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Homeostatic Intracellular-Free Ca²⁺ Is Permissive for Rap1-Mediated Constitutive Activation of ₄ Integrins on Eosinophils¹

Laurien H. Ulfman^2,*, Vera M. Kamp^*, Corneli W. van Aalst^*, Liesbeth P. Verhagen, Marjolein E. Sanders, Kris A. Reedquist, Miranda Buitenhuis and Leo Koenderman^*

^* Department of Respiratory Medicine and Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; and Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, The Netherlands

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 ₄β₁ 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 ₄ integrin activity in nonactivated eosinophils. Basal ₄ 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 ₄ integrin activity. Thus, residual Rap activity or constitutively active Rap activity in Ca²⁺-depleted cells is not sufficient to induce ₄ integrin activation. These data suggest that activation of functional ₄ integrin activity in resting eosinophils is mediated by Rap1 provided that the intracellular-free Ca²⁺ is at a normal homeostatic concentration.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Netherlands Organisation for Scientific Research Grants NWO nr 916.036.051 (to L.H.U.) and UU2005-3659 (to M.B.).

² Address correspondence and reprint requests to Dr. Laurien H. Ulfman, Department of Experimental Pulmonary Diseases, University Medical Center, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail address: L.Ulfman{at}umcutrecht.nl

³ Abbreviations used in this paper: GEF, guanine nucleotide exchange factor; PLC, phospholipase C; EGFP, enhanced GFP; DAG, diacylglycerol; CalDAG-GEF, calcium DAG-GEF; PKC, protein kinase C; [Ca²⁺]_i, intracellular Ca²⁺ concentration.