Development and characterization of a mouse cell line expressing the human V2 vasopressin receptor gene.
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 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 Gs-coupled human receptor of low abundance and poor accessibility.
Birnbaumer
M
M
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Hinrichs
V
V
Themmen
A P
AP
Themen
A P
AP
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D002460:Cell Line; D002461:Cell Line, Transformed; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure*; D015870:Gene Expression; D008024:Ligands; D051379:Mice; D010641:Phenotype; D011945:Receptors, Angiotensin / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D017483:Receptors, Vasopressin; D014162:Transfection; D014170:Transformation, Genetic
D008024:Ligands; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D001127:Arginine Vasopressin; D000262:Adenylyl Cyclases