Herein we report that interferon‐gamma (IFN gamma) induces the rapid and reversible tyrosine phosphorylation of the IFN gamma receptor. Using a panel of receptor intracellular domain mutants, we show that a membrane‐proximal LPKS sequence (residues 266‐269) is required for ligand‐induced tyrosine kinase activation and/or kinase‐receptor association and biological responsiveness, and a functionally critical membrane‐distal tyrosine residue (Y440) is a target of the activated enzyme. The biological significance of Y440 phosphorylation was demonstrated by showing that a receptor‐derived nonapeptide corresponding to receptor residues 436‐444 and containing phosphorylated Y440 bound specifically to p91, blocked p91 phosphorylation and inhibited the generation of an active p91‐containing transcription factor complex. In contrast, nonphosphorylated wild‐type, phosphorylated mutant, or phosphorylated irrelevant peptides did not. Moreover, the phosphorylated Y440‐containing peptide did not interact with a related but distinct latent transcription factor (p113) which is activatible by IFN alpha but not IFN gamma. These results thus document the specific and inducible association of p91 with the phosphorylated IFN gamma receptor and thereby elucidate the mechanism by which ligand couples the IFN gamma receptor to its signal transduction system.

We compared and contrasted the mechanism of action for the cysteine knot protein subfamily, Wise and Sost (Sclerostin). Our data suggest that functional interactions between Sost or Wise and LRP5/ LRP6 have the potential to regulate bone deposition by modulating the Wnt pathway.Introduction: The human disease sclerosteosis exhibits an increase in bone mass thought to be caused by hyperactive osteoblasts. Sclerostin, SOST, the gene affected in this disease, has been postulated to exert its activity by functioning as a BMP antagonist. However, recent evidence indicates that SOST is highly related to Wise, which can also modulate the Wnt pathway by binding to LRP5 and LRP6. Materials and Methods: For this study, we used cell culture to test the BMP and Wnt activity function of both Wise and Sost. In addition, we used Xenopus in vivo Wnt assays along with Xenopus in vitro Wnt assays to support our cell culture results. Epitope tagged cell supernatants containing either Sost or soluble mutant or wildtype LRP5/LRP6 were used for immunoprecipitation. Sost immunoprecipitation results were confirmed in vivo using cell culture. Finally, to support our in vitro data, we co-localized Sost, Wise, LRP5, and LRP6 in mouse long bone sections. Results: In this study, we report in vitro and in vivo evidence to show that Sost physically interacts with Lrp5 and Lrp6 and inhibits the canonical Wnt signaling pathway. Furthermore, using in vitro and in vivo assays, we showed that a variant of LRP5 (LRP5

Herein, we demonstrate that purified Stat1 binds to its tyrosine-phosphorylated docking site on the IFN gamma receptor alpha chain in a direct, specific, and reversible manner. Using surface plasmon resonance, we determine the affinity (KD = 137 nM) and specificity of the interaction and define the minimum affinity needed for receptor-mediated Stat1 activation. In addition, we quantitate the relative ability of purified Stat1 to interact with tyrosine-phosphorylated binding sites on other Stat proteins. Finally, we describe experiments that imply that the unidirectional release of activated Stat1 from the IFN gamma receptor reflects the preference of free tyrosine-phosphorylated Stat1 monomers to form high avidity reciprocal homodimers rather than reassociating with the receptor binding site. Our results demonstrate that IFN gamma-induced Stat1 activation is an ordered and affinity-driven process and we propose that this process may serve as a paradigm for Stat activation by other cytokine receptors.

We have reported previously that Noggin is a heparinbinding protein and associates with the cell surface through heparan sulfate proteoglycans, where it remains functional for the binding of bone morphogenetic proteins (BMPs). Here we report that the binding of Noggin to the cell surface is highly selective for heparan sulfate and that specific structural features are required for the interaction. Noggin binds most efficiently to heparin sequences composed of 10 or more monosaccharides; N-, 6-O-, and 2-O-sulfates contribute to this interaction. In addition, we have shown that the developmentally regulated endosulfatase Qsulf1 selectively removes sulfate groups from the 6-O position of sugars within the most highly sulfated S domains of heparan sulfate, whereas 6-O-sulfates in the NA/NS domains are not substrates for the enzyme. The activity of Qsulf1 in cells in culture results in the release of Noggin from the cell surface and a restoration of BMP responsiveness to the cells. This shows that Noggin binds to the S domains of heparan sulfate and provides evidence that, in addition to modulating Wnt signaling in vivo by the release of heparan sulfate bound Wnt, Qsulf1 also modulates BMP signaling by the release of surface-bound Noggin.Heparan sulfate proteoglycans are found ubiquitously both on the surface of cells as well as within the extracellular matrix, where they bind and modify the functions of a diverse array of ligands (1). Loss of function mutations in enzymes of the heparan sulfate biosynthetic pathway have confirmed that this polysaccharide has essential roles during development. Foremost among these is the regulation of cellular responsiveness to a number of growth factors and morphogens that control patterning events (2). Mutations in members of the glypican family of cell-surface heparan sulfate proteoglycans, in both vertebrates and invertebrates, have been specifically associated with loss of bone morphogenetic protein (BMP) 1 activities (3,4). Although the precise molecular mechanisms by which these specific heparan sulfate proteoglycans regulate cellular responsiveness to BMPs in vivo is not known, recent in vitro data does support the conclusion that heparan sulfate can directly augment signaling of BMPs through their receptors (5, 6).Previously we have shown that the BMP antagonist Noggin remains associated with the surface of cells by binding to heparan sulfate, where it remains functional for the inhibition of BMPs (7). We have proposed that the interaction with heparan sulfate in vivo is likely to regulate the range of influence of Noggin by restricting its diffusion, thus providing another mechanism by which cellular responsiveness to BMPs can be regulated by heparan sulfate in vivo (7). This hypothesis supposes that the interaction between Noggin and heparan sulfate is sequence specific and implies that there should be a developmentally regulated mechanism in vivo for the specification of heparan sulfate structures that modulate Noggin binding to the cell surface.Heparan sulfate sequences...

Glypicans represent a family of six cell surface heparan sulfate proteoglycans in vertebrates. Although no specific in vivo functions have thus far been described for these proteoglycans, spontaneous mutations in the human and induced deletions in the mouse glypican-3 (Gpc3) gene result in severe malformations and both pre- and postnatal overgrowth, known clinically as the Simpson-Golabi-Behmel syndrome (SGBS). Mice carrying mutant alleles of Gpc3 created by either targeted gene disruption or gene trapping display a wide range of phenotypes associated with SGBS including renal cystic dysplasia, ventral wall defects, and skeletal abnormalities that are consistent with the pattern of Gpc3 expression in the mouse embryo. Previous studies in Drosophila have implicated glypicans in the signaling of decapentaplegic, a BMP homolog. Our experiments with mice show a significant relationship between vertebrate BMP signaling and glypican function; GPC3-deficient animals were mated with mice haploinsufficient for bone morphogenetic protein-4 (Bmp4) and their offspring displayed a high penetrance of postaxial polydactyly and rib malformations not observed in either parent strain. This previously unknown link between glypican-3 and BMP4 function provides evidence of a role for glypicans in vertebrate limb patterning and skeletal development and suggests a mechanism for the skeletal defects seen in SGBS.