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|
|Date Published:||June 01, 1997|
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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)
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:
- They prepared a longer, though still truncated V2R, calling it G345ter.
- 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.