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Development and Characterization of a Mouse Cell Line Expressing the Human V2 Vasopressin Receptor Gene

Department of Cell Biology, Baylor College of Medicine Houston, Texas 77030

Address requests for reprints to: Dr. Mariel Birnbaumer, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.

Abstract

Human genomic DNA and the HSV tk gene were cotransfected into mouse Ltk^– cells and assayed for the acquisition of a G_s-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 µM hypoxanthine, 1 µM aminopterine, and 10 µM 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 downregulation of the cell authentic prostaglandin E₁ receptor was observed. The vasopressin receptor 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 G_s-coupled human receptor of low abundance and poor accessibility.

FOOTNOTES

This work was supported in part by NIH Grant HD-09581 and Center Grants DK-27685 and HD-07945.

Received for publication October 17, 1989. Revision received November 20, 1989. Accepted for publication November 20, 1989.

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