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(Journal of Leukocyte Biology. 2003;73:243-252.)
© 2003 by Society for Leukocyte Biology

Differential recruitment of {alpha}2ß1 and {alpha}4ß1 integrins to lipid rafts in Jurkat T lymphocytes exposed to collagen type IV and fibronectin

Brian J. Holleran*, Élie Barbar*, Marcel D. Payet{dagger} and Gilles Dupuis*,{ddagger}

* Signal Transduction Laboratory, Graduate Program in Immunology, Clinical Research Center, and Departments of
{dagger} Physiology and Biophysics and
{ddagger} Biochemistry, Faculty of Medicine, University of Sherbrooke, Quebec, Canada

Collagen type IV (CnIV) and fibronectin (Fn) were used as ligands to study the distribution of {alpha}2ß1 and {alpha}4ß1 integrins in low-density, detergent-resistant microdomains (DRM) of Jurkat lymphocytes. CnIV-coated microspheres induced (optical trapping) the redistribution of GM1-associated fluorescence from the cell periphery to the area of contact. This was not observed in cells treated with ß-methyl cyclodextrin (MCD). Fn- or bovine serum albumin-coated microspheres did not modify the peripheral distribution of fluorescence. These observations were confirmed by confocal microscopy. Western blot analysis of cells exposed to surfaces coated with CnIV revealed that the {alpha}2-subunit was initially present at low levels in DRM, became strongly associated after 40 min, and returned to basal levels after 75 min. Fn induced a slight recruitment of the ß1-integrin {alpha}4-subunit in DRM after 5 and 10 min, followed by a return to basal levels. Neither CnIV nor Fn triggered significant changes in the distribution of the ß1-subunit in DRM. Fn- and CnIV-coated microspheres or surfaces coated with these ligands triggered a MCD-sensitive mobilization of Ca2+. MCD did not alter the state of the Ca2+ reserves. The differential distributions of the {alpha}2ß1 and {alpha}4ß1 integrins in DRM may provide one additional step in the regulation of outside-in signaling involving these integrins.

Key Words: optical trapping • Western blots • confocal microscopy • cholera toxin • GM1 ganglioside • fluorescence • calcium mobilization




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