RT Journal Article SR Electronic T1 Biochemistry and Pharmacology of Epitope-Tagged α1-Adrenergic Receptor Subtypes JF Journal of Pharmacology and Experimental Therapeutics JO J Pharmacol Exp Ther FD American Society for Pharmacology and Experimental Therapeutics SP 58 OP 65 DO 10.1124/jpet.302.1.58 VO 302 IS 1 A1 Vicentic, Aleksandra A1 Robeva, Anna A1 Rogge, George A1 Uberti, Michelle A1 Minneman, Kenneth P. YR 2002 UL http://jpet.aspetjournals.org/content/302/1/58.abstract AB Human α1A-, α1B-, and α1D-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 α1A-, and to a lesser extent α1B- and α1D-adrenergic receptors. Radioligand binding site density was highest for α1A- and much lower for α1B- and α1D-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. The American Society for Pharmacology and Experimental Therapeutics