The normal function of kidney cells requires the regulated insertion and retrieval of proteins and lipids at the plasma membrane. For example, in principal cells water channels are moved to and from the apical membrane in response to the hormone vasopressin. Because perturbations in endocytic traffic can lead to significant kidney and heart disease (e.g., Liddle's syndrome or familial hypercholesterolemia) it is important to understand how these endocytic pathways are distributed in cells and how they are regulated. One group of proteins that governs endocytosis in kidney cells is the Rho family of small GTPases. This proposal has three aims. The first is to test the hypothesis that the Rho GTPases RhoA and Raci regulate, via activation of downstream effector pathways, distinct steps in receptor-mediated endocytosis and also modulate clathrin-independent endocytosis. Although RhoA and Raci regulate clathrin-dependent internalization at the apical and basolateral pole of polarized kidney cells, the actual step in clathrin-coated vesicle formation modulated by these GTPases is unknown. Quantitative ultrastructural analysis will be used to identify the actual step(s) involved in endocytosis at the apical and basolateral poles of Madin-Darby canine kidney cells. Because the molecular machinery that governs clathrin-independent endocytosis is poorly characterized it will be determined whether this form of endocytosis is modulated by RhoA and Raci. Lastly, it will be defined whether clathrin-dependent and -independent endocytosis is modulated by the effector proteins Rho kinase alpha, p21-activated kinase 1, and partner of Rac1. RhoB is a RhoA subfamily member that is found on endosomes, but whose function remains elusive. The hypothesis that RhoB is associated in part with early endosomes and regulates traffic through these compartments will be tested in the second specific aim. In vivo and in vitro assays will be used to measure the effects of wild-type and mutant RhoB expression on endocytosis, and early endosome fusion, maturation, and production of transcytotic vesicles. The final goal of this proposal is to use co-immunoprecipitations and affinity chromatography to identify novel downstream effectors of RhoB function. It is hypothesized that these novel effectors will regulate endocytic traffic downstream of RhoB.