Antibodies raised against purified β-adrenergic receptors specifically bind β-adrenergic ligands

Abstract

Antibodies raised against purified β-adrenergic receptors themselves specifically bind β-adrenergic ligands. Digitonin-solubilized frog (Rana pipiens) erythrocyte β-adrenergic receptors, purified 100- to 200-fold by adsorption to an alprenolol-agarose affinity support and specifically eluted from the affinity resin by 1-100 mM (±)-isoproterenol, were used to immunize six rabbits. All immune sera, in contrast to preimmune sera, bound the β-adrenergic antagonist [³H]Dihydroalprenolol binding activity was due to immunoglobulins. By competition studies, antibody [³H]dihydroalprenolol binding was found to display a specificity and stereoselectivity resembling that of the β-adrenergic receptor, [i.e., (-)-isoproterenol > (-)-epinephrine > (-)-norepinephrine; alprenolol ≈ propranolol ≫ phentolamine = aloperidol; and (-) isomers of both agonists and antagonists 10-100 times more potent than (+) isomers]. A portion of the [³H]dihydroalprenolol binding antibodies could be specifically adsorbed onto purified frog erythrocyte membranes, whereas Xenopus and human erythrocyte membranes, both of which are almost devoid of β-adrenergic receptors, were ineffective in adsorbing [³H]dihydroalprenolol binding antibodies. We suggest that the likely immunogen was a β-adrenergic receptor-isoproterenol complex and that immunization with drugs noncovalently bound to their receptors might be a means of raising antibodies to biologically active otherwise nonimmunogenic small molecules. Such antibodies, whose specificity mimics that of a receptor, should also provide useful models for the study of the structure of the receptor binding sites.

• agarose-alprenolol
• affinity chromatography
• catecholamines
• immunogen
• model receptor binding site