Functional Studies of Twelve Mutant V2 Vasopressin Receptors Related to Nephrogenic Diabetes Insipidus: Molecular Basis of a Mild Clinical Phenotype
| Title: | Functional Studies of Twelve Mutant V2 Vasopressin Receptors Related to Nephrogenic Diabetes Insipidus: Molecular Basis of a Mild Clinical Phenotype |
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| Authors: | Ala, Youssef; Morin, Denis; Mouillac, Bernard; Sabatier, Nancy; Vargas-Poussou, Rosa; Cotte, Nathalie; Dechaux, Michele; Antignac, Corinne; Arthus, Marie-Francoise; Lonergan, Michele; Turner, Maria S.; Balestre, Marie-Noelle; Alonso, Gerard; Hibert, Marcel; Barberis, Claude; Hendy, PhD, Geoffrey N.; Bichet, Daniel G.; Jard, Serge |
| Publisher: | Journal of the American Society of Nephrology |
| Date Published: | October 01, 1998 |
| Reference Number: | 339 |
X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine vasopressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. To study the cause of loss of function of mutant V2 receptors, we expressed 12 mutations (N55H, L59P, L83Q, V88M, 497CC-->GG, deltaR202, I209F, 700delC, 908insT, A294P, P322H, P322S) in COS-7 cells. Eleven of these, including P322H, were characterized by a complete loss of function, but the mutation P322S demonstrated a mild clinical and in vitro phenotype. This was characterized by a late diagnosis without any growth or developmental delay and a significant increase in urine osmolality after intravenous 1-deamino[D-Arg8]AVP administration. In vitro, the P322S mutant was able to partially activate the Gs/adenylyl cyclase system in contrast to the other V2R mutants including P322H, which were completely inactive in this regard. This showed not only that Pro 322 is important for proper V2R coupling, but also that the degree of impairment is strongly dependent on the identity of the substituting amino acid. Three-dimensional modeling of the P322H and P322S mutant receptors suggested that the complete loss of function of the P322H receptor could be due, in part, to hydrogen bond formation between the His 322 side chain and the carboxyl group of Asp 85, which does not occur in the P322S receptor.
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)
Normally, the AVPR2 is a protein consisting of 371 amino acids arranged in a precise sequence. Mutations in the AVPR2 gene can disrupt this sequence in a variety of ways, depending on the mutation. Some mutations result in a single amino acid being replaced by another amino acid. Other mutations interrupt the development of the sequence, preventing the full development of the sequence from occurring. Researchers can discover the structural alterations of the AVPR2 produced by mutations in the AVPR2 gene that codes for it, and see how these structural alterations alter the function of that AVPR2.
Ala, et al., report on their study of 12 mutated AVPR2 genes. After determining the structure of each AVPR2 produced by each mutated gene, they injected the AVPRs into laboratory cell cultures to determine how the mutation affected their function. 11 of the mutations resulted in a complete loss of function of the AVPR2s. They could not do their job at all.
Normally, the AVPR2 receives the antidiuretic hormone, arginine vasopressin (AVP). That is, it forms a weak, reversible chemical bond with AVP. Once bound with AVP, it stimulates the Gs/adenylyl cyclase system, which produces an elevation in cAMP. cAMP interacts with protein kinase A (PKA). In turn, PKA induces aquaporin-2 (AQP2) to travel to and insert itself into the apical membrane of the cell, thus increasing its water permeability.
Of the 12 mutated AVPR2s, nine failed to reach the cell membrane. Since the cell membrane is the place where the AVPR2 must be in order to form a bond with AVP, not being able to reach the cell membrane explains its failure to bind with AVP.
Mutations R202, P322H and P322S did reach the cell surface but the R202 and P322H mutations were still nonfunctional. R202 could not bind with AVP and P322H could not stimulate the Gs/adenylyl cyclase complex. P322S, however, demonstrated partial ability to couple with Gs/adenylyl cyclase.
This mutation was of particular interest to the researchers. First, because the NDI patient who bore it displayed only mild symptoms of NDI. The patient, a male, was not diagnosed with NDI until he was ten years old. He showed no developmental or growth delay and, after administration of DDAVP, he responded with more concentrated urine. Normally, a person with NDI manifests its symptoms in the first days of life, and if they go undiagnosed or untreated, the patient could experience physical and mental retardation. Further, NDI patients generally cannot increase the concentration of their urine in response to DDAVP.
The second reason of interest is that the P322S mutation, which resulted in mild symptoms, is similar to the P322H mutation, which resulted in full symptoms. P322S means that the 322cnd amino acid in the 371-long amino acid chain that makes up the APVR2, which normally is a proline amino acid, is replaced by a serine amino acid. P322H means that the proline is replaced by a histidine amino acid. So there is only a difference of a single amino acid between the two mutations, yet there is a wide functional difference.
To further understand the functional difference between the two mutations, the authors constructed a three-dimensional model of a normal AVPR2 receptor and the mutant P322H and P322S receptors. The model of the normal AVPR2 showed that the protein is so configured that the proline that is the 322cnd amino acid in the chain (Pro 322) is situated close to the aspartic acid amino acid that is the 85th amino acid in the chain (Asp 85). So, Pro 322 is close to ASP 85. When proline is replaced by histidine as it is in the P322H mutation, the authors speculate that the histidine and aspartic acid form a hydrogen bond between them. The reason for this is that histidine has a long side chain that can link up to the carboxyl group of Asp 85. This hydrogen bond changes the shape of the P322H AVPR2 receptor. This is why, the authors speculate, it can't couple with the Gs/AdC complex.
In the P322S AVPR2 receptor, the serine that takes the place of Pro322 has too small a side chain to form a hydrogen bond with Asp85. This small difference could be the reason the P322H mutation causes the full symptoms of NDI and the P322S mutation only results in mild ones.