Heterodimers of alpha1B- and alpha1D-adrenergic receptors form a single functional entity

Heterologous expression of alpha(1D)-adrenergic receptors (alpha(1D)-ARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with alpha(1B)-ARs promotes surface localization of alpha(1D)-ARs. Here, we report that the alpha(1B)-/alpha(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 alpha(1B)-ARs and epitope-tagged alpha(1D)-ARs in both human embryonic kidney 293 and DDT(1)MF-2 cells, alpha(1D)-AR binding sites were not detectable with the alpha(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 alpha(1D)-AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the alpha(1B)-AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in alpha(1B)/alpha(1D)-AR-expressing cells, revealing that alpha(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 alpha(1B)/alpha(1D)-AR-coexpressing cells. Stable coexpression of an uncoupled mutant alpha(1B)-AR (Delta12) with alpha(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 alpha(1B)/alpha(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 alpha(1D)-ARs are often pharmacologically undetectable in native tissues when they are coexpressed with alpha(1B)-ARs.