Talin-1 and Kindlin-3 Regulate α4β1 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 α4β1 integrin affinity and consequent adhesive events. Affinity upregulation of α4β1 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 α4β1 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 α4β1 and kindlin-3 in microridges may explain why kindlin-3 rapidly associates with α4β1 after G protein-coupled receptor signaling and contributes to adhesion strengthening. Talin-1 contributed to α4β1-dependent chemotaxis, suggesting that it participates in a later stage of the leukocyte adhesion cascade when the leukocyte cytoskeleton undergoes dramatic rearrangement.