An X-linked NDI Mutation Reveals a Requirement for Cell Surface V2R Expression

Title: An X-linked NDI Mutation Reveals a Requirement for Cell Surface V2R Expression
Authors: Sadeghi, Hamid; Innamorati, Giulio; Birnbaumer, Mariel
Publisher: Molecular Endocrinology
Date Published: June 01, 1997
Reference Number: 150
Function and biochemical properties of the V2 vasopressin receptor (V2R) mutant R337ter, identified in patients suffering from X-linked recessive nephrogenic diabetes insipidus, were investigated by expression in COS.M6 or HEK293 cells. Binding assays and measurements of adenylyl cyclase activitiy failed to detect function for the truncated receptor, although metabolic labeling demonstrated normal levels of protein synthesis. ELISA assays performed on cells expressing the receptors tagged at the amino terminus with the HA epitope failed to detect V2R R337ter on the plasma membrane. Treatment with endoglycosidase H revealed that the receptor was present only as a precursor form because the mature R337ter V2R, resistant to endoglycosidase H treatment, was not detected. The precursor of V2R-R337ter had a longer half-life than that of the wild type V2R, suggesting that arrested maturation may slow the degradation of the precursor. Unrelated experiments had demonstrated that V2R-G345ter, containing eight additional amino acids, was expressed on the plasma membrane and functioned normally. Receptor truncations longer than 337ter revealed that four of the eight amino acids identified initially provided the minimum length required for the protein to acquire cell surface expression. This was shown by the production of mature receptor (V2R-341ter) detectable in SDS-PAGE, which mediated arginine vasopressin stimulation of adenylyl cyclase activity and bound ligand. In addition, the identity of amino acid 340 was found to play a role in this phenomenon. In conclusion, these data demonstrate that the V2R R337ter is nonfunctional because it does not reach the plasma membrane and that the minimal protein length required for translocation of the V2R to the cell surface is sufficient to confer function to the receptor protein. They also suggest the existence of a protein quality control in the endoplasmic reticulum independent of glycosylation.
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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)

X-linked recessive nephrogenic diabetes insipidus (NDI) is caused when the vasopressin-2 receptor (V2R) does not function properly and fails to bind with and respond to the antidiuretic hormone, arginine vasopressin (AVP). Mutations of the V2 receptor are responsible for this. The V2R is located within the cell membrane of the principal cells of the kidney collecting duct. The cell membrane is a thin strip that encircles the cell, separating the inside of the cell from the outside.

The V2R is a chain of polypeptides, and if you imagine it as a beaded string, most of it sits within the membrane in seven folded clumps known as transmembrane helices or domains. Part of the V2R snakes outside the membrane, forming three curves called extracellular loops 1, 2 and 3. Part of it snakes inside the cell forming three curves called intracellular loops 1, 2 and 3. One end of the V2R, called the amino-terminus, is outside the cell with the extracellular loops. The other end is called the carboxyl-terminal and is inside the cell with the intracellular loops. (Please see diagram of V2R.)

The V2R is made up of many amino acids in specific sequences. Any rearrangement or interruption in its complex arrangement is a mutation which could produce a defective V2R incapable of doing its job, as in the case of X-linked NDI. Sadeghi, et al., examined one such mutation in the V2R. This mutation, called mutant R337ter, resulted in a truncated V2R, that is, one that stopped developing early and is therefore not as long as a normal V2R because it is missing part of its sequence. This mutant receptor was missing the last 35 amino acids of the carboxyl-terminus, which means its tail end was cut short after the seventh transmembrane region.

This segment of the receptor has been shown through previous research to play an important role in allowing the receptor to rise to the cell surface from where it waits within a part of the cell called the endoplasmic reticulum (ER). It also plays an important role in enabling the V2R to couple with G proteins, in allowing phosphate groups to be introduced into its make-up, and in desensitizing V2R to AVP at the proper time.

Sadeghi, et al., investigated the function and biochemical properties of the truncated V2R-R337ter. The researchers expressed the mutant V2R in laboratory cell cultures and found that it did not bind at all with AVP. They speculated that the V2R's missing segment was crucial to the receptor's hormone-binding capability, and caused a complete lack of binding. Alternatively, the lack of binding may have indicated that the mutated V2R could not get to the cell surface where V2R normally binds with AVP.

To examine if the V2R-R377ter was missing some amino acids crucial for allowing it to get to the cell surface, the researchers did two things:

  1. They prepared a longer, though still truncated V2R, calling it G345ter.
  2. And they added the amino-terminal portion of another type of receptor, the V1a vasopressin receptor, to the end of the V2R-R337ter in order to make it a full-length receptor.

They tested their new receptors for the ability to bind to AVP, to stimulate adenylyl cyclase activity (an important step in the urine concentrating process), and to rise to the cell surface.

The researchers found that the full-length and G345ter were able to get to the cell surface, bind with AVP and stimulate adenylyl cyclase activity, whereas the V2R-R337ter could not. All three forms of the V2R produced comparable quantities of their immature stages of development, but only the G345ter and the full-length form developed into mature receptor forms. This indicates that the lack of function of the V2R-R337ter was due to its absence from the cell membrane, which indicates that it could not get out of the ER, perhaps because it was misfolded.

Sadeghi, et al., postulate the existence of a V2R quality control system in the ER that inhibits the exit of incorrectly or incompletely shaped receptors. This suggests that the mutated V2R was synthesized in sufficient quantities, but its truncated form inhibited its ability to mature. The maturation process is likely to require the presence of more amino acids after transmembrane domain seven than those found in the truncated V2R. However, since the G345ter truncation could bind with AVP and couple with G proteins to produce adenylyl cyclase, it is clear that these functions are not influenced by the last 27 amino acids of the V2R, which the G345ter is missing. The researchers were able to find that a minimum length of 340 amino acids was required to obtain a functional (though not maximally functional) V2 receptor that could get to the cell surface.