This Article Full Text Full Text (PDF) All Versions of this Article: 2003-0079OCv1 30/2/233    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sitrin, R. G. Articles by Blackwood, R. A. Search for Related Content PubMed PubMed Citation Articles by Sitrin, R. G. Articles by Blackwood, R. A.

Lipid Raft Compartmentalization of Urokinase Receptor Signaling in Human Neutrophils

Pulmonary and Critical Care Medicine Division, Department of Internal Medicine, Department of Pediatrics and Communicable Diseases, and Department of Ophthalmology, University of Michigan, Ann Arbor, Michigan

Address correspondence to: Robert G. Sitrin, M.D., 6301 MSRB III, Box 0642, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0642. E-mail: rsitrin{at}umich.edu

Urokinase plasminogen activator (uPA) receptors (uPAR) can be engaged for activation signaling either by aggregation or by binding exogenous uPA. These signaling mechanisms require uPAR to associate with two distinct adhesion proteins, L-selectin and complement receptor 3 (CR3), respectively. uPAR contains a glycosylphosphatidylinositol anchor, suggesting that it is concentrated within glycosphingolipid-enriched microdomains, or "lipid rafts". This study was undertaken to determine the extent to which uPAR-mediated signaling is compartmentalized to lipid rafts. Human neutrophil uPAR was cross-linked or stimulated with uPA after pretreatment with the lipid raft–disrupting agents, methyl-ß-cyclodextrin or filipin III. Both agents suppressed increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) triggered by cross-linking, but did not affect [Ca²⁺ ]_i in response to uPA. Neutrophil membranes were separated into lipid raft and non-raft fractions, revealing the presence of uPAR and L-selectin, but the virtual absence of CR3 chain in lipid rafts, either constitutively or in response to uPAR aggregation. Fluorescence resonance energy transfer experiments confirmed close proximity of a lipid raft marker to both uPAR and L-selectin, but not CR3. We conclude that uPAR can engage distinct signaling pathways involving different partner proteins that are functionally and physically segregated from one another in both lipid raft and non-raft domains of the plasma membrane.

Abbreviations: intracellular calcium concentration, [Ca²⁺]_i • complement receptor 3, CR3 • fluorescein isothiocyanate, FITC • glycosylphosphatidylinositol, GPI • high molecular weight uPA, HMW-uPA • horseradish peroxidase, HRP • methyl-ß-cyclodextrin, MßCD • tetramethylrhodamine isothiocyanate, TRITC • urokinase plasminogen activator, uPA • urokinase plasminogen activator receptor, uPAR

This article has been cited by other articles:

				M. Carlile-Klusacek and V. Rizzo Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H366 - H375. [Abstract] [Full Text] [PDF]

				L. A. McVoy and R. R. Kew CD44 and Annexin A2 Mediate the C5a Chemotactic Cofactor Function of the Vitamin D Binding Protein J. Immunol., October 1, 2005; 175(7): 4754 - 4760. [Abstract] [Full Text] [PDF]

				P. E. Mattila, C. E. Green, U. Schaff, S. I. Simon, and B. Walcheck Cytoskeletal interactions regulate inducible L-selectin clustering Am J Physiol Cell Physiol, August 1, 2005; 289(2): C323 - C332. [Abstract] [Full Text] [PDF]

				Y. Wei, R.-P. Czekay, L. Robillard, M. C. Kugler, F. Zhang, K. K. Kim, J.-p. Xiong, M. J. Humphries, and H. A. Chapman Regulation of {alpha}5{beta}1 integrin conformation and function by urokinase receptor binding J. Cell Biol., January 31, 2005; 168(3): 501 - 511. [Abstract] [Full Text] [PDF]

				M. B. Fessler, P. G. Arndt, S. C. Frasch, J. G. Lieber, C. A. Johnson, R. C. Murphy, J. A. Nick, D. L. Bratton, K. C. Malcolm, and G. S. Worthen Lipid Rafts Regulate Lipopolysaccharide-induced Activation of Cdc42 and Inflammatory Functions of the Human Neutrophil J. Biol. Chem., September 17, 2004; 279(38): 39989 - 39998. [Abstract] [Full Text] [PDF]

				J. D. van Buul and P. L. Hordijk Signaling in Leukocyte Transendothelial Migration Arterioscler. Thromb. Vasc. Biol., May 1, 2004; 24(5): 824 - 833. [Abstract] [Full Text]

				A. L. Kindzelskii, R. G. Sitrin, and H. R. Petty Cutting Edge: Optical Microspectrophotometry Supports the Existence of Gel Phase Lipid Rafts at the Lamellipodium of Neutrophils: Apparent Role in Calcium Signaling J. Immunol., April 15, 2004; 172(8): 4681 - 4685. [Abstract] [Full Text] [PDF]