Structure-function of muscarinic receptor coupling to G proteins. Random saturation mutagenesis identifies a critical determinant of receptor affinity for G proteins
| Autor: | E S, Burstein, T A, Spalding, D, Hill-Eubanks, M R, Brann |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Alanine
Binding Sites Molecular Sequence Data 3T3 Cells Transfection Receptors Muscarinic Protein Structure Secondary Recombinant Proteins Cell Line Kinetics Mice Random Allocation Structure-Activity Relationship GTP-Binding Proteins Chlorocebus aethiops Mutagenesis Site-Directed Animals Humans Point Mutation Carbachol Amino Acid Sequence |
| Zdroj: | The Journal of biological chemistry. 270(7) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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