dopamine D 5 receptors (Tiberi and Caron, 1994), serotonin 5-HT 2C recep-tors

Abstract

ABSTRACT 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-hydroxytryptamine 2A (5-HT 2A ) 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-HT 2A 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 M 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-HT 2A 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. The third intracellular loop of GPCR has been identified as a region that is crucial for receptor/G protein interactions (Strader et al., 1987; Recently, several GPCR have exhibited constitutive activity in vivo. Naturally occurring amino acid mutations in the luteinizing hormone recepto