Functional Activity of Antibodies at the Bovine ß2 -Adrenoceptor

Abstract

Antibodies that can activate β2-adrenoceptors (β2-AR) have the potential to mimic the anabolic effects of β-agonist drugs, such as clenbuterol. In this study, antibodies were raised in rabbits against two peptide analogues of the human β2-adrenoceptor (β2-AR): One peptide corresponded to the complete second outer loop of the receptor (24 amino acids; H24T), and the second peptide was a truncated version of the first (13 amino acids; H13C). Following affinity purification, the antibodies were screened to detect interaction with β2-AR in vitro. Membrane proteins from transformed Escherichia coli that express the β2-AR were separated using SDS PAGE and transferred to nitrocellulose sheets. Immunoblotting revealed a single protein band (39,000 Da) that was recognized by the affinity-purified anti-H24T antibodies. However, the anti-H13C antibodies did not recognize any protein bands in immunoblots. In ligand binding studies, anti-H24T antibodies at a concentration of 50 nM, increased the affinity (KD) of the radiolabeled antagonist [125I]iodocyanopindolol for the bovine β2-AR from 31.7 pM to 25.3 pM (P < .05) without changing the receptor number. Anti-H13C antibodies had no effect on ligand binding. In competitive ligand binding experiments, there was no effect of antibodies on the affinity of bovine β2-AR for the agonist (−)-isoproterenol. However, functional activity of anti-H24T antibodies was demonstrated in an organ bath study. The presence of antibodies caused a leftward shift in the concentration-response curve for (−)-isoproterenol-induced relaxation of isolated bovine smooth muscle strips. Values for pD2 (-log EC50) were reduced in the presence of 10 nM antibody (8.62 ± .11) compared to controls (8.30 ± .08; P < .05). Anti-H13C antibodies had no effect on (−)-isoproterenol-induced smooth muscle relaxation. These studies have demonstrated recognition, interaction, and functional activity of site-directed antibodies at the β2-AR. Further studies will determine whether antibodies that potentiate activity at the β2-AR may be evoked by the active immunization of cattle with the peptide H24T, and if so, whether this will cause the repartitioning of nutrients in a manner analogous to conventional β2-agonists and thus provide an alternative to the use of xenobiotic compounds.