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Vol. 286, Issue 1, 85-90, July 1998
Department of Pharmacology and Neuroscience, Albany Medical
College, Albany, New York
Single amino acid mutations in the third intracellular loop, as well as
other domains of G protein-coupled receptors, have been shown to confer
drastic changes in receptor properties and have been postulated to be
responsible for various disease states. To determine whether an amino
acid mutation can confer dramatic alterations in the
5-hydroxytryptamine2A (5-HT2A) receptor, we mutated amino acid 322 to lysine (C322K), glutamate (C322E) or arginine
(C322R). Transient expression of the mutant receptors revealed
properties associated with constitutive activity. Radioligand binding
studies revealed an increase in 5-HT affinity from 293 nM (native) to
86 nM (C322E), 25 nM (C322K) and 11 nM (C322R). 5-HT potency for
stimulation of inositol phosphate production increased from 152 nM
(native) to 61 nM (C322E) and 25 nM (C322K). Basal inositol phosphate
levels in COS-7 cells expressing C322K and C322E mutant receptors were
8-fold and 4-fold higher, respectively, than cells expressing native
5-HT2A receptors. Basal levels of inositol phosphate
stimulated by C322K receptors represented 48% of total inositol
phosphate production stimulated by native receptors in the presence of
10 µM 5-HT. Antipsychotic drugs (chlorpromazine, clozapine,
haloperidol, loxapine and risperidone) displayed inverse agonist
activity by inhibiting C322K constitutive activation of phosphatidylinositol hydrolysis. These data indicate that amino acid
322 in the 5-HT2A receptor plays an important role in
maintaining the inactive conformation and provide further evidence that
amino acid mutations can produce profound alterations in G
protein-coupled receptor activity.
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