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Division of Physical Biochemistry, Medical Research Council
National Institute for Medical Research, London NW7 1AA, UK (Z.-L.L.,
C.A.C., E.C.H.),
Wyeth-Ayerst Research, CN8000, Princeton, New Jersey
08543-8000 (P.G.J.), and
Department of Pharmacology, Malaga University
School of Medicine, Campus Teatinos, 29080 Malaga, Spain (J.P.)
An Asp-Arg-Tyr triad occurs in a majority of rhodopsin-like G
protein-coupled receptors. The fully conserved Arg is critical for G
protein activation, but the function of the flanking residues is not
well understood. We expressed in COS-7 cells m1 muscarinic receptors
that were mutated at Asp122 and Tyr124. Most mutations at either
position strongly attenuated or prevented the expression of binding
sites for the antagonist
[3H]N-methylscopolamine. However, sites
that were expressed displayed unaltered affinity for the antagonist.
Receptor protein, visualized with a carboxyl-terminally directed
antibody, was reduced but never completely abolished. The effects of
these mutations were partially reversed by the deletion of 129 amino
acids from the third intracellular loop of the receptor. In several
cases, comparison of immunocytochemistry with binding measurements
suggested the presence of substantial amounts of inactive, presumably
misfolded, receptor protein. Some of the variants that bound
[3H]N-methylscopolamine underwent small
changes in their affinities for acetylcholine. All retained nearly
normal abilities to mediate an acetylcholine-induced phosphoinositide
response. We propose that Asp122 and Tyr124 make intramolecular
contacts whose integrity is important for efficient receptor folding
but that they do not participate directly in signaling. The role of
these residues is completely distinct from that of Arg123, whose
mutation abolishes signaling without diminishing receptor expression.
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