Eukaryotic cells are equipped with molecular machinery enabling them to internalize and translocate portions of their environment and to deliver these to specific intracellular destinations. One manifestation of this machinery is receptor-mediated endocytosis (RME) along the coated vesicle (CV) pathway. This pathway is of fundamental importance to organisms in a number of processes, including nutrition, cholesterol metabolism, hormone responsiveness, and in passive acquisition of immunity, to name a few. In addition, certain toxins and a number of types of viruses pirate the coated vesicle pathway to gain entry into cells. The proposed studies will investigate the interactions among key molecules involved in the coated vesicle patway using immunocytochemistry at both the light microscope and ultrastructural levels. In addition, a detailed characterization and quantitation of the proteins, antigenic composition and sidedness of isolated plasma membrane, coated vesicle membrane, and coat material (clathrin), during specific ligant challenge, will be carried out. What are the structural relationships among receptors, molecules of the endocytic machinery and regulatory components of endocytosis, such as the calcium dependent regulator protein calmodulin? Are these relationships dynamic? Do distinct ligant challenges result in entry of unique membrane compooents into the CV patway? The cultured human lymphoblastoid cell line Wil2 is a model system of choice for these studies. Wil2 sells have receptor IgM as intergral components of their plasma membrane which when crosslinked by anti-IgM antibodies will stimulate and enter the CV pathway. Other ligands that enter the CV pathway of Wiol2 cells include anti-IgD and concanavalin A. the fundamental importance of RME is evident when we consider the diversity of ligands which are processed along the CV pathway. In addition, since this pathway may have direct clinical valule, for example in delivery of pharmacological agents into specific cell populations, our complete understanding of this process at the molecular level is essential for the design of such applications.