Functional Rescue of the Nephrogenic Diabetes Insipidus-Causing Vasopressin V2 Receptor Mutants G185C and R202C by a Second Site Suppressor Mutation
| Title: | Functional Rescue of the Nephrogenic Diabetes Insipidus-Causing Vasopressin V2 Receptor Mutants G185C and R202C by a Second Site Suppressor Mutation |
|---|---|
| Authors: | Schulein, Ralf; Zuhlke, Kerstin; Krause, Gerd; Rosenthal, Walter |
| Publisher: | Journal of Biological Chemistry |
| Date Published: | March 16, 2001 |
| Reference Number: | 509 |
Mutations in the gene of the G protein-coupled vasopressin V2 receptor (V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of the missense mutations on the extracellular face of the receptor introduce additional cysteine residues. Several groups have proposed that these residues might disrupt the conserved disulfide bond of the V2 receptor. To test this 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 address this question experimentally, we used the NDI-causing mutant receptors G185C and R202C. Their Cys-195 residues were replaced by alanine to eliminate the hypothetical second disulfide bonds. This second site mutation led to functional rescue of both NDI-causing mutant receptors, strongly suggesting that the second disulfide bonds are indeed formed. Furthermore we show that residue Cys-195, which is sensitive to "additional cysteine" mutations, is not conserved among the V2 receptors of other species and that the presence of an uneven number of extracellular cysteine residues, as in the human V2 receptor, is rare among class I G protein-coupled receptors.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)
Most of the V2R mutations occur in the section of the V2R located within the cell membrane. Only a few are located in the portion that extends outside the cell (the extracellular portion of the V2R.)
Most of the mutations in the extracellular portion of the V2R introduce additional cysteine residues into this region of the V2R. That is, the mutation results in a cysteine taking the place of the residue that would normally be there. E.g., mutation G185C results in a cysteine residue as the 185th residue in the V2R, a position normally occupied by a glycine residue.
Researchers have previously hypothesized that these extra cysteines might prevent the chemical bridge between cysteine 112 and cysteine 192 from forming. This is important because the bridge between cys-112 and cys-192 pulls the V2R into a specific shape - the correct shape. If the bridge does not form, it is likely the V2R will not be properly shaped and therefore unable to function properly.
Schulein, et al., tested this hypothesis in two ways. First, they calculated the shape of the extracellular portions of the V2R in order to get a clearer idea whether additional cysteine amino acids might cause the V2R to have an irregular shape by forming a bond with either cys-112 or cys-192. Their calculations indicated the mutation-induced extra cysteine residues would more likely form a bond with cys-195, thus leaving the important bridge between cys-112 and cys-192 intact. But the bonding of cys-195 with the mutation-induced cysteine would still distort the shape and function of the V2R.
To test this, they worked with two V2R mutations that cause an extra-cysteine in the extracellular portion of the V2R: G185C and R202C. They manipulated both of these mutations so that an alanine amino acid would take the place of the cysteine that normally occurs as the 195th amino acid in the string of amino acid residues that make up the V2R. The researchers made, in effect, a mutation within a mutation to prevent the hypothesized bonding between cys-195 and the extra cysteine.
This second site mutation actually allowed the mutant V2Rs to function properly. This suggests that the extra cysteine introduced by mutants G185C and R202C leave the important bridge between C112 and C192 intact, but do form a second with cys-195, a bond that distorts the mutant V2Rs' shape and function. When these V2Rs are manipulated to produce an extra alanine instead of cys-195, they are unable to form this second bond with cys-195 and their resulting shape allows them to function more normally.