MUSCARINIC ACETYLCHOLINE-RECEPTORS - STRUCTURAL BASIS OF LIGAND-BINDING AND G-PROTEIN COUPLING

Muscarinic acetylcholine receptors (m1-m5) were studied by a combined molecular genetic/pharmacologic approach to elucidate the molecular ch aracteristics of the ligand binding site and of the receptor domains i nvolved in G protein coupling. Site-directed mutagenesis studies of th e rat m3 muscarinic receptor suggest that the acetylcholine binding do main is formed by a series of hydrophilic amino acids located in the ' 'upper'' half of transmembrane domains (TM) III, V, VI, and VII. Moreo ver, we showed that mutational modification of a TM VI Asn residue (As n507 in the rat m3 receptor sequence) which is characteristic for the muscarinic receptor family has little effect on high-affinity acetylch oline binding and receptor activation, but results in dramatic reducti ons in binding affinities for certain subclasses of muscarinic antagon ists. The N-terminal portion of the third intracellular loop (i3) of m uscarinic and other G protein-coupled receptors has been shown to play a central role in determining the G protein coupling profile of a giv en receptor subtype. Insertion mutagenesis studies with the rat m3 mus carinic receptor suggest that this region forms an amphiphilic alpha-h elix and that the hydrophobic side of this helix represents an importa nt G protein recognition surface. Further mutational analysis of this receptor segment showed that Tyr254 located at the N-terminus of the i 3 loop of the m3 muscarinic receptor plays a key role in muscarinic re ceptor-induced Gq activation. The studies described here, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.