Research ArticleIntegrins

Kindlin-3 disrupts an intersubunit association in the integrin LFA1 to trigger positive feedback activation by Rap1 and talin1

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Science Signaling  08 Jun 2021:
Vol. 14, Issue 686, eabf2184
DOI: 10.1126/scisignal.abf2184

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Linking inside-out and outside-in signaling

Integrins link the cytoskeleton to the extracellular matrix and mediate both outside-in signaling, which transduces cellular responses to ligands, and inside-out signaling, which controls ligand affinity. Talins and kindlins associate with the intracellular domains of integrins and coordinate bidirectional signaling. Using single-molecule imaging of lymphocytes, Kondo et al. found that ligand binding to the integrin LFA1 induced transient interactions with talin1, which recruited kindlin-3, led to a conformational change in LFA1, and stabilized talin1 binding. These effects stimulated the inside-out signaling mediator Rap1, causing LFA1 to adopt an open, high-affinity conformation. In migrating lymphocytes, the association of talin1 and kindlin-3 with LFA1 corresponded to LFA1 adopting the high-affinity conformation in the body of the cell but remaining in a low-affinity conformation at the leading edge. Thus, kindlin-3 mediates positive feedback in the ligand-induced conversion of LFA1 from a low-affinity conformation to a high-affinity conformation.

Abstract

Integrin activation by the intracellular adaptor proteins talin1 and kindlin-3 is essential for lymphocyte adhesion. These adaptors cooperatively control integrin activation through bidirectional (inside-out and outside-in) activation signals. Using single-molecule measurements, we revealed the distinct dynamics of talin1 and kindlin-3 interactions with the integrin LFA1 (αLβ2) and their functions in LFA1 activation and LFA1-mediated adhesion. The kinetics of talin1 binding to the tail of the β2 subunit corresponded to those of LFA1 binding to its ligand ICAM1. ICAM1 binding induced transient interactions between the membrane-proximal cytoplasmic region of the β2 subunit with an N-terminal domain of kindlin-3, leading to disruption of the association between the integrin subunits (the α/β clasp) and unbending of the ectodomains of the α/β heterodimer. These conformational changes promoted high-affinity talin1 binding to the β2 tail that required the talin rod domain and the actomyosin cytoskeleton. Inside-out signaling induced by the GTPase Rap1 did not markedly stabilize the binding of talin1 and kindlin-3 to LFA1. In contrast, ligand-induced outside-in signaling, the stabilization of open LFA1 conformers, or shear force substantially altered the dynamics of talin1 and kindlin-3 association with LFA1 and enhanced both Rap1 and LFA1 activation. In migrating lymphocytes, asymmetrical distribution of talin1 and kindlin-3 correlated with the maturation of LFA1 from a low-affinity conformation at the leading edge to a high-affinity conformation in the adherent mid-body. Our results suggest that kindlin-3 spatiotemporally mediates a positive feedback circuit of LFA1 activation to control dynamic adhesion and migration of lymphocytes.

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