The glycoprotein VI (GPVI)-Fc receptor (FcR)␥The adhesion and activation of platelets by subendothelial collagen fibers initiates aggregate formation at sites of vessel damage. Glycoprotein (GP) 1 VI plays a critical role in the activatory events induced by collagen as shown by the lack of response to collagen in human and mice platelets deficient in the glycoprotein (1, 2). A collagen-related peptide and a snake venom toxin, convulxin, interact specifically with GPVI and mimic many of the responses to collagen (3-5).Because of the physiological importance of GPVI, the mechanism of the GPVI-mediated signaling system has been extensively investigated (6 -8). GPVI is present as a complex with Fc receptor (FcR) ␥-chain in the platelet membrane (8 -10). The Src family kinases, Fyn and Lyn, are associated with GPVIFcR ␥-chain complex in platelets and initiate activation through phosphorylation of the immunoreceptor tyrosinebased activation motif (ITAM) in the FcR ␥-chain leading to binding and activation of the tyrosine kinase Syk. A series of adapter molecules including LAT and SLP76 orchestrate a carefully regulated signaling network leading to activation of PLC␥2, phosphoinositol 3-kinase, and small molecular weight G proteins, leading to platelet activation (6, 7).The cloning of GPVI (11-14) has revealed it to be a member of the immunoglobulin (Ig) superfamily, showing close homology to Fc␣RI. GPVI has a charged arginine residue in its transmembrane domain. This arginine, together with elements within the cytoplasmic domain, is crucial for association of GPVI with FcR ␥-chain and GPVI-mediated signal transduction (15,16). In addition, the cytoplasmic tail of GPVI has a cluster of 6 proline residues of unknown function (11)(12)(13)(14). This sequence of GPVI, RPLPPLPPLP, contains a consensus Src family kinase-SH3 recognition motif (RPLPPLP) (17,18), and provides a potential site of interaction with Fyn and Lyn via their SH3 domains.In this study, we demonstrate that depletion of the prolinerich domain in GPVI abolishes the association with Fyn and Lyn and prevents tyrosine phosphorylation of FcR ␥-chain and downstream responses. From these findings, we suggest that Fyn/Lyn directly bind the proline-rich domain of GPVI and that this association is necessary for phosphorylation of the ITAM and downstream signals.

Summary. Background: Glycoprotein VI (GPVI) is a physiologic receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerization of GPVI has been proposed as being necessary for the interaction with collagen, although direct evidence of dimerization has not been reported in cell lines or platelets. Objectives: To investigate oligomerization of GPVI in transfected cell lines and in platelets under non-stimulated conditions. Methods and results: By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extracellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerization of GPVI under these conditions. A chemical crosslinker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the Fc receptor (FcR) c-chain. Conclusions: The present results directly demonstrate GPVI-FcR c-chain oligomerization at the surface of the platelet, and thereby add to the growing evidence that oligomerization of GPVI may be a prerequisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage.

Summary. Background: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcRc-chain, which has an immunoreceptor tyrosinecontaining activation motif (ITAM). Objective: To determine why GPVI-FcRc signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin. Methods and results: Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen-induced GPVIFcRc signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin. Conclusion: The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR-1. In platelets, we propose that GPVI-FcRc has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery.

Misfolded proteins are caused by genomic mutations, aberrant splicing events, translation errors or environmental factors. The accumulation of misfolded proteins is a phenomenon connected to several human disorders, and is managed by stress responses specific to the cellular compartments being affected. In wild-type cells these mechanisms of stress response can be experimentally induced by expressing recombinant misfolded proteins or by incubating cells with large concentrations of amino acid analogues. Here, we report a novel approach for the induction of stress responses to protein aggregation. Our method is based on engineered transfer RNAs that can be expressed in cells or tissues, where they actively integrate in the translation machinery causing general proteome substitutions. This strategy allows for the introduction of mutations of increasing severity randomly in the proteome, without exposing cells to unnatural compounds. Here, we show that this approach can be used for the differential activation of the stress response in the Endoplasmic Reticulum (ER). As an example of the applications of this method, we have applied it to the identification of human microRNAs activated or repressed during unfolded protein stress.