STRUCTURE-FUNCTION OF MUSCARINIC RECEPTOR COUPLING TO G-PROTEINS - RANDOM SATURATION MUTAGENESIS IDENTIFIES A CRITICAL DETERMINANT OF RECEPTOR AFFINITY FOR G-PROTEINS

To derive structure-function relationships for receptor-G protein coup ling, libraries were created of human m5 muscarinic acetylcholine rece ptors (m5) randomly mutated in the C-terminal region of the third intr acellular loop. Functional receptors were identified based on their ab ility to amplify NIH 3T3 cells in a ligand-dependent manner, These rec eptors either had wildtype phenotypes (Group 1) or were functionally i mpaired (Group 2). No ''activated receptors'' were identified, Tolerat ed substitutions in Group 2 receptors were randomly distributed and fr equently included prolines and glycines. In contrast, tolerated substi tutions in Group 1 receptors exhibited a periodicity proximal to trans membrane domain 6 where proline and glycine substitutions were not obs erved. These observations are consistent with a short alpha-helical ex tension of the C-terminal region of the third intracellular loop from transmembrane domain 6. Mutations at Ala-441 were most commonly associ ated with impaired function of Group 2 receptors. Twelve point mutatio ns at Ala-441 were tested, and all caused marked increases in EC(50) v alues with little effect on maximal response or agonist binding affini ty. These results indicate that Ala-441 is a key determinant of m5 rec eptor affinity for G proteins and exists within the structural context of a short alpha-helix.