Interaction of Linker for Activation of T Cells with Multiple Adapter Proteins in Platelets Activated by the Glycoprotein VI-selective Ligand, Convulxin

Abstract

The snake venom toxin convulxin activates platelets through the collagen receptor glycoprotein VI (GPVI)/Fc receptor ␥-chain (FcR ␥-chain) complex leading to tyrosine phosphorylation and activation of the tyrosine Syk and phospholipase C␥2 (PLC␥2). In the present study, we demonstrate that convulxin is a considerably more powerful agonist than collagen or the GPVI-selective collagen-related peptide (CRP). Confirmation that the response to convulxin is mediated solely via Syk was provided by studies on Syk-deficient platelets. The increase in phosphorylation of the FcR ␥-chain is associated with marked increases in tyrosine phosphorylation of downstream proteins including Syk, linker for activation of T cells (LAT), SLP-76, and PLC␥2. The transmembrane adapter LAT coprecipitates with SLP-76 and PLC␥2, as well as with a number of other adapter proteins, some of which have not been previously described in platelets, including Cbl, Grb2, Gads, and SKAP-HOM. Gads is constitutively associated with SLP-76 and is probably the protein bridging its association with LAT. There was no detectable association between Grb2 and SLP-76 in control or stimulated cells, suggesting that the interaction of LAT with Grb2 is present in a separate complex to that of LAT-Gads-SLP-76. These results show that the trimeric convulxin stimulates a much greater phosphorylation of the FcR ␥-chain and subsequent downstream responses relative to CRP and collagen, presumably because of its ability to cause a greater degree of cross-linking of GPVI. The adapter LAT appears to play a critical role in recruiting a number of other adapter proteins to the surface membrane in response to activation of GPVI, presumably at sites of glycolipidenriched microdomains, enabling an organized signaling cascade that leads to platelet activation.Platelets express a remarkably large number of receptors for collagen, including the integrin ␣ 2 ␤ 1 and the immunoglobulin receptor glycoprotein VI (GPVI) 1 (1). It is generally accepted that ␣ 2 ␤ 1 is the major receptor supporting adhesion of platelets to collagen, while GPVI mediates activation. Evidence for this includes the observation that the GPVI-selective agonists collagen-related peptide (CRP) and convulxin are powerful platelet agonists, yet do not bind ␣ 2 ␤ 1 . GPVI is associated with the Fc receptor (FcR) ␥-chain in the membrane (2, 3). Cross-linking of GPVI induces tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic tail of FcR ␥-chain leading to a tyrosine phosphorylation-regulated cascade that involves Syk and PLC␥2 (4). The sequence of events that underlie phosphorylation of PLC␥2 and subsequent platelet activation, however, are not established. The adapter proteins LAT (linker for activation of T cells) and SLP-76 undergo tyrosine phosphorylation downstream of Syk and have critical, but undefined roles in the regulation of PLC␥2 (5-7).LAT is a 36 -38 kDa integral membrane adapter protein that was first identified in T cells (8). Activation of ZAP-70 ...

TULA-2 Protein Phosphatase Suppresses Activation of Syk through the GPVI Platelet Receptor for Collagen by Dephosphorylating Tyr(P)346, a Regulatory Site of Syk

“…The activation of Syk is expected to increase phosphorylation of its direct targets, such as the adaptor protein SLP-76, followed by all downstream elements of the GPVI signaling pathway. An increase in the tyrosine phosphorylation of SLP-76 and other proteins is thought to lead to the assembly of protein-protein complexes that play a key role in GPVI signaling (41). However, Syk phosphorylation/dephosphorylation might affect protein complex formation not only by up-regulating the enzymatic activity of Syk, but by directly enhancing protein-protein interactions of this PTK; thus, binding of Syk to PLC␥1 was shown to be Tyr(P) 346 -dependent (42).…”

The role of Gads in hematopoietic cell signalling

“…It stands to reason that Gads has a role in these cell types similar to the one it plays in T cells, linking SLP-76 to membrane-associated LAT upon antigen receptor activation. In fact, the Gads-SLP-76-LAT complex can be detected upon activation of the GPVI Ig receptor of platelets (Asazuma et al, 2000) or the Fce receptor of primary mast cells and cells of the RBL-2H3 mast cell line (unpublished observations, S Liu, D Berry and CJ McGlade). However, the functional signi®cance of the SLP-76-Gads-LAT complex has not been completely evaluated in these cell types.…”

The Toll-like receptor 2/1 (TLR2/1) complex initiates human platelet activation via the src/Syk/LAT/PLCγ2 signalling cascade

“…One eminent characteristic of collagen/convulxin-initiated platelet activation compared to that by thrombin or ADP is the prominent phosphorylation of the adaptor protein LAT [21][22][23].…”