Integrins are a/? heterodimeric cell adhesion receptors that play essential roles in the developing and adult kidney, through tightly regulated metal-dependent interactions with the extracellular matrix (ECM) that modulate cell migration, proliferation, differentiation, matrix deposition and remodeling. Binding of integrins to ECM ligands requires an activation step initiated upon binding of cytoskeletal talin or kindlin to the ?-subunit cytoplasmic domain. This interaction breaks an intracellular a/? salt bridge in the cytoplasmic domains, triggering a conformational wave that travels through the a/? transmembrane (TM) and lower leg domains to activate the ligand-binding head domain. Binding of ECM ligands then triggers structural changes in the integrin that travel in the opposite direction to the cell interior, modifying cell behavior. Crystal structures of unliganded ?TM-aV?3 and ?TM-aII?3 ectodomains and structures of these ectodomains bound to short peptide ligands- diffused into the preformed protein crystals- identified an unexpected bent conformation of the ectodomain and elucidated the structural basis of metal ion dependency for ligand binding. But as these structures are lacking critical exofacial residues, as well as the TM and cytoplasmic domains, and were unoccupied by macromolecular ECM ligands de novo, a comprehensive understanding of bidirectional integrin signaling at the atomic level remains elusive. In preliminary studies, we have determined the crystal structure of 1TM-aV?3, which encodes the complete sequence of the ectodomain plus an a/? TM fragment. Second, we produced the first electron cryomicroscopy structure of a full-length integrin in a bent conformation with resolved features of the TM domains. Third, we obtained diffraction quality crystals of the integrin in complex with a macromolecular ligand and with an activating mAb. Fourth, we demonstrate that a/? TM-cytoplasmic domains expressed in bacteria can form heterodimers in the absence of the ectodomain, through one of two TM interfaces, one of which is found in the inactive full-length integrin. Fifth, we show the feasibility of assessing integrin conformational states in live mammalian cells by fluorescence lifetime imaging microscopy. These findings form the basis of new hypotheses that are tested in three specific aims. The results derived from the proposed studies should answer central questions in the biology of integrin-mediated cell adhesion and define the structural basis of bidirectional signaling.

Public Health Relevance

Formation and maintenance of kidney structure and function critically depends on the normal function of a family of cell adhesion receptors named integrins. Kidney agenesis, proteinuria, harmful inflammation and graft rejection are manifestations of abnormal integrin function. Elucidating how integrins are regulated at an atomic level is essential in understanding their biologic role, information that could be harnessed in devising new therapeutics.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Cellular and Molecular Biology of the Kidney Study Section (CMBK)
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Ketchum, Christian J
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Massachusetts General Hospital
United States
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