BINDING AND SIGNAL TRANSDUCTION OF THE CLONED VASCULAR ANGIOTENSIN-II (AT1A) RECEPTOR CDNA STABLY EXPRESSED IN CHINESE-HAMSTER OVARY CELLS

The vascular angiotensin (A) II receptor cDNA (AT1a) was transfected into Chinese hamster ovary (CHO) cells to generate the stable cell line CHO-AT1a. This cell line was used to investigate the binding and signal transduction properties of the cloned vascular AT1 receptor. Specific binding of sarcosine1-[I-125]tyrosine4-isoleucine8-AII ([I-125 ]SI-AII) to CHO-AT1a membranes reached equilibrium after 1 h at 25-degrees-C and was consistently greater than 95% of total binding. Saturation binding analyses demonstrated [I-125] SI-AII bound to a saturable population of sites on membranes with an equilibrium dissociation constant (K(D)) of 0.7 nM and a binding site maximum of 1.2 pmol/mg protein. [I-125] SI-AII binding to CHO cells was inhibited by the following compounds with a rank order of potency of SI-AII>AII>losartan>AI> >PD 123,177. AII (1 muM) treatment of CHO-AT1a cells caused an increase in inositol phosphates and intracellular calcium relative to basal levels. These responses were blocked by losartan but not by PD 123,177. AII (1 muM) did not effect adenylate cyclase activity in CHO-AT1a cells, whereas the agonist inhibited adenylate cyclase activity in rat liver cell membranes. These effects were blocked by 10 muM losartan. These results indicate that CHO-AT1a Cells express functional AT1a receptors which stimulate phospholipase C activity but not adenylate cyclase activity. CHO-AT1a cells should provide a useful model for studies of AT1a receptor domains which are critical to signaling pathways.