Muscarinic receptors are purified to apparent homogeneity from the both porcine brain and heart. Sufficient peptide sequence is obtained from these preparations to allow the cloning of the ml and m2 muscarinic receptor subtypes from these tissues. Using homology cloning, the human and rat forms of these receptors as well as three additional subtypes (m3-m5) are identified. Based on the sequence of the five cloned muscarinic receptor subtypes (ml-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues. The distributions of these subtypes and their potential physiological roles are discussed. By the use of molecular genetic manipulation of cloned muscarinic receptor cDNAs, the regions of muscarinic receptors that specify G-protein coupling and ligand binding have been defined in several recent studies. Overall, these studies have shown that amino acids within the third cytoplasmic loop of the receptors define their selectivities for different G-proteins and that multiple discontinuous epitopes contribute to their selectivity for different ligands. In this chapter, the residues that contribute to ligand binding and G-protein coupling are described, as well as the implied structures of these functional domains.
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