Functional characterization of AVPR2 mutants found in Turkish patients with nephrogenic diabetes insipidus

Abstract

Diabetes insipidus is a rare disorder characterized by an impairment in water balance because of the inability to concentrate urine. While central diabetes insipidus is caused by mutations in the AVP, the reason for genetically determined nephrogenic diabetes insipidus can be mutations in AQP2 or AVPR2. After release of AVP from posterior pituitary into blood stream, it binds to AVPR2, which is one of the receptors for AVP and is mainly expressed in principal cells of collecting ducts of kidney. Receptor activation increases cAMP levels in principal cells, resulting in the incorporation of AQP2 into the membrane, finally increasing water reabsorption. This pathway can be altered by mutations in AVPR2 causing nephrogenic diabetes insipidus. In this study, we functionally characterize four mutations (R68W, ΔR67-G69/G107W, V162A and T273M) in AVPR2, which were found in Turkish patients. Upon AVP stimulation, R68W, ΔR67-G69/G107W and T273M showed a significantly reduced maximum in cAMP response compared to wild-type receptor. All mutant receptor proteins were expressed at the protein level; however, R68W, ΔR67-G69/G107W and T273M were partially retained in the cellular interior. Immunofluorescence studies showed that these mutant receptors were trapped in ER and Golgi apparatus. The function of V162A was indistinguishable from the indicating other defects causing disease. The results are important for understanding the influence of mutations on receptor function and cellular trafficking. Therefore, characterization of these mutations provides useful information for further studies addressing treatment of intracellularly trapped receptors with cell-permeable antagonists to restore receptor function in patients with nephrogenic diabetes insipidus.

Chemical Chaperone Effects on Arginine-Vasopressin Receptor 2 Mutants

“…Using the site directed mutagenesis method as described in our previous studies, R68W, ΔR67_G69/G107W, ΔR67_G69, G107W, V162A, T273M, V88M, R106C, G12E mutations were introduced into the pLV2R plasmid [8]. All mutant constructs were confirmed by DNA sequencing.…”

“…Therefore, monoclonal anti-HA High Affinity, HRP-conjugated (Roche Applied Science, Mannheim, Germany) were used for the ELISA experiments. The protocol was described in our previous studies [8].…”

“…By this binding, secondary messenger cAMP increase in the cell and leads to protein kinase A (PKA) activation. PKA activation lead to phosphorylation of water channel aquaporin 2 (AQP2) which finally reabsorbs the water into the collecting duct cells [7,8]. In this flow of the reabsorption of free water from urine, V2R receptor proteins play important roles.…”

“…Pharmacological chaperones are non-peptide and cell-permeable ligands and specifically bind to the related target proteins unlike chemical chaperones [10]. We have previously identified several mutations on AVPR2 gene, causing the NDI disease in Turkish families and we also studied functional characterization of these mutations [7,8]. In this study we aimed to determine the rescue potentials of these chemical chaperones on mutant proteins.…”

Clinical and Functional Characterization of a Novel Mutation in AVPR2 Causing Nephrogenic Diabetes Insipidus in a Four-Generation Chinese Family

“…The detection of a missense variation in AVPR2 gives a hint for a possible pathogenic cause of CNDI. Additionally, functional analysis is necessary to verify the impact of the mutation on receptor function and may suggest possible therapeutic strategies for specific functional defects (12)(13)(14)(15)(16)(17). Therefore, it is important to understand the effects caused by a given variation in detail.…”