Muscarinic acetylcholine receptors consist of five genetic subtypes that mediate signal transduction by coupling with G-proteins. The structures of G-protein-coupled receptors are not known in detail, as no high resolution structural information is available from physical measurements. These receptors are known to be integral membrane proteins, that have seven hydrophobic regions that form seven transmembrane (TM) domains (TM1-TM7) connected by three outer (o1-o3) and three intracellular loops (i1-i3). Studies will a variety of mutant receptors have indicated that the amino acids adjacent to the TM domains on the intracellular face are essential for G-protein coupling. Studies with chimeric receptors have implicated both the i2 and i3 loops in defining the selectivity of receptors for distinct G-proteins. And in the case of muscarinic receptor subtypes, amino acids on the N-terminal side of the i3 loop (Ni3) are critical determinants that define subtype preferences for the G-proteins Gq versus Gi. In the case of the alpha adrenergic receptors, amino acids in the c- terminal region of the i3 loop (Ci3) have been implicated in receptor activation and allosteric regulation of agonist binding. To gain insight into the structural requirements of receptor/G-protein coupling, we will subject the i2, Ni3 and Ci3 regions of the m5 muscarinic receptor to random-saturation mutagenesis. Mutant muscarinic receptors with a variety of functional phenotypes will be identified by screening all recombinants via rapid functional assays that we have developed. Our screens are designed to identify the range of amino acid substitutions that allow retention of overall receptor function, activate the receptor in the absence of agonist, change the affinity of the receptor for G-protein, and change receptor/G-protein coupling efficiency. When combined with molecular modeling and data from physical measurements, we anticipate that our experiments will provide empirical data defining the structural basis of ligand binding and activation of a muscarinic receptor.