Characterization and Signaling of the AT4 Receptor in Human Proximal Tubule Epithelial (HK-2) Cells

Abstract

Abstract. 125I-divalinal-angiotensin IV (metabolically resistant analog of angiotensin IV) was used as a receptor ligand to identify the expression and properties of the angiotensin AT4 receptor in epithelial HK-2 cells (an immortalized cell line derived from adult human proximal tubules). Saturation binding isotherms revealed that HK-2 cells contain a saturable 125I-divalinal-angiotensin IV binding site with an affinity of 3 nmol/L and a density of 508 fmol/mg protein. An analysis of ligand specificity showed that only angiotensin AT4 receptor ligands (angiotensin IV and divalinal-angiotensin IV) competed with both a high- and low-affinity binding site. GTPγS and dithiothreitol did not affect 125I-Ang IV or 125I-divalinal-Ang IV binding, suggesting that the AT4 receptor was not G-protein coupled and did not require sulfhydryl bonds for receptor affinity. Activation of the AT4 receptor caused a complex concentration-dependent rise in [Ca2+]i, an elevation in [Na+]i, and increased mitogen-activated protein kinase activity. These results suggest that human proximal tubule epithelial cells contain functional AT4 receptors that are pharmacologically similar to the AT4 receptor described in more distal segments of the nephron. Furthermore, the AT4 receptor uses several intracellular signaling pathways to convey information.