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J. Biol. Chem., Vol. 279, Issue 37, 39139-39145, September 10, 2004

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Aggregation of Integrins and RhoA Activation Are Required for Thy-1-induced Morphological Changes in Astrocytes^*

Ana María Avalos, William T. Arthur, Pascal Schneider¶, Andrew F. G. Quest||, Keith Burridge, and Lisette Leyton**

From the Programs of Morphology and ^||Cell and Molecular Biology, FONDAP Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Independencia 1027-A, Santiago, Chile, Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and ^¶Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a ₃-containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a ₃-containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC₁ astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominant-negative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein A-Sepharose beads. The same was shown by cross-linking ₃-containing integrin with anti-₃ antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on ₃ integrin clustering and the resulting increase in RhoA activity.

Received for publication, March 29, 2004 , and in revised form, June 24, 2004.

^* This work was supported by Fogarty-National Institutes of Health Grant 1-R03-TW06024-01 (to K. B. and L. L.), Parent National Institutes of Health Grant GM29860 (to K. B.), Grant 1040390 (to L. L.), Grants 1020585 and 15010006 (to A. F. G. Q.) from Fondecyt (Chile), Collaborative Wellcome Trust Project 064911/Z/01/z, Grant CRP/CH100-05 from the International Centre for Genetic Engineering and Biotechnology (to A. F. G. Q.), and a Ph.D. fellowship and Grant AT-403113 from Conicyt and Grant PG/110/02 from the Postgraduate Department of the University of Chile (to A. M. A.). 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.

^** To whom correspondence should be addressed. Tel./Fax: 56-2-738-2015; E-mail: lleyton{at}med.uchile.cl.

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