The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conse rved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors, Here we have assessed the properties of the V2 receptor after site-directed mutagen esis of its conserved cysteine residues in transiently transfected human em bryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C 192A) were non-functional and located mostly in the cell's interior. The co nserved cysteine residues of the V2 receptor are thus not only important fo r the structure of the ligand binding domain but also for efficient intrace llular receptor transport. In addition to the functional significance of th e conserved cysteine residues, pie have also analyzed the defects of two mu tant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extrac ellular loop (mutants G185C, R202C), These mutations are assumed to impair normal disulfide bond formation, Mutant receptor G185C and R202C were effic iently transported to the plasma membrane but mere defective in ligand bind ing. Only in the case of the mutant receptor R202C, the more sensitive aden ylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC50 value and with a reduced ECmax, indicating th at ligand binding is not completely abolished. Taking the unaffected intrac ellular transport of both NDI-causing mutant receptors into account, our re sults indicate that the observed impairment of ligand binding by the additi onal cysteine residues is not due to the prevention of disulfide bond forma tion between the conserved cysteine residues. (C) 2000 Federation of Europe an Biochemical Societies.