Biochemistry and pharmacology of epitope-tagged alpha(1)-adrenergic receptor subtypes.

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Vicentic A, Robeva A, Rogge G, Uberti M, Minneman KP

Biochemistry and pharmacology of epitope-tagged alpha(1)-adrenergic receptor subtypes.

J Pharmacol Exp Ther. 2002 Jul;302(1):58-65.

PubMed ID

12065700 [ View in PubMed

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Abstract

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.

DrugBank Data that Cites this Article

Binding Properties

Drug	Target	Property	Measurement	pH	Temperature (°C)
Prazosin	Alpha-1B adrenergic receptor	Ki (nM)	0.62	N/A	N/A	Details
Prazosin	Alpha-1D adrenergic receptor	Ki (nM)	0.38	N/A	N/A	Details