Characterization of Vasopressin V2 Receptor Mutants in Nephrogenic Diabetes Insipidus in a Polarized Cell Model

Title: Characterization of Vasopressin V2 Receptor Mutants in Nephrogenic Diabetes Insipidus in a Polarized Cell Model
Authors: Robben, Joris; Knoers, Nine; Deen, Peter M.T.
Publisher: American Journal of Physiology: Renal Physiology
Date Published: August 01, 2005
Reference Number: 694
X-linked nephrogenic diabetes insipidus (NDI) is caused by mutations in the gene encoding the vasopressin V2 receptor (V2R). For the development of a tailored therapy for NDI, knowledge of the cellular fate of V2R mutants is needed. It would be useful when this fate could be predicted from the location and type of mutation. To identify similarities and differences in localization, maturation, stability, and degradation of COOH-terminal GFP-tagged V2R mutants, we stably expressed nine mutants in polarized Madin-Darby canine kidney cells. The mutants V2R-L44P, -Delta62-64, -I130F, -S167T, -S167L, and -V206D were mainly expressed in the endoplasmic reticulum (ER) as immature proteins. These mutants had relatively short half-lives due to proteasomal degradation, except for V2R-Delta62-64. In contrast, V2R-R113W, -G201D, and -T204N were expressed in the ER and in the basolateral membrane as immature, high-mannose glycosylated, and mature complex-glycosylated proteins. The immature forms of V2R-R113W and -T204N, but not V2R-G201D, were rapidly degraded. The mature forms varied extensively in their stability and were degraded by only lysosomes (V2R-T204N and wild-type V2R) or lysosomes and proteasomes (V2R-G201D, -R113W). These data reveal that most missense V2R mutations lead to retention in the ER and suggest that mutations that likely distort a transmembrane domain or introduce a charged amino acid close to it make a V2R mutant more prone to ER retention. Because six of the mutants tested showed significant increases in intracellular cAMP levels on transient expression in COS cells, activation of these six receptors following rescue of cell-surface expression might provide a cure for NDI patients.
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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)

Mutations in the vasopressin-2 receptor (V2R) gene are responsible for about 90% of the cases of inherited NDI. Currently, there have been over 180 different mutations of the V2R gene that have been described by researchers. Most of the mutations are of the type called missense mutations. These entail a change in a codon of the V2R gene that results in a different amino acid than is normal in the chain of 371 amino acid residues that make up the V2R protein.

The normal V2R protein is a chain of amino acid residues that has a specific shape that loops in and out of the membrane of the cell it works in. The part of the protein that is located outside the cell membrane is a series of three loops called the extracellular loops. The parts of the V2R that forms three loops inside the cell are called the intracellular loops. Part of the V2R is inside the cell membrane, forming seven distinct clumps called the transmembrane domains. The head of the V2R is extracellular; the tail is inside the cell.

Deen, et al., studied how the cell deals with 9 different V2R mutants. To do this, they stably expressed the mutants in a laboratory cell culture of polarized cells. Stably expressing these mutants in a polarized cell culture much more closely mimics the conditions the mutant proteins would experience in the body than transiently expressing the mutants in unpolarized cells. This is important because Deen, et al., posited that the development of an effective therapy, tailored to the specific V2R gene mutation (and there are over 180 of them), depends on what happens to the V2R mutants in the kidney principal cells. Specifically, the research team wanted to know for each of the 9 mutant V2Rs tested:

  1. Where did they end up in the cell, i.e., what was their location?

  2. How mature did each one get? I.e., the protein synthesis process takes place in succeeding stages from the very immature state to the mature state. How far in their maturation process did each mutant get?

  3. How stable was each mutant? I.e., how long did they exist before being dismantled by the cell’s quality control systems?

  4. When the mutant was degraded, by what means or pathway in the cell was it degraded (dismantled)? I.e., V2Rs can either be degraded by the proteasomal pathway or the lysosomal pathway. (Both proteasomes and lysosomes are cellular enzymes.)

The researchers’ data indicate that six of the V2R mutants tested were located in the cell’s endoplasmic reticulum (ER), the quality control mechanism of the cell that can recognize misshapen proteins and not let them proceed to their job site. These six mutants only developed to an immature stage, had relatively short half lives compared to normal V2Rs, and were degraded along the proteasomal pathway. The exception in this group was the mutant V2R-D62-64, which had a much longer half-life than the rest, though still short when compared to a normal V2R half-life. The three remaining mutant V2Rs were expressed in both the ER and the basolateral section of the cell membrane. Some of each of these three types of V2R mutation reached only immature stages of maturation, some reached the high-mannose glycosylated stage, and some reached the mature complex glycosylated stage. The immature forms of two of the three were rapidly degraded, whereas for the complex-glycosylated forms, the stability and degradation patterns varied considerably, and they were subject to degradation only by lysosomes or lysosomes and proteasomes. The researchers reasoned that the observed differences in degradation and stability among the mutants could be due to the different impacts of the mutations of the shape of the resultant V2R protein.

Six of the nine mutants showed they were capable of performing their function (binding with AVP) under special laboratory cell culture conditions. The researchers speculated that if they were able to be rescued from the ER, they could adequately perform their job on the cell membrane. The findings reveal that most missense mutations are retained in the ER due to a distortion of their shape caused by the V2R gene mutation. The distortions are likely to occur in one of the transmembrane domains. The ER could also detain the mutant V2R because of the introduction of a charged amino acid caused by the mutation.