(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-
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...
Lethal yellow (AY) is a mutation at the mouse agouti locus in chromosome 2 that causes a number of dominant pleiotropic effects, ieluding a completely yellow coat color, obesity, an insuinnt ype I diabetic condition, and an increased propensity to develop a variety ofspontaneous and induced tumors. Additionally, homozygosity for Ay results in preimplantation lethality, which terminates development by the blastocyst stage. The A' mutation Is the result of a 170-kb deletion that removes all but the promoter and noncoding first exon of another gene called Raly, which lies in the same transriptional orientation as agouti and maps 280 kb proximal to the 3' end of the agonti gene. We present a model for the structure of the A' allele that can explain the dominant pleiotropic effects assocated with this mutation, as well as the recessive lethality, which Is unrelated to the agouti gene.
We examined the role of Fc;R in antibody therapy of metastatic melanoma in wild-type and different Fc;R knockout mice. Treatment of B16F10-challenged wild-type mice with TA99 antibody specific for the gp75 tumor antigen resulted in a marked decrease in numbers of lung metastases. Treatment of individual Fc;R knock-out mice revealed the high-affinity IgG receptor, Fc;RI (CD64), to represent the central Fc;R for TA99-induced antitumor effects. The potential of immunemodulating agents to further enhance the protective effect induced by monoclonal antibody (mAb) TA99 was examined in combination treatments consisting of mAb TA99 and a TLR-4 agonist, monophosphoryl lipid A (MPL). MPL did potently boost TA99 antibody-induced effects, and combination therapy was, again, found to be dependent on the presence of
Most Ig receptors exist as hetero-oligomeric complexes with separate ligand binding (alpha) and signal transducing (beta, gamma, or zeta) subunits. For Fc gamma RIIIa and Fc epsilon RI, association with the FcR gamma-chain is essential for surface expression. However, the human high affinity IgG receptor, hFc gamma RI, was found to be surface- expressed by itself in transient transfection models. We have now analyzed the integrity of hFc gamma RI expression in more detail in stable transfectants. In vitro we noted that, in the absence of FcR gamma-chain, surface expression of hFc gamma RI rapidly declined to background levels, in both IIA1.6 B cells and NIH3T3 fibroblasts. The effect of FcR gamma-chain on hFc gamma RI surface expression in vivo was evaluated by using two newly generated transgenic mouse lines, selectively expressing hFc gamma RI on myeloid cells. These transgenic mice were crossed with FcR gamma-chain-deficient mice. Analysis of blood monocytes and peritoneal macrophages showed that surface expression of hFc gamma RI was reduced by approximately 80%. The remaining approximately 20% of receptors were still capable of binding IgG-opsonized RBC, suggesting FcR gamma-chain not to be critical for hFc gamma RI ligand-binding capacity. Importantly, however, hFc gamma RI signaling capacity was lost in FcR gamma-chain-deficient cells. No phagocytosis could be observed using either ligand sensitized (EA- IgG2a) or CD64-targeted erythrocytes (using a bispecific antibody) in both hFc gamma RI transgenic lines. This documents the FcR gamma-chain to be indispensable for both surface membrane expression and function of human Fc gamma RI in vivo.
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