Neuronal Thy-1 induces astrocyte adhesion by engaging syndecan-4 in a cooperative interaction with αvβ3 integrin that activates PKCα and RhoA

Abstract

Clustering of αvβ3 integrin after interaction with the RGD-like integrin-binding sequence present in neuronal Thy-1 triggers formation of focal adhesions and stress fibers in astrocytes via RhoA activation. A putative heparin-binding domain is present in Thy-1, raising the possibility that this membrane protein stimulates astrocyte adhesion via engagement of an integrin and the proteoglycan syndecan-4. Indeed, heparin, heparitinase treatment and mutation of the Thy-1 heparin-binding site each inhibited Thy-1-induced RhoA activation, as well as formation of focal adhesions and stress fibers in DI TNC1 astrocytes. These responses required both syndecan-4 binding and signaling, as evidenced by silencing syndecan-4 expression and by overexpressing a syndecan-4 mutant lacking the intracellular domain, respectively. Furthermore, lack of RhoA activation and astrocyte responses in the presence of a PKC inhibitor or a dominant-negative form of PKCα implicated PKCα and RhoA activation in these events. Therefore, combined interaction of the astrocyte αvβ3-integrin–syndecan-4 receptor pair with Thy-1, promotes adhesion to the underlying matrix via PKCα- and RhoA-dependent pathways. Importantly, signaling events triggered by such receptor cooperation are shown here to be the consequence of cell-cell rather than cell-matrix interactions. These observations are likely to be of widespread biological relevance because Thy-1–integrin binding is reportedly relevant to melanoma invasion, monocyte transmigration through endothelial cells and host defense mechanisms.

Dynamic catch of a Thy-1–α5β1+syndecan-4 trimolecular complex

“…3). It was reported that Thy-1 contains a putative polycationic heparin-binding sequence, which mediates binding to the HSPG, Syn4, and activation of PKCa 7 . When K562 cells were pretreated with heparinase (HPSE) to cleave heparin sulphate glycosaminoglycans (GAGs) from the cell surface, adhesions were reduced; Syn4 knockdown by small interfering RNA (siRNA) showed similar reduction (Fig.…”

“…Thy-1 (CD90) is a heavily N-glycosylated single v-type immunoglobulin (Ig) domaincontaining glycoprotein, probably of primordial origin within the immunoglobulin superfamily 2 . It is linked to the outer leaflet of the plasma membrane via a carboxy-terminal glycophosphatidylinositol anchor, where it binds integrins in a variety of tissue-specific contexts, including a M b 2 (Mac-1) on leukocytes 3,4 , a v b 3 on melanoma cells 5 and neurons 6,7 , and a v b 5 on lung fibroblasts 8 . For a full review, see ref.…”

“…9. Binding to a v integrins is mediated by Thy-1's Arg-Leu-Asp (RLD) motif, as mutation to non-functional Arg-Leu-Glu abolishes binding [6][7][8] . Thy-1 also binds heparin sulphate proteoglycans (HSPGs), including syndecan-4 (Syn4), through a conserved polycationic motif in the C-C' strand loop motif 7,10 .…”

“…Binding to a v integrins is mediated by Thy-1's Arg-Leu-Asp (RLD) motif, as mutation to non-functional Arg-Leu-Glu abolishes binding [6][7][8] . Thy-1 also binds heparin sulphate proteoglycans (HSPGs), including syndecan-4 (Syn4), through a conserved polycationic motif in the C-C' strand loop motif 7,10 . In patients with metastatic melanoma, Thy-1 is highly expressed on both the venous and lymphatic endothelium, where it facilitates metastasis in experimental models of melanoma 11,12 .…”

ATP release due to Thy-1–integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

“…Thy-1, an abundant mammalian glycoprotein described initially 50 years ago (Barker and Hagood, 2008;Morris, 1992), is expressed in T cells, activated endothelial cells and neurons (Lee et al, 1998;Williams, 1976), and acts in trans as a ligand for avb3 integrin and syndecan-4 on astrocytes (Avalos et al, 2004;Avalos et al, 2009;Leyton et al, 2001). Binding of Thy-1 to both avb3 integrin and syndecan-4 in astrocytes triggers tyrosine phosphorylation of focal adhesion (FA) proteins, activation of protein kinase Ca (PKCa), RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix (Avalos et al, 2004;Avalos et al, 2002;Avalos et al, 2009;Hermosilla et al, 2008;Leyton et al, 2001).…”