Heterodimers of α1B- and α1D-Adrenergic Receptors Form a Single Functional Entity

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Abstract
Heterologous expression of α1D-adrenergic receptors (α1D-ARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with α1B-ARs promotes surface localization of α1D-ARs. Here, we report that the α1B-/α1D-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 α1B-ARs and epitope-tagged α1D-ARs in both human embryonic kidney 293 and DDT1MF-2 cells, α1D-AR binding sites were not detectable with the α1D-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 α1D-AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the α1B-AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in α1B/α1D-AR-expressing cells, revealing that α1D-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 α1B/α1D-AR-coexpressing cells. Stable coexpression of an uncoupled mutant α1B-AR (Δ12) with α1D-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 α1B/α1D-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 α1D-ARs are often pharmacologically undetectable in native tissues when they are coexpressed with α1B-ARs.

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