Abstract

Human α_1A-, α_1B-, and α_1D-adrenergic receptors were tagged at their amino termini with FLAG epitopes and stably expressed in human embryonic kidney (HEK)293 cells. Tagged receptors demonstrated a wild-type pharmacology and mobilization of intracellular Ca²⁺. After solubilization and immunoprecipitation, monomers, dimers, and trimers of each subtype were apparent on Western blots. Further denaturation with 6 M urea reduced most oligomers to monomers. Deglycosylation reduced the molecular size of α_1A-, and to a lesser extent α_1B- and α_1D-adrenergic receptors. Radioligand binding site density was highest for α_1A- and much lower for α_1B- and α_1D-adrenergic receptors, but did not correlate with protein expression. Commercial anti-α₁-adrenergic receptor antibodies did not recognize the tagged receptors in Western blots of cell lysates, and substantial cross-reactivity was still observed after solubilization and immunoprecipitation. Surprisingly, only receptor monomers were apparent after photoaffinity labeling with ¹²⁵I-arylazidoprazosin, and the intensity of photoaffinity-labeling correlated with the density of radioligand binding sites. We conclude that epitope-tagged α₁-adrenergic receptors exist as both monomers and oligomers in HEK293 cells, but there is substantial discrepancy between protein and binding site expression. Because only monomers are detected by photoaffinity labeling, dimers and trimers observed on Western blots may be pharmacologically inactive.