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Here, we investigated the biologic effect of introducing a proline mutation in the \u03b22 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 \u03b22 integrin alpha chains, \u03b1L, \u03b1M, \u03b1X, and \u03b1D, all contain an inserted (I) domain with homology to von Willebrand factor A domain. \u03b22 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 \u03b22 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of \u03b22 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 \u03b22, limiting the appearance of extended high-affinity (E+H+) \u03b22. 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 \u03b22 integrin extension, cell arrest, and early spreading.<\/jats:p>","DOI":"10.1002\/jlb.1hi0219-073rr","type":"journal-article","created":{"date-parts":[[2019,9,2]],"date-time":"2019-09-02T06:24:31Z","timestamp":1567405471000},"page":"175-183","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Frontline Science: A flexible kink in the transmembrane domain impairs \u03b22 integrin extension and cell arrest from rolling"],"prefix":"10.1093","volume":"107","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4790-8847","authenticated-orcid":false,"given":"Hao","family":"Sun","sequence":"first","affiliation":[{"name":"Department of Medicine, University of California, San Diego , La Jolla, California, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhichao","family":"Fan","sequence":"additional","affiliation":[{"name":"Division of Inflammation Biology, La Jolla Institute for Immunology , La Jolla, California, USA"},{"name":"Department of Immunology, University of Connecticut Health Center , Farmington, Connecticut, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandre R.","family":"Gingras","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of California, San Diego , La Jolla, California, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miguel A.","family":"Lopez-Ramirez","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of California, San Diego , La Jolla, California, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark H.","family":"Ginsberg","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of California, San Diego , La Jolla, California, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Klaus","family":"Ley","sequence":"additional","affiliation":[{"name":"Division of Inflammation Biology, La Jolla Institute for Immunology , La Jolla, California, USA"},{"name":"Department of Bioengineering, University of California, San Diego , La Jolla, California, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2019,9,1]]},"reference":[{"issue":"1","key":"2023012312512305300_jlb10480-bib-0001","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1182\/blood.V88.1.146.146","article-title":"Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b\/CD18","volume":"88","author":"Diacovo","year":"1996","journal-title":"Blood"},{"key":"2023012312512305300_jlb10480-bib-0002","article-title":"Leukocyte arrest: biomechanics and molecular mechanisms of beta2 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