title: Nitric oxide/cGMP pathway signaling actively down-regulates α4β1-integrin affinity: an unexpected mechanism for inducing cell de-adhesion.
abstract: BACKGROUND
  Integrin activation in response to inside-out signaling serves as the basis for rapid leukocyte arrest on endothelium, migration, and mobilization of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule, which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic progenitors, stem cells, hematopoietic cancer cells, and others. VLA-4 conformation is rapidly up-regulated by inside-out signaling through Gαi-coupled GPCRs and down-regulated by Gαs-coupled GPCRs. However, other signaling pathways, which include nitric oxide-dependent signaling, have been implicated in the regulation of cell adhesion. The goal of the current report was to study the effect of nitric oxide/cGMP signaling pathway on VLA-4 conformational regulation.
  
  
  RESULTS
  Using fluorescent ligand binding to evaluate the integrin activation state on live cells in real-time, we show that several small molecules, which specifically modulate nitric oxide/cGMP signaling pathway, as well as a cell permeable cGMP analog, can rapidly down-modulate binding of a VLA-4 specific ligand on cells pre-activated through three Gαi-coupled receptors: wild type CXCR4, CXCR2 (IL-8RB), and a non-desensitizing mutant of formyl peptide receptor (FPR ΔST). Upon signaling, we detected rapid changes in the ligand dissociation rate. The dissociation rate after inside-out integrin de-activation was similar to the rate for resting cells. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by nitric oxide had a statistically significant effect on real-time cell aggregation.
  
  
  CONCLUSIONS
  We conclude that nitric oxide/cGMP signaling pathway can rapidly down-modulate the affinity state of the VLA-4 binding pocket, especially under the condition of sustained Gαi-coupled GPCR signaling, generated by a non-desensitizing receptor mutant. This suggests a fundamental role of this pathway in de-activation of integrin-dependent cell adhesion.
authors.affiliation: Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA. achigaev@salud.unm.edu
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chigaev
authors.forename: Yelena
authors.initials: Y
authors.lastname: Smagley
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
mesh_terms: D002448:Cell Adhesion / Q000276:immunology; D002450:Cell Communication / Q000276:immunology; D002465:Cell Movement / Q000276:immunology; D006152:Cyclic GMP / Q000378:metabolism*; D015536:Down-Regulation / Q000276:immunology; D004730:Endothelium, Vascular / Q000276:immunology; D005456:Fluorescent Dyes; D006801:Humans; D039041:Integrin alpha4beta1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009000:Monocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology; D020298:U937 Cells
chemical_list: D005456:Fluorescent Dyes; D039041:Integrin alpha4beta1; D009569:Nitric Oxide; D006152:Cyclic GMP