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(Received for publication, October 25, 1994) 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 where
proline and glycine substitutions were not observed. These observations
are consistent with a short
Volume 270,
Number 7,
Issue of February 17, 1995 pp. 3141-3146
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
RANDOM SATURATION MUTAGENESIS IDENTIFIES A CRITICAL DETERMINANT OF
RECEPTOR AFFINITY FOR G PROTEINS
-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 EC
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 -helix.
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