title: Talin-1 and kindlin-3 regulate alpha4beta1 integrin-mediated adhesion stabilization, but not G protein-coupled receptor-induced affinity upregulation.
abstract: Chemokine/chemoattractant G protein-coupled receptors trigger an inside-out signaling network that rapidly activates integrins, a key step in inflammatory leukocyte recruitment. Integrins mediate leukocyte arrest and adhesion to endothelium through multivalent binding, and they transmit outside-in signals to stabilize adhesion and coordinate cell spreading and migration. In the present study, we used RNA interference in the U937 monocytic cell line to investigate the role of talin-1, kindlin-3, and α-actinin-1 in the fMLF- and SDF-1α-induced upregulation of α(4)β(1) integrin affinity and consequent adhesive events. Affinity upregulation of α(4)β(1) integrin was not impaired by small interfering RNA knockdown of talin-1, kindlin-3, or α-actinin-1. Only kindlin-3 knockdown increased flow-induced detachment from VCAM-1-coated surfaces in response to fluid flow, whereas knockdown of either talin-1 or kindlin-3 increased detachment from ICAM-1-coated surfaces. Biochemical analyses revealed that α(4)β(1) expression was highly enriched in U937 cell microridges and murine lymphocyte microvilli. Kindlin-3 was present throughout the cell, whereas talin-1 was largely excluded from microridges/microvilli. The subcellular colocalization of α(4)β(1) and kindlin-3 in microridges may explain why kindlin-3 rapidly associates with α(4)β(1) after G protein-coupled receptor signaling and contributes to adhesion strengthening. Talin-1 contributed to α(4)β(1)-dependent chemotaxis, suggesting that it participates in a later stage of the leukocyte adhesion cascade when the leukocyte cytoskeleton undergoes dramatic rearrangement.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L7, Canada. sharon.hyduk@utoronto.ca
authors.forename: Sharon J
authors.initials: SJ
authors.lastname: Hyduk
authors.forename: Jacob
authors.initials: J
authors.lastname: Rullo
authors.forename: Adrianet Puig
authors.initials: AP
authors.lastname: Cano
authors.forename: Haiyan
authors.initials: H
authors.lastname: Xiao
authors.forename: Mian
authors.initials: M
authors.lastname: Chen
authors.forename: Markus
authors.initials: M
authors.lastname: Moser
authors.forename: Myron I
authors.initials: MI
authors.lastname: Cybulsky
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion / Q000276:immunology; D002634:Chemotaxis, Leukocyte / Q000276:immunology*; D006801:Humans; D047468:Immunoprecipitation; D039041:Integrin alpha4beta1 / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008855:Microscopy, Electron, Scanning; D009363:Neoplasm Proteins / Q000276:immunology / Q000378:metabolism*; D034622:RNA Interference; D043562:Receptors, G-Protein-Coupled / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology*; D016608:Talin / Q000276:immunology / Q000378:metabolism*; D020298:U937 Cells; D015854:Up-Regulation
chemical_list: C477322:FERMT3 protein, human; D039041:Integrin alpha4beta1; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D043562:Receptors, G-Protein-Coupled; C518832:TLN1 protein, human; D016608:Talin