ACTIVATING MUTATIONS OF THE SEROTONIN 5-HT2C RECEPTOR

Site-directed mutagenesis was performed to create a mutant serotonin 5 -HT2C receptor that would mimic the active conformation of the native receptor, Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K), After expression in COS-7 cells, the binding affinity of 5-HT for [H-3]-mesulergine-labeled 5-HT2C receptors increased from 203 nM (native) to 76 nM for S312F and 6.6 nM for S312K mutant recept ors. 5-HT potency for stimulation of phosphatidylinositol (PI) hydroly sis increased from 70 nM (native) to 28 nM for S312F and 2.7 nM for S3 12K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five-fold increases, respectively, i n basal levels of PI hydrolysis. Mianserin and mesulergine displayed i nverse agonist activity by decreasing basal levels of PI hydrolysis st imulated by S312K mutant receptors. [H-3]5-HT and [H-3]-mesulergine la beled the same number of S312K mutant receptors and 5'-guanylylimidodi phosphate had no effect on [H-3]5-HT binding. These results indicate t hat serine --> lysine mutation at amino acid no. 312 produces an agoni st high-affinity slate of the 5-HT2C receptor that spontaneously coupl es to G proteins and stimulates PI hydrolysis in the absence of agonis t.