Functional activity of antibodies at the bovine beta2-adrenoceptor.
Antibodies that can activate beta2-adrenoceptors (beta2-AR) have the potential to mimic the anabolic effects of beta-agonist drugs, such as clenbuterol. In this study, antibodies were raised in rabbits against two peptide analogues of the human beta2-adrenoceptor (beta2-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 beta2-AR in vitro. Membrane proteins from transformed Escherichia coli that express the beta2-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 beta2-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 beta2-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 beta2-AR. Further studies will determine whether antibodies that potentiate activity at the beta2-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 beta2-agonists and thus provide an alternative to the use of xenobiotic compounds.
Hill
R A
RA
Tropical Beef Centre: a joint venture of Queensland Department of Primary Industries, Rockhampton, Australia.
Hoey
A J
AJ
Sillence
M N
MN
D000318:Adrenergic beta-Agonists / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000502:physiology*; D001667:Binding, Competitive; D002417:Cattle / Q000254:growth & development / Q000502:physiology*; D005260:Female; D007106:Immune Sera / Q000502:physiology; D015151:Immunoblotting; D007545:Isoproterenol / Q000378:metabolism; D008969:Molecular Sequence Data; D009126:Muscle Relaxation / Q000502:physiology; D009130:Muscle, Smooth / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D011817:Rabbits; D011869:Radioligand Assay; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000276:immunology* / Q000502:physiology; D012508:Sarcolemma / Q000502:physiology; D014132:Trachea / Q000502:physiology
D000318:Adrenergic beta-Agonists; D000906:Antibodies; D007106:Immune Sera; D010446:Peptide Fragments; D018343:Receptors, Adrenergic, beta-2; D007545:Isoproterenol