The composition and function of the plasma membrane is maintained by a complex intracellular traffic moving cell surface glycoproteins between organelles. This requires the recognition and sorting of different classes of proteins, not only during biosynthesis, but also during redistributive processes, such as the internalization and recycling of receptors during receptor-mediated endocytosis, or during the down-regulation of hormone receptors. Disease can result form the failure of any one of these processes, or from its subversion for use by a pathogen such as a virus. Experiments described in this application are designed to reveal molecular details of the cellular mechanisms that control the intracellular transport of a major class of cell surface glycoproteins. Through genetic engineering, gain-of-function mutants have been produced that are sorted into pathways from which they were previously excluded. This allows features of proteins important for intracellular sorting events to be distinguished from those important for the structure of the protein as a whole. Six experiments are proposed (1) to determine the number and relationship of independent mechanisms for recognition of proteins by coated pits during endocytosis; (2) to identify the proteins controlling sorting in the endocytic pathway; (3) to identify the proteins responsible for sorting transmembrane glycoproteins in polarized epithelial cells; (4) to determine the role of the transmembrane domain in the intracellular transport of glycoproteins. Mutants made previously will be used to establish in vitro assays for internalization and recycling for the purpose of identifying essential cellular components of these events. Permanent cell lines expressing these mutants will be used to determine which proteins compete for recognition by components of coated pits. Genetic engineering will be used to introduce progressively smaller mutations into glycoproteins to identify the amino acid sequences responsible for interesting changes in their intracellular traffic. The secondary, tertiary and quarternary structure of features important for sorting will be investigated. Synthetic peptides that mimic transport signals will be used to identify cellular proteins that interact with them in experiments employing chemical cross-linking, direct affinity chromatography, or anti-idiotype antibodies.
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