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Vol. 302, Issue 1, 58-65, July 2002
1-Adrenergic Receptor Subtypes
Department of Pharmacology, Emory University School of Medicine,
Atlanta Georgia
Human 
1A-, 1B-, and
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 1A-, and to a lesser extent 1B- and 1D-adrenergic receptors.
Radioligand binding site density was highest for 1A- and
much lower for 1B- and 1D-adrenergic
receptors, but did not correlate with protein expression. Commercial
anti-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 125I-arylazidoprazosin,
and the intensity of photoaffinity-labeling correlated with the density
of radioligand binding sites. We conclude that epitope-tagged
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.
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