Endocytosis in mammalian cells permits the efficient uptake of extracellular macromolecules which bind to specific plasma membrane receptors. Typically, ligand-receptor complexes dissociate shortly after internalization, facilitating the recycling of free receptors for reuse and the transport of ligand to lysosomes for degradation. This sorting of receptor from ligand occurs in endosomes, prelysosomal vesicles whose acidic pH facilitates the disruption of many ligand-receptor complexes. In some cases, however, the complex fails to dissociate in endosomes, causing transport of both ligand and receptor to lysosomes. While endosomes clearly play a central role in targeting receptors to their appropriate destinations, the mechanisms controlling these transport events are poorly understood. This proposal is aimed at elucidating some of the molecular determinants of membrane transport. As in the previous grant period, study of the mouse macrophage IgG Fc receptor (FcR) will be emphasized. This receptor, which recycles from endosomes when bound to monovalent ligand, is redirected to lysosomes by multivalent ligands. Using specific anti-FcR antibodies, biochemical, and immunocytochemical techniques, the relationship between ligand valency, receptor clustering, and the modulation of receptor transport will be investigated in detail. Endosome subpopulations involved in FcR recycling or transport to lysosomes will also be analyzed following isolation by free flow electrophoresis and immunoadsorption. Using an FcR cDNA clone, we will identify receptor domains involved in controlling intracellular transport as well as other domains involved in mediating the FcR's transmembrane signaling and ion channel functions. We will also compare macrophage FcR structure with other related receptors. We also plan to study the signals controlling transport to lysosomes in the biosynthetic pathway. Using organelle specific antibodies, the biogenesis, processing, and transport of lysosomal membrane proteins will be directly compared with plasma membrane proteins (e.g. FcR). Using a cDNA clone for one of these lysosomal proteins, structural, mutagenesis, and expression studies will help define the molecular determinants governing the selective transport of newly synthesized membrane proteins to lysosomes.
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