Cellular compartmentation effects in receptor-mediated signal transduction

Cells survey their environment primarily through the engagement of specific cell surface receptor proteins. Ligated receptors participate in signal transduction by initiating intracellular reactions involving heterologous proteins and metobolites. A quantitative understanding of these signaling processes is the key to controlling cell functional responses, including survival and growth, migration, differentiation, and secretion. One issue of particular importance is how the structural organization of the cell can affect these events, by regulating the subcellular localization of signaling molecules. For example, many signaling receptors are internalized into the cell upon activation, leading to their delivery from the plasma membrane to intracellular organelles called endosomes. Receptors in complex with cytosolic proteins almost invariably target membraneassociated molecules, including certain membrane lipids, Ras and related small GTPases, heterotrimeric G-proteins, and Src family tyrosine kinases, to carry out signaling functions. Based on theoretical modeling efforts, it is asserted that the membrane localization of these molecules impacts the organization of signaling interactions in two major ways: 1) it allows amplification of signaling via recruitment of enzymes from the cytosol to the membrane, which is expected to completely synergize with allosteric effects and covalent modifications like phosphorylation, and 2) it allows variations in membrane component concentrations and formation of microdomains to arise based on the chemical interactions between different membrane lipids. The latter would also affect the composition of endosomes relative to the plasma membrane, environments that are physically separated. For these reasons, unstructured models of intracellular signal transduction pathways are likely to be inadequate. Experimental work involved the investigation of how the internalization of epidermal growth factor receptor (EGFR), a prominent receptor tyrosine kinase in mammals, affects the magnitude of signaling through distinct pathways involving phospholipase C (PLC) and the Ras GTPase. For both pathways, the binding and tyrosine phosphorylation of cytosolic proteins (PLC-yl for the PLC pathway and Shc for the Ras pathway) were not affected by receptor compartmentation in endosomes for a given level of total receptor activation. However, at the level of membrane target modification, endosome-associated PLC-yl could not hydrolyze its lipid substrate phosphatidylinositol (4,5)-bisphosphate (PIP,), while the membrane-associated protein Ras was efficiently activated by internal EGFR in the same cell line. This provides evidence that receptors in different compartments may not have access to the same membrane-associated signaling molecules, and that internalization can select for the activation of certain pathways. Thesis Supervisor: Douglas A. Lauffenburger Title: Professor of Chemical Engineering