Development and Characterization of a Mouse Cell Line Expressing the Human V2 Vasopressin Receptor Gene
| Title: | Development and Characterization of a Mouse Cell Line Expressing the Human V2 Vasopressin Receptor Gene |
|---|---|
| Authors: | Birnbaumer, Mariel; Hinrichs, Victoria; Themmen, Axel P.N. |
| Publisher: | Molecular Endocrinology |
| Date Published: | February 01, 1990 |
| Reference Number: | 245 |
Human genomic DNA and the HSV tk gene were cotransfected into mouse Ltk- cells and assayed for the acquisition of a Gs-coupled receptor to obtain cell lines expressing human receptors that are so far unavailable. The transfected cells were distributed into 96-well microtitration plates at a density such that after HAT (100 microM hypoxanthine, 1 microM aminopterin, and 10 microM thymidine) selection each well contained, on the average, two to three tk+ cell clones. After replication, half of them were tested for expression of a new phenotype: an adenylyl cyclase stimulatory receptor not normally expressed in the Ltk- recipient cell. The screen yielded a positive result on testing cells arising from the third transfection, the newly expressed receptor is that for arginine vasopressin, commonly referred to as type 2 or V2. DNA from primary transformants (HTB-1 cells) served to obtain secondary transformants by the same technique (HTB-2 cells). Pharmacological properties confirmed that this new receptor, which stimulates adenylyl cyclase activity 7- to 10-fold, is the human V2 receptor and not the activated homologous murine gene. The new cell line provides a permanent accessible source to study the human receptor, by-passing the need for human kidneys. The V2 receptor was susceptible to homologous down-regulation in the HTB-2 cell, but no down-regulation of the cell authentic prostaglandin E1 receptor was observed. The vasopressin did not modify phospholipase-C activity in these cells as expected from V2 receptors. Thus, we successfully applied genomic DNA-mediated gene transfer and were able to develop a cell line expressing a Gs-coupled human receptor of low abundance and poor accessibility.
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.
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Efficient methods for studying human cell surface receptors that bind with hormones have been lacking. Birnbaumer, et al., experimented with one approach to make laboratory study of human cell surface receptors easier. They attempted to place a receptor gene in a laboratory cell culture by injecting human DNA taken from white blood cells into the laboratory cell culture. In this sort of DNA-mediated gene transfer into a cell culture, researchers look to see if the human DNA will produce the biological artifact they desire. Birnbaumer, et al., were looking to see if the human DNA they transferred into the laboratory cell culture (a mouse cell line called Ltk-) would produce a human cell surface hormone receptor that was coupled to a Gs-protein.
The researchers developed a new strategy to determine if the Ltk- cells acquired the ability to produce a human, Gs-coupled cell surface receptor: they measured the cell line's ability to elevate its adenylyl cyclase (AdC) activity. Mouse Ltk- cells possess an AdC system, but they do not increase AdC activity in response to AdC stimulating hormones. The authors placed the human DNA-injected Ltk- cells culture into 96-well microtitration plates, added a mixture of 12 AcD stimulating hormones, and measured to see if any of the 96 samples showed an increase in AdC activity. An increase in AdC activity would show that the mouse Ltk- cell line had incorporated a part of the human DNA that would bind with one of the AdC stimulating hormones. This would indicate that the cell line had developed a new ability: the ability to express a human cell surface hormone receptor, which a hormone must bind with before it can stimulate AdC.
The cell cultures in the well plates that showed significant increases in AdC activity were replicated and again tested for increases in AdC activity in response to the hormone mixture. One of these cell plates continually exhibited higher AdC activity than the rest. The researchers called the cells in this sample HTB cells.
The human cell surface receptor that the HTB cells were expressing were vasopressin-2 receptors (V2Rs), receptors which bind with the hormone, arginine vasopressin (AVP).
Thus, Birnbaumer, et al., developed a mouse cell line (HTB) capable of expressing the human V2R. This new cell line provides an efficient, reliable source from which to study the human V2R receptor, rendering it unnecessary to use human kidneys for this purpose.