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Outside-In Signal Transmission by Conformational Changes in Integrin Mac-1¹

Craig T. Lefort,^2* Young-Min Hyun,^2* Joanne B. Schultz, Foon-Yee Law,^* Richard E. Waugh, Philip A. Knauf, and Minsoo Kim^3*

^*Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, Department of Biomedical Engineering, and Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY 14642

Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils. Mac-1 conformational activation induced by ligand occupancy or activating Ab binding, but not integrin clustering, triggered similar patterns of intracellular protein tyrosine phosphorylation, including Akt phosphorylation, and inhibited spontaneous neutrophil apoptosis, indicating that global conformational changes are critical for Mac-1-dependent outside-in signal transduction. In neutrophils and myeloid K562 cells, ligand ICAM-1 or activating Ab binding promoted switchblade-like extension of the Mac-1 extracellular domain and separation of the _M and β₂ subunit cytoplasmic tails, two structural hallmarks of integrin activation. These data suggest the primacy of global conformational changes in the generation of Mac-1 outside-in signals.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants IH HL087088 (to M.K.) and HL18208 (to M.K. and R.E.W.).

² These authors contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Minsoo Kim, Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, Rochester, NY 14642. E-mail address: minsoo_kim{at}urmc.rochester.edu

⁴ Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; PVP, polyvinylpyrrolidone; ORB, octadecylrhodamine B; FP, fluorescent protein; CFP, cyan FP; mCFP, monomeric CFP; YFP, yellow FP; mYFP, monomeric YFP; EM, electron microscopy; FRET, fluorescence resonance energy transfer.

⁵ The online version of this article contains supplemental material.