Vital cellular processes such as nutrient uptake, receptor signaling, and cell migration are controlled by a balance between cell surface receptor internalization and recycling. Clathrin-mediated endocytosis is the major mechanism of receptor internalization in which cargo-enriched endocytic vesicles form at, and are released from, the plasma membrane before maturing into early endosomes. The receptors can then be sorted into fast and slow recycling pathways that replenish receptor levels at the cell surface. A major fast recycling pathway is controlled by the small GTPase Rab4a, which plays a central role in cell migration and cancer cell invasion through regulation of integrin αvβ3 recycling.
Recent studies have discovered a family of clathrin-coated vesicle proteins, known as adaptin-ear-binding coat-associated proteins (NECAPs), that consists of two family members, NECAP1 and NECAP2. NECAP1 functions in endocytosis and cooperates with the clathrin adaptor AP-2 to control endocytic vesicle size, number and cargo. Importantly, NECAP2 did not rescue the knock-down phenotype of NECAP1, revealing that NECAPs are not functionally redundant. The studies described in this dissertation show that NECAP2 controls the fast recycling of epidermal growth factor receptor and transferrin receptor. Furthermore, NECAP2 specifically functions in Rab4a-mediated fast recycling together with the clathrin adaptor AP-1. In contrast, NECAP2 has no effect on AP-1-mediated transport from the Golgi or on other Rab4a-dependent sorting events that utilize additional clathrin adaptors and effector proteins. Thus, NECAP2 regulates a sub-route within the Rab4a recycling pathway and, in fact, is the first protein known to date to show this level of specificity. NECAP2 knock-down revealed that this sub-route controls cell migration and cancer cell invasion. Specifically, NECAP2 knock-down impaired the recycling of integrin αvβ3 to the cell surface, leading to decreased Rac1 activation and integrin αvβ3-dependent persistent cell migration. NECAP2 depletion also alleviated the inhibitory effect on integrin α5β1 recycling, switching cells to integrin α5β1-dependent cell migration. Notably, loss of NECAP2 function in breast cancer cells inhibited invasive migration in a 3D invasion model system. Therefore, the NECAP2 pathway may provide a therapeutic target, in particular for the 25% of breast cancers with amplification of Rab4a.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/32975 |
| Date | 24 October 2018 |
| Creators | Chamberland, John |
| Contributors | Ritter, Brigitte |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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