Biochemistry and pharmacology of epitope-tagged alpha(1)-adrenergic receptor subtypes.
Human alpha(1A)-, alpha(1B)-, and alpha(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 Ca(2+). 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 alpha(1A)-, and to a lesser extent alpha(1B)- and alpha(1D)-adrenergic receptors. Radioligand binding site density was highest for alpha(1A)- and much lower for alpha(1B)- and alpha(1D)-adrenergic receptors, but did not correlate with protein expression. Commercial anti-alpha(1)-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 (125)I-arylazidoprazosin, and the intensity of photoaffinity-labeling correlated with the density of radioligand binding sites. We conclude that epitope-tagged alpha(1)-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.
Vicentic
Aleksandra
A
Department of Pharmacology, 5017 Rollins Research Center, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.
Robeva
Anna
A
Rogge
George
G
Uberti
Michelle
M
Minneman
Kenneth P
KP
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004229:Dithiothreitol / Q000494:pharmacology; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D009532:Nickel / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D019920:Photoaffinity Labels; D011233:Precipitin Tests; D011869:Radioligand Assay; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects* / Q000378:metabolism*; D012116:Resins, Plant; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D000939:Epitopes; D019920:Photoaffinity Labels; D018340:Receptors, Adrenergic, alpha-1; D012116:Resins, Plant; D013439:Sulfhydryl Reagents; D009532:Nickel; D014508:Urea; D009571:Nitrilotriacetic Acid; D002118:Calcium; D004229:Dithiothreitol