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Departments of Psychiatry and Biochemistry, Case Western Reserve
University Medical School, Cleveland, Ohio 44106-4935
Several models of agonist binding to G protein-coupled
5-hydroxytryptamine [5-HT] (serotonin) receptors have highlighted the potential importance of highly conserved aromatic residues for ligand
binding and agonist efficacy. In this study, we tested these models by
constructing and characterizing a number of point mutations of
conserved and nonconserved aromatic residues using the
5-HT2A receptor as a model system. Mutations of three
highly conserved tryptophans (W200A, W336A, and W367A) proposed to
reside near the binding pocket markedly reduced agonist affinity and efficacy at 5-HT2A receptors. Mutations of two other highly
conserved aromatic residues postulated to be near the agonist binding
site (F340L and Y370A) also had dramatic effects on agonist binding and
efficacy. Point mutations of neighboring conserved phenylalanines (F339L and F365L) had minimal effects on agonist binding, although the
F365L mutation diminished agonist efficacy. Finally, mutations of two
nonconserved aromatic residues (F125L and F383A) not predicted to be
near the binding pocket had no effects on agonist binding, potency, or
efficacy. Our results are best explained by models that suggest that
helices III, V, VI, and VII can form a unit of interacting helices in
which highly conserved aromatic residues are oriented toward the center
of the helical aggregate to form an aromatic pocket. In addition, our
novel results identify a series of aromatic residues essential for
agonist-induced second messenger production. These results demonstrate
that highly conserved aromatic residues residing in neighboring helices
provide the optimum environment for both agonist binding and activation
of 5-HT2A receptors.
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