CREATION OF A CONSTITUTIVELY ACTIVATED STATE OF THE 5-HYDROXYTRYPTAMINE(2A) RECEPTOR BY SITE-DIRECTED MUTAGENESIS - INVERSE AGONIST ACTIVITY OF ANTIPSYCHOTIC-DRUGS

Single amino acid mutations in the third intracellular loop, as well a s other domains of G protein-coupled receptors, have been shown to con fer drastic changes in receptor properties and have been postulated to be responsible for various disease states. To determine whether an am ino acid mutation can confer dramatic alterations in the 5-hydroxytryp tamine(2A) (5HT(2A)) receptor, we mutated amino acid 322 to lysine (C3 22K), glutamate (C322E) or arginine (C322R). Transient expression of t he mutant receptors revealed properties associated with constitutive a ctivity. Radioligand binding studies revealed an increase in 5-HT affi nity from 293 nM (native) to 86 nM (C322E), 25 nM (C322K) and 11 nM (C 322R). 5-HT potency for stimulation of inositol phosphate production i ncreased from 152 nM (native) to 61 nM (C322E) and 25 nM (C322K). Basa l inositol phosphate levels in COS-7 cells expressing C322K and C322E mutant receptors were 8-fold and 4-fold higher, respectively, than cel ls expressing native 5-HT2A receptors. Basal levels of inositol phosph ate stimulated by C322K receptors represented 48% of total inositol ph osphate production stimulated by native receptors in the presence of 1 0 mu M 5-HT. Antipsychotic drugs (chlorpromazine, clozapine, haloperid ol, loxapine and risperidone) displayed inverse agonist activity by in hibiting C322K constitutive activation of phosphatidylinositol hydroly sis. These data indicate that amino acid 322 in the 5-HT2A receptor pl ays an important role in maintaining the inactive conformation and pro vide further evidence that amino acid mutations can produce profound a lterations in G protein-coupled receptor activity.