Clathrin-coated vesicles are surrounded by a polyhedral lattice of polymerized clathrin, composed of a 180 KD heavy chain and two light chain subunits. LCa and LCb (25-29 KD). The clathrin lattice is attached to the vesicle membrane by interaction with adaptor molecules, which sequester receptors with particular motifs in their cytoplasmic domains, allowing them to be sorted from other proteins in the membrane. The clathrin heavy chain is known to provide the backbone for the clathrin lattice. While functions for the clathrin light chains have not yet been completely established, it is known that they contain regulatory domains that influence the recruitment, assembly and disassembly of clathrin within the cell. All cell types express both forms of clathrin light chain and in most cells the LCa form is dominant. In contrast, cells with a regulated secretory pathway contain more LCb than LCa, suggesting that LCb may play a role in the specialized clathrin functions in these cells. The proposed studies investigate potential functional differences between the LCa and LCb light chains, using PC12 secretory cells as a model system. A method for changing ratios of clathrin light chains within transfected PC12 cells has been established and the effect of this manipulation of light chain levels on secretory and endocytic processes will be determined. A distinguishing feature of the LCb light chain is its ability to be phosphorylated by a casein kinase, at a site which is not present in the LCa sequence. The role of phosphorylation in clathrin function in secretory cells and in cells exposed to growth factors and hormones which stimulate kinase activity will be investigated. In all cell types, clathrin-coated vesicles are responsible for receptor-mediated endocytosis at the plasma membrane and for the receptor-mediated sorting of lysosomal enzymes from trans-Golgi membranes to a pre-lysosomal compartment. In cells with a regulated secretory pathway, clathrin-coated membranes are involved in the formation of secretory granules and in the rapid uptake of plasma membrane following degranulation. Thus, in addition to their constitutive functions of bringing nutrients into the cell and maintaining the degradative pathway of the cell, clathrin- coated vesicles play an important role in the secretion and mechanism of action of hormones. These studies will provide insight into functional differences between the clathrin light chains and how they influence clathrin-mediated membrane traffic during secretion and hormonal stimulation.
