Nitric oxide/cGMP pathway signaling actively down-regulates α4β1-integrin affinity: an unexpected mechanism for inducing cell de-adhesion

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.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.Integrins are ubiquitous cell adhesion molecules that play an essential role in the regulation of leukocyte traffic, stem cell mobilization and homing, immune responses, development, hemostasis, and cancer [1-3]. On the cell surface at rest, a variety of integrin exhibit a non-adhesive inactive state and multiple signaling cascades are capable of rapidly and reversibly regulating integrin-dependent cell adhesion. Typically, this regulation is achieved without altering the integrin expression level. Conformational changes within the molecule, together with a spatial reorganization of integrins, are responsible for the rapid modulation of cell adhesion [1,4-6]. Understanding signaling pathways that regulate activation and, especially, inactivation of integrin-mediated cell adhesion is crucial, as integrins a