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

Heterologous expression of α 1D ‐adrenergic receptors (α 1D ‐ARs) in most cell types results in intracellular retention and little or no functionality. We previously showed that heterodimerization with α 1B ‐ARs promotes surface localization of α 1D ‐ARs. Here, we report that the α 1B ‐/α 1D ‐AR interaction has significant effects on the pharmacology and signaling of the receptors, in addition to the previously‐described effects on trafficking. Upon co‐expression of α 1B ‐ARs and epitope‐tagged α 1D ‐ARs in both HEK293 and DDT 1 MF‐2 cells, α 1D ‐AR binding sites were not detectable with the α 1D ‐AR selective antagonist BMY 7378, despite the ability to detect α 1D ‐AR protein using confocal microscopy, immunoprecipitation and a luminometer cell‐surface assay. However, the α 1B ‐AR selective F18A conotoxin showed a striking biphasic inhibition in α 1B ‐/α 1D ‐AR expressing cells, revealing that α 1D ‐ARs were expressed but not binding BMY 7378 with high affinity. Studies of norepinephrine‐stimulated inositol phosphate formation showed that maximal responses were greatest in α 1B ‐/α 1D ‐AR co‐expressing cells. Stable co‐expression of an uncoupled mutant α 1B ‐AR (Δ12) with α 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 α 1B ‐/α 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 α 1D ‐ARs are often pharmacologically undetectable in native tissues when they are co‐expressed with α 1B ‐ARs.