Modulation of the M2 muscarinic acetylcholine receptor activity with monoclonal anti-M2 receptor antibody fragments.
Antibodies directed against the second extracellular loop of G protein-coupled receptors are known to have functional activities. From a partial agonist monoclonal antibody directed against the M2 muscarinic receptor, we constructed and produced a single chain variable fragment with high affinity for its target epitope. The fragment is able to recognize its receptor on Chinese hamster ovary cells transfected with the M2 muscarinic acetylcholine receptor to block the effect of carbachol on this receptor and to exert an inverse agonist activity on the basal activity of the receptor. The antibody fragment is also able to increase the basal rhythm of cultured neonatal rat cardiomyocytes and to inhibit in a non-competitive manner the negative chronotropic effect of carbachol. This antibody fragment is able to exert its inverse agonist activity in vivo on mouse heart activity. The immunological strategy presented here could be useful to develop specific allosteric inverse agonist reagents for G protein-coupled receptors.
Peter
Jean-Christophe
JC
CNRS, Unité Propre de Recherche 9021, Institut de Biologie Moléculaire et Cellulaire, Laboratory of Therapeutical Chemistry and Immunology, 15 rue René Descartes, F-67084 Strasbourg, France.
Wallukat
Gerd
G
Tugler
Jean
J
Maurice
Damien
D
Roegel
Jean-Christophe
JC
Briand
Jean-Paul
JP
Hoebeke
Johan
J
D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments; D007150:Immunohistochemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D043585:Receptor, Muscarinic M2 / Q000737:chemistry* / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D009711:Nucleotides; D010455:Peptides; D043585:Receptor, Muscarinic M2; D011950:Receptors, Cholinergic; D002217:Carbachol