(FcR γ-chain), the non-receptor tyrosine kinase Syk and Bristol BS8 1TD, UK phospholipase Cγ2 (PLCγ2) (Blake et al., 1994; Daniel et al., 1994a; Fujii et al., 1994;Yanaga et al., 1995; 5 Corresponding author Asazuma et al., 1996;Gibbins et al., 1996). Syk assembles A.Poole, J.M.Gibbins and M.Turner contributed equally to this work into signalling complexes at the plasma membrane via interaction between its tandem Src homology 2 (SH2) Activation of mouse platelets by collagen is associated domains and a tyrosine-phosphorylated immunoreceptor with tyrosine phosphorylation of multiple proteins tyrosine-based activation motif (ITAM). We recently proincluding the Fc receptor γ-chain, the tyrosine kinase posed a model for collagen-induced signalling in human Syk and phospholipase Cγ2, suggesting that collagen platelets in which receptor clustering induced by collagen signals in a manner similar to that of immune receptors.leads to tyrosine phosphorylation of the FcR γ-chain, This hypothesis has been tested using platelets from possibly by a Src family kinase, allowing binding of Syk, mice lacking the Fc receptor γ-chain or Syk. Tyrosine which becomes tyrosine phosphorylated and activated phosphorylation of Syk and phospholipase Cγ2 by (Gibbins et al., 1996). This initiates a series of events collagen stimulation is absent in mice lacking the Fc which may involve other kinases and adapter proteins receptor γ-chain. Tyrosine phosphorylation of phospholeading to tyrosine phosphorylation and activation of lipase Cγ2 by collagen stimulation is also absent in mice PLCγ2. This model has been evaluated in the present platelets which lack Syk, although phosphorylation of study using platelets from genetically modified mice which the Fc receptor γ-chain is maintained. In contrast, lack the FcR γ-chain or Syk. tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor Results γ-chain or Syk. The absence of Fc receptor γ-chain or Syk is accompanied by a loss of secretion and aggrega-Tyrosine phosphorylation in collagen-and tion responses in collagen-but not thrombin-stimulated thrombin-stimulated platelets platelets. These observations provide the first direct Collagen and thrombin stimulated distinct but overlapping evidence of an essential role for the immunoreceptor increases in whole cell tyrosine phosphorylation in platetyrosine-based activation motif (ITAM) in signalling lets (Figures 1 and 2A). Both agonists stimulated increases by a non-immune receptor stimulus.in tyrosine phosphorylation of proteins of~42, 70-75, Keywords: collagen/Fc receptor γ-chain/ITAM/platelets/ 100, 110 and 130 kDa in platelets from B6 mice; the 42 Syk and 110 kDa proteins were more heavily phosphorylated in thrombin-stimulated cells. The largest increase in tyrosine phosphorylation was in the 70-75 kDa proteins. Collagen but not thrombin also stimulated marked tyrosine phos-

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The agouti gene normally confers the wild-type coat color of mice. Dominant mutations at the agouti locus result in a pleiotropic syndrome that is characterized by excessive amounts of yellow pigment in the coat, obesity, a non-insulin-dependent diabetic-like condition, and the propensity to form a variety of tumors. Here, we describe a new dominant mutation at the agouti locus in which an intracisternal A-particle (IAP) has integrated in an antisense orientation immediately 5' of the first coding exon of the gene. This mutation, which we have named Aiapy, results in the ectopic expression of the agouti gene through the utilization of a cryptic promoter within the IAP 5' long terminal repeat (LTR). The coat color of Aiapy/-mice ranges from solid yellow to a pigment pattern that is similar to wild type (pseudoagouti), and the expressivity of this mutant phenotype varies with parental inheritance. Those offspring with a yellow coat ectopically express agouti mRNA at high levels and exhibit marked obesity, whereas pseudoagouti mice express agouti mRNA at a very low level and their weights do not differ from wild-type littermates. Data are presented to show that the differential expressivity of the Aiapy allele is correlated with the methylation status of the inserted IAP 5' LTR. These data further support the hypothesis that in dominant yellow mutations at the agouti locus, it is the ubiquitous expression of the wild-type agouti coding sequence that is responsible for the yellow coat color, obesity, diabetes, and tumorigenesis.

There is extensive evidence to show that phosphatidylinositol 3-kinase plays an important role in signaling by the immune family of receptors, which has recently been extended to include the platelet collagen receptor, glycoprotein VI. In this report we present two potential mechanisms for the regulation of this enzyme on stimulation of platelets by collagen. We show that on stimulation with collagen, the regulatory subunit of phosphatidylinositol 3-kinase associates with the tyrosinephosphorylated form of the adapter protein linker for activator of T Cells (LAT) and the tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif of the Fc receptor ␥-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor ␥-chain and LAT with p85 are rapid and supported by the Src-homology 2 domains of the regulatory subunit. We did not obtain evidence to support previous observations that the regulatory subunit of phosphatidylinositol 3-kinase is regulated through association with the tyrosine kinase Syk. The present results provide a molecular basis for the regulation of the p85/110 form of phosphatidylinositol 3-kinase by GPVI, the collagen receptor that underlies activation.Subendothelial collagens are primary platelet agonists and are thereby essential components of the hemostatic system. At sites of vascular damage, platelets adhere to exposed collagen fibers and undergo activation via a tyrosine kinase-dependent signaling pathway. Activation causes an increase in the binding capability of the fibrinogen receptor, integrin ␣ IIb ␤ 3 , and the secretion of various mediators that culminate in the formation of an irreversible platelet aggregate, or hemostatic plug. The integrin ␣ 2 ␤ 1 is expressed on the platelet surface and has been shown to support adhesion to collagen, although increasing evidence suggests that a second platelet collagen receptor underlies platelet activation.The collagen receptor that underlies activation comprises the uncharacterized platelet glycoprotein VI (GPVI) 1 (1, 2), which is noncovalently associated with the Fc receptor (FcR) ␥-chain (3). Additional components may also exist. The FcR ␥-chain is recognized for its role in the expression of, and signaling by, the high affinity receptor for IgE (Fc⑀RI) (4 -6) and IgG (Fc␥RI) (7,8) and the low affinity IgG receptor (Fc␥RIII) (8). The FcR ␥-chain is a transmembrane protein that is expressed as a homodimer. The cytoplasmic tail of the protein contains a consensus motif termed an immunoreceptor tyrosine-based activation motif (ITAM),which is defined as YXXLX (6 -8) YXXL where X represents any amino acid (9). This motif, which is also present in subunits of the T and B cell antigen receptors, becomes phosphorylated on the conserved tyrosine residues on ligand binding, enabling association of members of the Syk/ Zap70 family of tyrosine kinases (3, 4, 10). Tyrosine phosphorylation of ITAMs has been demonstrated to involve the activity of Src-family kinases, and two recent report...

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