Homeostatic intracellular-free Ca²⁺ is permissive for Rap1-mediated constitutive activation of α₄ integrins on eosinophils

Although much progress has been made in understanding the molecular mechanisms underlying agonist-induced "inside-out" activation of integrins, little is known about how basal levels of integrin function are maintained. This is particularly important for nonactivated eosinophils, where intermediate activation of α₄β₁ integrin supports recruitment to endothelial cells under flow conditions. Depletion of intracellular Ca²⁺ and pharmacological inhibition of phospholipase C (but not other intracellular signaling molecules, including PI3K, ERK1/2, p38 MAPK, and tyrosine kinase activity) abrogated basal α₄ integrin activity in nonactivated eosinophils. Basal α₄ integrin activation was associated with activation of the small GTPase Rap1, a known regulator of agonist-induced integrin function. Basal Rap activation was dependent upon phospholipase C, but not intracellular Ca²⁺. However, depletion of intracellular Ca²⁺ in CD34⁺ hematopoietic progenitor cells abolished RapV12-mediated induction of α₄ integrin activity. Thus, residual Rap activity or constitutively active Rap activity in Ca²⁺-depleted cells is not sufficient to induce α₄ integrin activation. These data suggest that activation of functional α₄ integrin activity in resting eosinophils is mediated by Rap1 provided that the intracellular-free Ca²⁺ is at a normal homeostatic concentration. The Journal of Immunology, 2008, 180: 5512-5519.