Expression Cloning of the Human V2 Vasopressin Receptor
| Title: | Expression Cloning of the Human V2 Vasopressin Receptor |
|---|---|
| Authors: | Seibold, Anita; Rosenthal, Walter; Birnbaumer, Mariel; Barberis, Claude; Ishido, Ph.D., Masami |
| Publisher: | Regulatory Peptides |
| Date Published: | April 29, 1993 |
| Reference Number: | 242 |
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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)
They injected a mouse cell line called Ltk- with human genetic material. The Ltk- cells were then placed in 96 different plates where it was determined that each plate contained two to three Ltk cell clones. Each of the plates was then tested for hormone stimulated adenylyl cyclase (AdC) activity. (When AVP binds with V2Rs, it stimulates AdC activity. Thus, plates which indicated hormone stimulated AdC, indicate the possibility of the presence of V2Rs.) The cells of the plates that indicated this were called HTB cells. The researchers showed that this elevated AdC activity of HTB cells was due to a property not found in Ltk- cells: the ability to respond to AVP. These cells had undergone a transformation, a change of structure in response to the human DNA material.
These cells were again cultured, and one of these subcultures consistently exhibited higher AdC activity in response to AVP than the others. These cells were called HTB-cells. The researchers extracted genomic DNA from the HTB-1 cells, on the basis that it now contained the V2R gene (as indicated by the AdC activity). This process was repeated. Then the researchers extracted the genomic DNA from the HTB-3 cell line and used the extraction to create a set of cloned DNA fragments that represent the complete human gene compliment.
They took the seven clones positive for DNA and injected them into mouse Ltk- cells. One of the clones was able to confer the ability to respond to AVP, indicating the presence of the V2R gene. These cells, in fact, were able to express very high levels of human V2R. Several further refinements enabled the researchers to find the sequence of the actual structure of the V2R. They predict the V2R is a protein made up of 371 amino acids.