ALTERED LIGAND DISSOCIATION RATES IN THYROTROPIN-RELEASING-HORMONE RECEPTORS MUTATED IN GLUTAMINE-105 OF TRANSMEMBRANE HELIX-III

Glutamine 105 in the third transmembrane helix of the thyrotropin-rele asing hormone receptor (TRH-R) occupies a position equivalent to a con served negatively charged residue in receptors for biogenic amines whe re it acts as counterion interacting with the cationic amine moiety of the ligand. Maximum levels of response to TRH in oocytes expressing w ild-type TRH-Rs were indistinguishable from those of oocytes expressin g receptors mutated to Glu, Asn, or Asp in position 105. However, the EC(50) values for activation of oocyte responses increased more than 5 00 times in oocytes expressing mutant Glu(105) receptors, in which the amido group of Gin(105) has been removed by site-directed mutagenesis . Charge effects do not seem to be involved in the huge effect of muta ting Gln(105) to Glu, since mutation of Gin(105) to Asp induces only a 15-fold increase in EC(50). Furthermore, no change in EC(50) is obser ved after mutation of Asn(110) to Asp. The affinity shift (identified by changes in EC(50) values for systems of comparable efficacy) in Glu (105) mutant receptors was partially recovered in oocytes expressing A sn(105) mutant receptors. These results and those obtained after subst itution of Lys, Leu, Tyr, and Ser for Gin(105) suggest that the presen ce and the correct position of the Gin hydrogen bond-donor amido group are important for normal functionality of the receptor. In wild type or Asp(105) mutant receptors showing the same maximal responses, decre ases in affinity with TRH and methyl-histidyl-TRH correlated with incr eased dissociation rates of hormone from the receptor. Rapid dilution experiments following subsecond stimulation indicate that the TRH-R is converted rapidly from a form showing fast dissociation kinetics to a form from which the hormone dissociates slowly. Mutation of residue 1 05 impairs the receptor shift between these two forms. This effect was demonstrated in a direct way by comparing [H-3]methyl-histidyl-TRH di ssociation rates in COS-7 cells transfected with either wild type or A sp(105) mutant TRH-Rs. Thus, residues located in transmembrane helix I II positions equivalent to those of the counterions for biogenic amine s, regulate hormone-receptor interactions in the TRH receptor (and per haps other receptors). Furthermore, the nature of the amino acid in th ese positions may also play a role, directly or indirectly, in conform ational changes leading to receptor activation, and hence to signal tr ansduction.