Frontline Science: A flexible kink in the transmembrane domain impairs β2 integrin extension and cell arrest from rolling

Abstract

β2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the β2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The β2 integrin alpha chains, αL, αM, αX, and αD, all contain an inserted (I) domain with homology to von Willebrand factor A domain. β2 activation was monitored in a homogenous binding assay of 2 reporter monoclonal antibodies: KIM127 reporting extension (E +) and mAb24 reporting the highaffinity (H +) conformation of the β2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of β2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of β2, limiting the appearance of extended high-affinity (E + H +) β2. This also resulted in a defect in early spreading (3 min after arrest), which could be overcome by forced integrin activation using Mn 2+. We conclude that the TMD proline mutation severely impairs β2 integrin extension, cell arrest, and early spreading.

β2 Integrin Signaling Cascade in Neutrophils: More Than a Single Function

“…Although recent technology can identify proteins associating with integrins ( 91 ), the heterogeneity among the different β integrin subunits as well as their diverse function in different cell types averts a general statement about integrin signaling and function. Although the proteins that can directly associate with β 2 integrins or regulate their affinity and function have been reviewed ( 92 ), their contribution to downstream signaling of β 2 integrins in the context of neutrophils is limited ( 38 , 93 – 97 ). Examples of proteins interacting directly with the cytoplasmic tail of the β 2 subunit and having a known function in neutrophils include Cytohesin-1 ( 98 , 99 ), SYK ( 100 , 101 ), and Filamin A ( 97 ) as we will also discuss below.…”

The Activation and Regulation of β2 Integrins in Phagocytes and Phagocytosis

“…If the β2 TMD is mutated (β2 L697P, right), talin-1 will still bind the intracellular domain and align the beginning of the TMD to an angle >25°, but the kink prevents this from being transmitted to the extracellular domain. The integrin stays bent, but the headpiece opens ( 60 ). Talin head domain (THD).…”

“…This alters the membrane-crossing angle of β TMD, thereby disrupting the outer membrane association of α and β TMDs, which is important for αIIbβ3 integrin activation ( 77 ). Studies showed that these transmitting conformation changes across the cell membrane are also important for both β7 ( 78 ) and β2 integrins ( 60 ). Blocking TMD topology transmission by introducing a TMD kink (L697P mutation) impairs chemokine-induced cell adhesion and β2 integrin extension, but not chemokine-induced β2 integrin high‐affinity confirmation and manganese-induced cell spreading ( 60 ).…”

“…Studies showed that these transmitting conformation changes across the cell membrane are also important for both β7 ( 78 ) and β2 integrins ( 60 ). Blocking TMD topology transmission by introducing a TMD kink (L697P mutation) impairs chemokine-induced cell adhesion and β2 integrin extension, but not chemokine-induced β2 integrin high‐affinity confirmation and manganese-induced cell spreading ( 60 ). As expected, talin-1 knockout cells showed a dramatic defect in chemokine-induced β2 integrin extension and high‐affinity confirmation as well as manganese-induced cell spreading ( Figure 1B ).…”

Super-STORM: Molecular Modeling to Achieve Single-molecule Localization with STORM Microscopy

“…The assembly of the microfluidic devices used in this study and the coating of coverslips with recombinant human P-selectin-Fc and ICAM-1-Fc has been described previously ( Fan et al., 2016 , Sundd et al., 2012 , Sundd et al., 2011 , Sundd et al., 2010 , Sun et al., 2020 ). Briefly, cleaned coverslips were coated with P-selectin-Fc (2 μg ml-1) and ICAM-1-Fc (10 μg ml-1) for 2 hours and then blocked for 1 hour with casein (1%) at 22-24°C.…”