Glutamine 105 in the third transmembrane helix of the thyrotropin-releasing hormone receptor (TRH-R) occupies a position equivalent to a conserved negatively charged residue in receptors for biogenic amines where it acts as counterion interacting with the cationic amine moiety of the ligand. Maximum levels of response to TRH in oocytes expressing wild-type TRH-Rs were indistinguishable from those of oocytes expressing receptors mutated to Glu, Asn, or Asp in position 105. However, the EC50 values for activation of oocyte responses increased more than 500 times in oocytes expressing mutant Glu105 receptors, in which the amido group of Gln105 has been removed by site-directed mutagenesis. Charge effects do not seem to be involved in the huge effect of mutating Gln105 to Glu, since mutation of Gln105 to Asp induces only a 15-fold increase in EC50. Furthermore, no change in EC50 is observed after mutation of Asn110 to Asp. The affinity shift (identified by changes in EC50 values for systems of comparable efficacy) in Glu105 mutant receptors was partially recovered in oocytes expressing Asn105 mutant receptors. These results and those obtained after substitution of Lys, Leu, Tyr, and Ser for Gln105 suggest that the presence and the correct position of the Gln hydrogen bond-donor amido group are important for normal functionality of the receptor. In wild type or Asp105 mutant receptors showing the same maximal responses, decreases in affinity with TRH and methyl-histidyl-TRH correlated with increased 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 105 impairs the receptor shift between these two forms. This effect was demonstrated in a direct way by comparing [3H]methyl-histidyl-TRH dissociation rates in COS-7 cells transfected with either wild type or Asp105 mutant TRH-Rs. Thus, residues located in transmembrane helix III positions equivalent to those of the counterions for biogenic amines, regulate hormone-receptor interactions in the TRH receptor (and perhaps other receptors). Furthermore, the nature of the amino acid in these positions may also play a role, directly or indirectly, in conformational changes leading to receptor activation, and hence to signal transduction.