SUBTYPE-SPECIFIC ANTIBODIES FOR MUSCARINIC CHOLINERGIC RECEPTORS .2. STUDIES WITH RECONSTITUTED CHICK HEART RECEPTORS AND THE GTP-BINDING PROTEIN-G0

The antibodies described in the accompanying paper were used to probe the interactions of the chick heart muscarinic acetylcholine receptors (mAChRs) with the GTP-binding protein G(o). The anti-m4b antibodies, which were made against a peptide from the amino-terminal portion of t he third cytoplasmic loop of the m4 mAChR subtype, were tested for the ir abilities to affect the coupling of the chick heart mAChR to the GT P-binding protein G(o). The purified chick heart mAChRs were reconstit uted with purified G(o) in phospholipid vesicles, and their interactio ns were monitored in the presence or absence of the antibodies. The an ti-m4b antibodies completely inhibited the ability of G(o) to promote high affinity agonist binding to the purified receptors. The anti-m4b antibodies also completely inhibited the agonist-stimulated binding of guanosine-5'-O-(3-thio)triphosphate (GTP-gamma-S) to G(o) and the rec eptor-stimulated GTPase activity of G(o). These findings indicate that the amino-terminal portion of the third cytoplasmic loop is an import ant determinant for G(o) to promote high affinity agonist binding to t he chick heart mAChR and also for the agonist-stimulated GTP-gamma-S b inding and GTPase activity. The anti-m4a, anti-m2, and anti-m1a antibo dies, which were made against centrally located peptides of the third cytoplasmic loop of the m4, m2, and m1 mAChR subtypes, respectively, w ere also tested for their effects in the reconstituted receptor/G(o) s ystem. The anti-m2 and anti-m4a antibodies also significantly reduced agonist-stimulated GTP-gamma-S binding, as well as GTPase activity, bu t did not completely abolish these functions, as was the case with ant i-m4b antibodies. However, the anti-m4a and anti-m2 antibodies shared with anti-m4b antibodies the ability to markedly inhibit the ability o f G(o) to promote high affinity agonist binding to the purified and re constituted receptors. In contrast to the results obtained with the an ti-m2 and anti-m4 antibodies, the anti-m1 a antibodies had smaller eff ects on the receptor/G(o) interactions. These results suggested that c entral portions of the loop can also influence mAChR/G(o) interactions . Studies were also performed to test the effects of the peptides that were used as antigens on receptor-mediated GTP-gamma-S binding to G(o ). Each of the peptides caused significant inhibition of this function , but the greatest inhibition was observed with the m4b peptide. In su m, the results suggest that multiple domains in the third cytoplasmic loop of chick heart mAChR can modulate interactions with G(o).