2000 Global Researcher Conference Proceeding

March 10 - 12, 2000

Conference: 2000 Global Researcher Conference
Title: Functional rescue of three vasopressin V2 receptor mutants causing nephrogenic diabetes insipidus by a second site suppressor mutation
Authors: Schulein, Ralf; Zuhlke, Kerstin; Rosenthal, Walter
Institutions: Forschungsinstitut fur Molekulare Pharmakologie (FMP), Forschungsinstitut fur Molekulare Pharmakologie, Charite - Universitatsmedizin Berlin
SchuleinMutations in the gene of the vasopressin V2 receptor (V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI), a disease which is characterized by the inability of the kidney to concentrate urine. We have analyzed the defects of three mutant V2 receptors which cause NDI by the introduction of cysteine residues into the second extracellular loop (mutants G185C, R202C and Y205C). These mutations are assumed to impair the formation of the normal disulfide bond between cysteine residues C112 and C192 of the V2 receptor. The pharmacological properties and the intracellular trafficking of the mutant receptors were analyzed in transiently transfected HEK 293 cells. Mutant receptors G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Mutant receptor Y205C was trapped in a nonfunctional form in the ER. Mutant receptors G185C and R202C thus seem to have a relatively localized defect in the ligand binding domain whereas mutant receptor Y205C may have a stronger defect in overall receptor folding which is recognized by the quality control system of the ER. For all three mutant receptors, the additional mutation of cysteine residue C195 led to functional rescue. The data obtained with this suppressor mutation strongly suggest that the additional cysteine residues of the three NDI-causing mutant receptors form a second disulfide bond with cysteine residue C195 rather than impairing formation of the normal disulfide bond between cysteine residues C112 and C192.

The vasopressin-2 receptor (V2R) is a protein made up of a specific sequence of amino acids configured in a specific shape. Amino acids that combine to form a protein undergo a slight chemical change and are called residues. Rosenthal, et al., examined three mutant V2Rs, all of which had a cysteine residue (C) where there should have been another type of residue. In each case, the C was assumed to alter the shape of the V2R by impairing the formation of the normal disulfide bond that occurs in normal V2Rs. This structural alteration affected the function of the three mutant V2Rs in different ways. In two of the mutants, it prevents the V2Rs from forming a bond with the antidiuretic hormone, arginine vasopressin (AVP). The other mutant was unable to travel to the cell membrane where it must be if it is to bind with AVP.

The researchers introduced a second mutation into each of these mutants, an additional A taking the place of the normally occurring C residue at position 195 of the amino acid sequence of the V2R. This manipulation allowed each of the mutants now to function normally. The researchers suggest the functional rescue occurs because the mutation-causing additional C in each of the three original mutants each form a second disulfide bond with the C at position 195 which was eliminated by the second mutation.