Abstract

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 the third cytoplasmic loop of the m4 mAChR subtype, were tested for their abilities to affect the coupling of the chick heart mAChR to the GTP-binding protein G(o). The purified chick heart mAChRs were reconstituted with purified G(o) in phospholipid vesicles, and their interactions were monitored in the presence or absence of the antibodies. The anti-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 receptor-stimulated GTPase activity of G(o). These findings indicate that the amino-terminal portion of the third cytoplasmic loop is an important determinant for G(o) to promote high affinity agonist binding to the chick heart mAChR and also for the agonist-stimulated GTP gamma S binding and GTPase activity. The anti-m4a, anti-m2, and anti-m1a antibodies, which were made against centrally located peptides of the third cytoplasmic loop of the m4, m2, and m1 mAChR subtypes, respectively, were also tested for their effects in the reconstituted receptor/G(o) system. The anti-m2 and anti-m4a antibodies also significantly reduced agonist-stimulated GTP gamma S binding, as well as GTPase activity, but did not completely abolish these functions, as was the case with anti-m4b antibodies. However, the anti-m4a and anti-m2 antibodies shared with anti-m4b antibodies the ability to markedly inhibit the ability of G(o) to promote high affinity agonist binding to the purified and reconstituted receptors. In contrast to the results obtained with the anti-m2 and anti-m4 antibodies, the anti-m1a antibodies had smaller effects on the receptor/G(o) interactions. These results suggested that central portions of the loop can also influence mAChR/G(o) interactions. Studies were als 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 sum, the results suggest that multiple domains in the third cytoplasmic loop of chick heart mAChR can modulate interactions with G(o).