Disulfide bond structure and accessibility of cyteines in the ectodomain of the cholecystokinin receptor: Specific mono-reactive receptor constructs examine charge-sensitivity of loop regions

Cysteine residues play a unique role in structural analysis. We examined endogenous cysteine residues in the cholecystokinin receptor to determine participation in disulfide bonds and accessibility to methanethiosulfonate (MTS) reagents. Bonds linking Cys¹¹⁴ to Cys¹⁹⁶ and Cys¹⁸ to Cys²⁹ were demonstrated, with the first functionally important and the amino-terminal bond having no apparent function. Cys⁹⁴, in the second transmembrane segment, was also accessible. Mutation of this residue to serine (C94S) was key for establishing a null cysteine-reactive pseudo - wild type receptor that could act as a template for insertion of a reactive cysteine (N102C, A204C, and T341C). Modification of T341C with a negatively charged MTS reagent reduced CCK agonist binding, while this binding was enhanced by a positively charged MTS reagent. This pattern was repeated in mutants having the same residue directly replaced with a charged residue.