A disulfide bond between conserved extracellular cysteines in the thyrotropin-releasing hormone receptor is critical for binding.

The assumption that a disulfide bond is present between two highly conserved cysteines in the extracellular loops of G protein-coupled receptors and is critical for receptor function has been cast in doubt. We undertook to determine whether a disulfide bond important for binding or activation is present in the thyrotropin-releasing hormone (TRH) receptor (TRH-R). Studies were performed with cells expressing wild-type (WT) and mutant receptors in the absence or presence of the reducing agent dithiothreitol (DTT). The affinity of WT TRH-R was 16-22-fold lower in the presence of DTT than in the absence of DTT. Mutant receptors were constructed in which Ala was substituted for conserved Cys-98 and Cys-179 of extracellular loops 1 and 2, respectively, and for the nonconserved Cys-100. C98A and C179A TRH-Rs did not exhibit high affinity binding. These mutant receptors were capable of stimulating inositol phosphate second messenger formation to the same extent as WT TRH-Rs but with a markedly lower potency. The affinities of C98A and C179A TRH-Rs, estimated from their potencies, were 4400- and 640-fold lower, respectively, than WT TRH-R. The estimated affinities of neither C98A nor C179A TRH-R were decreased by DTT. In contrast, the estimated affinity of C100A TRH-R was not different from WT TRH-R and was DTT sensitive. Moreover, the effect of mutating both Cys-98 and Cys-179 was not additive with the effects of the individual mutations. These data provide strong evidence that Cys-98 and Cys-179 form a disulfide bond. This interaction is not involved in receptor activation but is critical for maintaining the high affinity conformation of TRH-R.