Characterization and signaling of the AT(4) receptor in human proximal tubule epithelial (HK-2) cells.

(125)I-divalinal-angiotensin IV (metabolically resistant analog of angiotensin IV) was used as a receptor ligand to identify the expression and properties of the angiotensin AT(4) 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 (125)I-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 AT(4) receptor ligands (angiotensin IV and divalinal-angiotensin IV) competed with both a high- and low-affinity binding site. GTPgammaS and dithiothreitol did not affect (125)I-Ang IV or (125)I-divalinal-Ang IV binding, suggesting that the AT(4) receptor was not G-protein coupled and did not require sulfhydryl bonds for receptor affinity. Activation of the AT(4) receptor caused a complex concentration-dependent rise in [Ca(2+)](i), an elevation in [Na(+)](i), and increased mitogen-activated protein kinase activity. These results suggest that human proximal tubule epithelial cells contain functional AT(4) receptors that are pharmacologically similar to the AT(4) receptor described in more distal segments of the nephron. Furthermore, the AT(4) receptor uses several intracellular signaling pathways to convey information.