␤ 2 AR) increased the surface localization of ␣ 2C AR in human embryonic kidney-293 cells. Coimmunoprecipitation of ␣ 2C AR with ␤ 2 AR confirmed a physical interaction between the two receptors. Confocal microscopy studies demonstrated that ␣ 2C AR expressed alone was mainly intracellular, whereas ␣ 2C AR coexpressed with ␤ 2 AR was predominantly localized to the plasma membrane. Ligand binding studies revealed a significant increase in ␣ 2C AR binding sites upon coexpression with ␤ 2 AR, with no apparent change in affinity for ␣ 2 AR ligands. Functional assays with the ␣ 2 AR-specific agonist brimonidine (UK 14,304) revealed that coexpression of ␤ 2 AR with ␣ 2C AR enhanced ␣ 2C AR-mediated activation of extracellular signalregulated kinase 1/2. Furthermore, analyses of agonist-promoted receptor endocytosis demonstrated enhanced ␣ 2C AR internalization in response to ␣ 2 AR agonists when ␣ 2C AR and ␤ 2 AR were coexpressed. In addition, substantial cointernalization of ␣ 2C AR in response to ␤AR agonists was observed when ␣ 2C AR was coexpressed with ␤ 2 AR. These data reveal that ␣ 2C AR can interact with ␤ 2 AR in cells in a manner that regulates ␣ 2C AR surface expression, internalization, and functionality.The adrenergic receptors are a family of cell-surface G protein-coupled receptors (GPCRs) that mediate the actions of the hormone epinephrine and the neurotransmitter norepinephrine. The three main adrenergic receptor (AR) classes (␣ 1 , ␣ 2 , and ␤ 2 ) can be further divided into three subtypes each, and all of these subtypes are excellent targets for therapeutic pharmaceuticals. The specific roles of the various adrenergic receptor subtypes is becoming increasingly clear through studies on knock-out mice (Philipp and Hein, 2004), and novel therapies making use of these insights await the development of more subtype-specific drugs. However, two of the adrenergic receptor subtypes, ␣ 2C AR and ␣ 1D

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