Abstractβ2 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.

01 Jan 2009·Journal of Biological ChemistryQ2 · BIOLOGY

Disruption of the Integrin αLβ2 Transmembrane Domain Interface by β2 Thr-686 Mutation Activates αLβ2 and Promotes Micro-clustering of the αL Subunits

Q2 · BIOLOGY

Article

Author: Vararattanavech, Ardcharaporn ; Tan, Suet-Mien ; Torres, Jaume ; Lin, Xin

Integrins are type I heterodimeric cell adhesion molecules that mediate a wide array of biological processes. Integrin bidirectional signaling allows communication between the cell interior with its microenvironment. The integrin transmembrane domains (TMs) are the transducers of activation signal that is relayed from the cytoplasmic domains to the distal ligand binding site located in the ectodomain of the integrin and vice versa. In this study, we showed that the disruption of the alphaLbeta2 TMs by mutation of a key interface residue Thr-686 in the beta2 TM promoted alphaLbeta2 activation with ICAMs binding properties that are reminiscent of an intermediate affinity receptor. The activated alphaLbeta2 TM mutants, however, showed minimal reactivity with the reporter mAb KIM127 that recognizes a highly extended alphaLbeta2. Two models of alphaLbeta2 TM interaction were proposed previously. One with GXXXG-type interaction, and another that is based on TM cysteine-scanning analyses. Our data are consistent with a GXXXG-type interaction of the alphaLbeta2 TMs. Finally, we observed by FRET analyses that perturbation of the alphaLbeta2 TMs by beta2 Thr-686 mutation facilitated alphaL micro-cluster formation. This was diminished by linking the alphaLbeta2 TMs with a disulfide bond, which served to clasp the TMs. These data suggest that disruption of the TM interface changes alphaLbeta2 ligand binding affinity, and it may contribute to alphaL micro-cluster formation.

01 Dec 2007·Scandinavian Journal of ImmunologyQ4 · MEDICINE

TNF‐α is Secreted by Monocytes in Transit to become Macrophages, but not by Peripheral Blood Monocytes, following OK‐432 (Lyophilized S. pyogenes) Stimulation

Q4 · MEDICINE

Article

Author: J. Olofsson ; C. Olsnes ; H. Stavang ; H. J. Aarstad

AbstractOK‐432, penicillin‐killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied proinflammatory interleukin (IL) secretion following OK‐432 stimulation of total blood, peripheral blood mononuclear cell (PBMC) and purified monocytes in vitro. OK‐432 stimulation of purified monocytes gave IL‐1β, IL‐1RA, IL‐6, IL‐12p40 and tumour necrosis factor (TNF)‐α response. OK‐432 stimulation of cells within blood did, however, not yield TNF‐α secretion. When PBMC or monocytes were cultured in low‐attachment wells a decreased IL secretion was observed compared to adherent cells. Inhibition of Syk kinase with piceatannol, only at high, non‐specific doses, but not PI3 kinase inhibition with LY294002 or Wortmannin, decreased monocyte IL response to OK‐432. This shows that β1–3‐integrin receptor function is not necessary for monocyte OK‐432‐stimulated TNF‐α secretion. Direct blockage of the β2‐integrin (CD18) receptor by anti‐CD18 antibody was also unable to prevent the stimulating effects of OK‐432 in human monocytes. On the other hand, Syk phosphorylation is elevated upon adherence of monocytes and this is further increased by OK‐432 stimulation, as shown by Western blot. The Fc‐receptor was also ruled out as a main receptor of the OK‐432 monocyte response. In conclusion, TNF‐α secretion is only found in monocytes removed from blood. This TNF‐α secretion is not mediated through the β1–3‐integrin receptors. OK‐432 may act as a target‐seeking substance whereby only monocytes adhered, e.g. to a tumour cell, become cytotoxic in part explaining why OK‐432 is well suited as a cancer treatment drug.

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