Functional rescue of the nephrogenic diabetes insipidus-causing vasopressin V-2 receptor mutants G185C and R202C by a second site suppressor mutation

Mutations in the gene of the G protein-coupled vasopressin V-2 receptor (V- 2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of th e missense mutations on the extracellular face of the receptor introduce ad ditional cysteine residues. Several groups have proposed that these residue s might disrupt the conserved disulfide bond of the V-2 receptor. To test t his hypothesis, we first calculated a structure model of the extracellular receptor domains. The model suggests that the additional cysteine residues may form a second disulfide bond with the free, nonconserved extracellular cysteine residue Cys-195 rather than impairing the conserved bond. To addre ss this question experimentally, we used the NDI-causing mutant receptors G 185C and R202C. Their Cys-195 residues were replaced by alanine to eliminat e the hypothetical second disulfide bonds. This second site mutation led to functional rescue of both NDI-causing mutant receptors, strongly suggestin g that the second disulfide bonds are indeed formed. Furthermore we show th at residue Cys-195, which is sensitive to "additional cysteine" mutations, is not conserved among the V-2 receptors of other species and that the pres ence of an uneven number of extracellular cysteine residues, as in the huma n V-2 receptor, is rare among class I G protein-coupled receptors.