Subtype-specific antibodies for muscarinic cholinergic receptors. I. Characterization using transfected cells and avian and mammalian cardiac membranes

Abstract

Polyclonal antibodies to a number of synthetic peptides corresponding to sequences of human muscarinic acetylcholine receptor (mAChR) subtypes m1, m2, and m4 have been prepared, in order to obtain specific tools with which to study the properties of the different receptors. Hydrophilic peptides from each subtype were chosen from the large intracellular loop between the fifth and the sixth transmembrane domains, because these loops are distinct in each of the mAChR subtypes and contain determinants for coupling to different GTP-binding proteins, as well as potential sites for phosphorylation. Five different antibodies were prepared and tested for their reactivity and specificity toward different mAChR subtypes by immunoprecipitation of ligand-binding activity and receptor protein and by immunoblot analysis. Each of the antisera immunoprecipitated its respective mAChR subtype, as evidenced by precipitation of 50-70% of the ligand-binding activity from stably transfected cells expressing the respective mAChR subtype. Very little cross-reactivity toward other subtypes was observed. We used these subtype-specific antibodies to probe the nature of the mAChR subtypes naturally expressed in avian and mammalian cardiac membranes. In tests using the antibodies with cardiac mAChRs, differences in the avian and mammalian mAChR subtypes were detected. Only the anti-m2 antibodies reacted with mAChR from porcine heart, confirming previous pharmacological and molecular biological studies that suggested that this tissue expressed only the m2 mAChR subtype. In contrast, both the anti-m2 and anti-m4 mAChR antibodies immunoprecipitated approximately 50% of the mAChRs solubilized from chick heart membranes. The combined or successive use of anti-m2 and anti-m4 antisera in immunoprecipitation and/or immunoblotting studies with chick heart mAChRs suggested that both antibodies appeared to recognize the same population of mAChRs from that tissue. Taken together, the results show that the subtype-specific antibodies developed in this work are useful in recognizing mAChR subtypes in cells and tissues. These antibodies should be valuable tools for further study of the properties and regulation of mAChR subtypes in various preparations. Evidence that the antibodies can modify mAChR/GTP-binding protein interactions is presented in the accompanying paper.