Angiotensin II (Ang II) is an octapeptide with varying effects in different organ systems. In addition to vasoconstrictive effects in the kidney, Ang II greatly influences proximal tubule function, stimulating Na+, HCO3, and fluid reabsorption. Unlike most G-protein-coupled receptors, Ang H receptors have been identified in a polar distribution in proximal tubule epithelium at brush border and basolateral membranes. We have developed a model of proximal tubule epithelium using LLCPKC14 cells, an LLCPK clone without endogenous Ang H receptors. LLCPKC14 cells transfected with a rabbit Ang H receptor transcript express Ang II receptors at apical and basolateral membranes. In this model, Ang H receptors in either domain may display different characteristics, e.g. rates of internalization. Furthermore, apical Ang Il receptors selectively may stimulate a calcium-independent phospholipase A2 (PLA2) in contrast to basolateral Ang II receptors. Yet, the factors influencing these receptor properties as well as the determinants for membrane targeting have not yet been defined. Site-directed mutagenesis will be employed to alter regions in the rabbit Ang H receptor transcript. LLCPKC14 cells transfected with these Ang II receptor mutants will be assayed for apical vs. basolateral Ang II binding, receptor-mediated endocytosis and recycling at either surface, and signal transduction including PLA2 activity, Ang II-mediated tyrosine phosphorylation, and phosphoinositide hydrolysis. Pulse-chase studies, photoaffinity labelling and immunoblotting will be employed to determine what aspects of the Ang H receptor sequence are important for receptor trafficking and Ang II receptor-protein interactions with G-proteins and/or proteins involved in ligand-induced internalization. Finally, cells expressing Ang II receptor mutants will be assayed for Na+ transport to determine the effects of these mutations on an important physiologic process mediated by Ang II in proximal tubule epithelium. It is important to understand factors which affect proximal tubule Ang II receptors given the extent of hypertension and renal disease in our present population. Advancing our knowledge of Ang II and how it affects proximal tubule function may enhance our understanding of kidney diseases affected by Ang II, e.g. hypertensive nephropathy.
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