TY - JOUR
T1 - Frontline Science
T2 - A flexible kink in the transmembrane domain impairs β2 integrin extension and cell arrest from rolling
AU - Sun, Hao
AU - Fan, Zhichao
AU - Gingras, Alexandre R.
AU - Lopez-Ramirez, Miguel A.
AU - Ginsberg, Mark H.
AU - Ley, Klaus
N1 - Funding Information:
This work was supported by grants from the National Institutes of Health, USA (HL078784 to M.H.G. and K.L., R01HL145454 to Z.F.) and WSA postdoctoral fellowships (17POST33660181 to H.S., 16POST31160014 to Z.F.) and the Career Development Award (18CDA34110426 to Z.F.) from the American Heart Association, USA.
Publisher Copyright:
©2019 Society for Leukocyte Biology
PY - 2020/2/1
Y1 - 2020/2/1
N2 - 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 high-affinity (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 Mn2+. We conclude that the TMD proline mutation severely impairs β2 integrin extension, cell arrest, and early spreading.
AB - 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 high-affinity (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 Mn2+. We conclude that the TMD proline mutation severely impairs β2 integrin extension, cell arrest, and early spreading.
KW - activation
KW - adhesion
KW - affinity
KW - spreading
KW - talin
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U2 - 10.1002/JLB.1HI0219-073RR
DO - 10.1002/JLB.1HI0219-073RR
M3 - Article
C2 - 31475386
AN - SCOPUS:85071623191
SN - 0741-5400
VL - 107
SP - 175
EP - 183
JO - Journal of Leukocyte Biology
JF - Journal of Leukocyte Biology
IS - 2
ER -