Characterization of anti-peptide antibodies directed against an extracellular immunogenic epitope on the human α1-adrenergic receptor

A synthetic peptide corresponding to amino acids 192-218 of the second extracellular loop of the human alpha 1A-adrenergic receptor was used to raise antibodies in rabbits. Affinity-purified antibodies specifically recognized main bands with a molecular weight of about 68, 40 and 37 kD on the electrotransferred membrane proteins of rat ventricle membranes. The incubation of these antibodies with rat myocardial membranes resulted in a decrease in the number of binding sites for the specific radiolabelled alpha 1-antagonist prazosin. These antibodies were also able to mimic the effects of agonist stimulation as demonstrated by a positive chronotropic effect on cultured cardiomyocytes. These results constitute the first immunochemical evidence of the presence of both the A and B subtypes of the alpha 1-adrenergic receptor in the heart. They also confirm that the second extracellular loop of the alpha 1-adrenergic receptors is an immunologically and functionally important domain.