A Disulfide Bond between Conserved Extracellular Cysteines in the Thyrotropin-releasing Hormone Receptor Is Critical for Binding

Jeffrey H Perlman,Wei Wang,Daniel R Nussenzveig,Marvin C Gershengorn

doi:10.1074/jbc.270.42.24682

Abstract

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.

Highlights

Seven transmembrane-spanning, guanine nucleotide-binding (G) protein-coupled receptors (GPCRs)1 constitute a large family of cell surface regulatory molecules [1, 2]
The EC50 values of C98A, C100A, and C179A thyrotropinreleasing hormone (TRH)-Rs were 4400, 2, and 640-fold higher than that of WT TRH receptor (TRH-R) (Fig. 2A, Table I). This indicated that C98A and C179A TRH-Rs are expressed on the cell surface and that Cys at position 98 (Cys-98) and Cys-179 are critical for high affinity binding
To determine whether there was an interaction between Cys-98 and Cys-179, the double mutant C98A/C179A TRH-R was expressed in COS-1 cells and the EC50 of TRH was measured

Summary

EXPERIMENTAL PROCEDURES

Materials—TRH was purchased from Calbiochem, MeTRH and DTT from Sigma, and [3H]MeTRH from DuPont NEN. Mutants were prepared by polymerase chain reaction, and plasmid sequences were confirmed by the dideoxy chain termination method. To study the effects of reduction of disulfide bonds on IP formation, cells expressing TRH-Rs were preincubated for 1 h at 37 °C in the absence or presence of 10 mM DTT. Receptor Binding Studies—Assays were performed as described [21]. To study the effects of reduction of disulfide bonds on binding, cells expressing TRH-Rs were preincubated for 1 h at 37 °C in the absence or presence of 10 mM DTT. [3H]MeTRH (1 nM, in the absence of DTT, or 5–10 nM, in the presence of DTT) and various concentrations of unlabeled MeTRH were added for an additional 1 h at 37 °C. Statistical Analyses—Curves were fitted by nonlinear regression and drawn with GraphPad Prism (GraphPad Software, San Diego, CA)

RESULTS

ϪDTT ϩDTT

DISCUSSION