SUBTYPE-SPECIFIC ANTIBODIES FOR MUSCARINIC CHOLINERGIC RECEPTORS .1. CHARACTERIZATION USING TRANSFECTED CELLS AND AVIAN AND MAMMALIAN CARDIAC MEMBRANES

Polyclonal antibodies to a number of synthetic peptides corresponding to sequences of human muscarinic acetylcholine receptor (mAChR) subtyp es m1, m2, and m4 have been prepared, in order to obtain specific tool s with which to study the properties of the different receptors, Hydro philic peptides from each subtype were chosen from the large intracell ular loop between the fifth and the sixth transmembrane domains, becau se these loops are distinct in each of the mAChR subtypes and contain determinants for coupling to different GTP-binding proteins, as well a s potential sites for phosphorylation. Five different antibodies were prepared and tested for their reactivity and specificity toward differ ent mAChR subtypes by immunoprecipitation of ligand-binding activity a nd receptor protein and by immunoblot analysis. Each of the antisera i mmunoprecipitated its respective mAChR subtype, as evidenced by precip itation of 50-70% of the ligand-binding activity from stably transfect ed cells expressing the respective mAChR subtype. Very little cross-re activity toward other subtypes was observed. We used these subtype-spe cific antibodies to probe the nature of the mAChR subtypes naturally e xpressed 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 wit h mAChR from porcine heart, confirming previous pharmacological and mo lecular biological studies that suggested that this tissue expressed o nly the m2 mAChR subtype. In contrast, both the anti-m2 and anti-m4 mA ChR antibodies immunoprecipitated approximately 50% of the mAChRs solu bilized from chick heart membranes. The combined or successive use of anti-m2 and anti-m4 antisera in immunoprecipitation and/or immunoblott ing studies with chick heart mAChRs suggested that both antibodies app eared to recognize the same population of mAChRs from that tissue. Tak en together, the results show that the subtype-specific antibodies dev eloped in this work are useful in recognizing mAChR subtypes in cells and tissues. These antibodies should be valuable tools for further stu dy of the properties and regulation of mAChR subtypes in various prepa rations. Evidence that the antibodies can modify mAChR/GTP-binding pro tein interactions is presented in the accompanying paper.