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 coupling, libraries were created of human m5 muscarinic acetylcholine receptors (m5) randomly mutated in the C-terminal region of the third intracellular loop. Functional receptors were identified based on their ability to amplify NIH 3T3 cells in a ligand-dependent manner. These receptors either had wild-type phenotypes (Group 1) or were functionally impaired (Group 2). No "activated receptors" were identified. Tolerated substitutions in Group 2 receptors were randomly distributed and frequently included prolines and glycines. In contrast, tolerated substitutions in Group 1 receptors exhibited a periodicity proximal to transmembrane domain 6 were proline and glycine substitutions were not observed. These observations are consistent with a short alpha-helical extension of the C-terminal region of the third intracellular loop from transmembrane domain 6. Mutations at Ala-441 were most commonly associated with impaired function of Group 2 receptors. Twelve point mutations at Ala-441 were tested, and all caused marked increases in EC50 values with little effect on maximal response or agonist binding affinity. These results indicate that Ala-441 is a key determinant of m5 receptor affinity for G proteins and exists within the structural context of a short alpha-helix.
Burstein
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Department of Psychiatry, University of Vermont, Burlington 05405.
Spalding
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Hill-Eubanks
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Brann
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D016475:3T3 Cells; D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002522:Chlorocebus aethiops; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011897:Random Allocation; D011976:Receptors, Muscarinic / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D002217:Carbachol; D019204:GTP-Binding Proteins; D000409:Alanine