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Vol. 53, Issue 1, 166-175, January 1998
Laboratory for Molecular Pharmacology (B.H., S.Z., C.E.E., S.A.H.,
T.W.S.), Pharmacological Institute, University of Copenhagen,
Rigshospitalet 6321, DK-2100, Copenhagen, Denmark, and
Department of
Protein Chemistry, Institute of Molecular Biology (T.W.S.), University
of Copenhagen, DK-1353, Copenhagen, Denmark
Residues in transmembrane domain (TM)-III, TM-V, TM-VI, and TM-VII
believed to be facing the deep part of the presumed main ligand-binding
pocket of the NK1 receptor were probed by alanine substitution and introduction of residues with larger and/or chemically distinct side chains. Unaltered or even improved binding affinity for
four peptide agonists, substance P, substance P-O-methyl
ester, eledoisin, and neurokinin A, as well as normal EC50
values for substance P in stimulating phosphatidylinositol turnover
indicated that these mutations did not alter the overall functional
integrity of the receptor. The alanine substitutions in general had
only minor effects on nonpeptide antagonist binding. However, the
introduction of the larger and polar aspartic acid and histidine
residues at positions corresponding to the monoamine binding aspartic
acid in TM-III of the 
2-adrenoceptor (ProIII:08, Pro112
in the NK1 receptor) and to the presumed monoamine
interacting "two serines" in TM-V (ThrV:09, Thr201; and IleV:12,
Ile204) impaired by >100-fold the binding of a group of nonpeptide
antagonists, including CP96,345, CP99,994, RP67,580, RPR100,893, and
CAM4092. In contrast, another group of nonpeptide antagonists,
LY303,870, FK888, and SR140,333, were little or not at all affected by
the space-filling substitutions. Two of these compounds, FK888 and
LY303,870, were those most seriously affected (75-89-fold) by alanine
substitution of PheVI:20 located in the upper part of the main
ligand-binding crevice. Surprisingly, substitution of AlaIII:11
(Ala115), which is located in the middle of TM-III, conceivably
pointing toward TM-VII, with a larger valine residue increased the
affinity for all 13 ligands tested, presumably by creating a closer
interhelical packing. It is concluded that the introduction of larger
side chains at positions at which molecular models indicate that this
is structurally allowed can be a powerful method of locating
ligand-binding sites due to the considerable difference between
positive and negative results. Such steric hindrance mutagenesis
strongly indicates that one population of nonpeptide antagonists bind
in the deep pocket of the main ligand-binding crevice of the
NK1 receptor, whereas another group of nonpeptide antagonists, especially SR140,333, was surprisingly resistant to
mutational mapping in this pocket.
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