Conserved helix 7 tyrosine functions as an activation relay in the serotonin 5HT(2C) receptor

The Function of the helix VII Tyr in the conserved Asn-Pro-X-X-Tyr segment of rhodopsin-like G protein coupled receptors has been investigated in many receptors. Various effects of site-directed mutation of this locus have be en found, including altered coupling, sequestration and agonist affinity. W e report the first constitutively active mutations of this Tyr. In the sero tonin 5HT(2C) receptor, substituting Ala or Cys for Tyr resulted in a marke d increase in the basal level of inositol phosphate accumulation in transfe cted COS-1 cells. This constitutive signaling was abolished by the inverse agonist SB206553. Introducing Phe at this locus eliminated both basal and a gonist-stimulated signaling. All three mutant receptors showed an increase in binding affinity for the structurally dissimilar agonists -hydroxytrypta mine (5HT), (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-ami (DOI), and quipazine , suggesting that both the activating and inactivating mutations stabilize a high affinity state. These results implicate the conserved Tyr in the con formational rearrangements that occur during agonist complexing and recepto r activation. (C) 2000 Elsevier Science B.V. All rights reserved.