Bone morphogenetic protein 4 (BMP4) is a potent growth factor that is involved in many important biological processes. Regulation of the level of secreted mature BMP4 determines the biological effects of BMP4 on cells in the local microenvironment. Previous studies suggested that Gremlin, a member of DAN family proteins, antagonizes BMP4 activity by sequestering extracellular BMP4. Herein, we report a novel intracellular regulatory mechanism by which Gremlin interacts with BMP4 precursor, prevents secretion of mature BMP4, and therefore inhibits BMP4 activity more efficiently. Furthermore, we also defined a 30-amino acid peptide sequence within the Gremlin DAN domain that is essential for BMP4 interaction. This novel Gremlin-mediated BMP4 posttranslational regulatory mechanism implies that the level of BMP4 mRNA expression does not truly reflect BMP4 activity when Gremlin and BMP4 are coexpressed within the same cell. Similar regulatory mechanisms may be utilized by other DAN family proteins.

The first cloned chain (IFNAR1) of the human interferon-alpha (IFN alpha) receptor acts as a species-specific transducer for type 1 IFN action when transfected into heterologous mouse cells. Stably transfected mouse L929 cell lines expressing truncation mutants of the intracellular domain of the human IFNAR1 chain were tested for biological responses to human IFN alpha. Deletion of the intracellular domain resulted in a complete loss of sensitivity to the biological activity of human IFN but markedly increased IFNAR1 cell surface expression, demonstrating that the intracellular domain is required for biological function and contains a domain that negatively regulates its cell surface expression. Removal of the conserved membrane distal 16-amino-acid IRTAM (Interferon Receptor Tyrosine Activation Motif) sequence: (1) increased sensitivity to IFN alpha's antiviral activity, (2) increased the rapid IFN alpha-dependent formation of STAT-containing DNA-binding complexes, (3) prolonged tyrosine phosphorylation kinetics of the JAK-STAT pathway, and (4) blocked the IFN-dependent down-regulation of the IFNAR1 chain. These results indicate that the IRTAM negatively regulates signalling events required for the induction of IFN's biological actions via regulating receptor down-regulation.

STAT (signal transducers and activators of transcription) proteins undergo cytokine-dependent phosphorylation on serine and tyrosine. STAT3, a transcription factor for acute phase response genes, was found to act as an adapter molecule in signal transduction from the type I interferon receptor. STAT3 bound to a conserved sequence in the cytoplasmic tail of the IFNAR1 chain of the receptor and underwent interferon-dependent tyrosine phosphorylation. The p85 regulatory subunit of phosphatidylinositol 3-kinase, which activates a series of serine kinases, bound to phosphorylated STAT3 and subsequently underwent tyrosine phosphorylation. Thus, STAT3 acts as an adapter to couple another signaling pathway to the interferon receptor.

The four mammalian Janus kinase (JAK) family members, JAK1, JAK2, JAK3 and TYK2, are non-receptor protein tyrosine kinases (PTKs) that are crucial for cytokine receptor signaling in blood formation and immune responses. Mutations and translocations in the JAK genes leading to constitutively active JAK proteins are associated with a variety of hematopoietic malignancies, including the myeloproliferative disorders (JAK2), acute lymphoblastic leukemia (JAK2), acute myeloid leukemia (JAK2, JAK1), acute megakaryoblastic leukemia (JAK2, JAK3) and T-cell precursor acute lymphoblastic leukemia (JAK1). In contrast, loss-of-function mutations of JAK3 and TYK2 lead to immunodeficiency. The role of JAKs as therapeutic targets is starting to expand, as more insights into their structure and activation mechanisms become available.

All the cloned subunits of interferon receptors (IFNRs) belong to the type II cytokine receptor family (CRF2), Although three members of CRF2 encoded on human chromosome 21 share a 50 amino acid cytoplasmic homology domain (the IRH2 domain), a fourth subunit, the second cloned chain of the type I IFNR (IFN(I)R-2), contains a juxtamembrane 20 amino acid stretch of high similarity to the IRH2 domain that stops abruptly. Comparison of the membrane-distal portion of the IFN(I)R-2 cytoplasmic domain with sequence databases revealed a very high similarity to Alu repeat sequences, We provide evidence that all but 18 amino acids of the predicted cytoplasmic domain of the IFN(I)R-2 chain are encoded by an Alu cassette in its antisense orientation. Incorporation of an Alu cassette into the receptor chain is proposed to occur by a splicing mechanism, All previous well-characterized examples of insertion of an antisense Alu cassette into an open reading frame have involved alternative splicing, Thus, we predict the existence of an alternatively spliced product of the IFN(I)R-2 chain with a substantially different cytoplasmic domain.

The IFNAR chain of the type I interferon (IFN) receptor (IFNIR) undergoes rapid ligand-dependent tyrosine phosphorylation and acts as a species-specific transducer for type I IFN action. Using the vaccinia/T7 expression system to amplify IFNAR expression, we found that human HeLa-S3 cells transiently express high levels of cell surface IFNAR chains (approximately 250,000 chains per cell). Metabolic labeling and immunoblot analysis of transfected HeLa cells show that the IFNAR chain is initially detected as 65-kDa and 98-kDa precursors, and then as the 130-kDa mature protein. Due to variation in N-glycosylation, the apparent molecular mass of the mature IFNAR chain varies from 105 to 135 kDa in different cells. IFNIR structure was characterized in various human cell lines by analyzing 125I-labeled IFN cross-linked complexes recognized by various antibodies against IFNIR subunits and JAK protein-tyrosine kinases. Precipitation of cross-linked material from Daudi cells with anti-IFNAR antibodies showed that IFNAR was present in a 240-kDa complex. Precipitation of cross-linked material from U937 cells with anti-TYK2 sera revealed a 240-kDa complex, which apparently did not contain IFNAR and was not present in IFN-resistant HEC1B cells. The tyrosine phosphorylation and down-regulation of the IFNAR chain were induced by type I IFN in several human cell lines of diverse origins but not in HEC1B cells. However, of type I IFNs, IFN-beta uniquely induced the tyrosine phosphorylation of a 105-kDa protein associated with the IFNAR chain in two lymphoblastoid cell lines (Daudi and U266), demonstrating the specificity of transmembrane signaling for IFN-beta and IFN-alpha through the IFNAR chain.

A previously cloned cDNA encodes one subunit of the human interferon alpha/beta receptor (IFN alpha R), denoted IFN alpha R1. To study the expression and signaling of IFN alpha R1, we used monoclonal antibodies (mAbs) generated against the baculovirus-expressed ectodomain of IFN alpha R1. Immunoprecipitation and immunoblotting of lysates from a variety of human cell lines showed that IFN alpha R1 has an apparent molecular mass of 135 kDa. Binding analysis with 125I-labeled mAb demonstrated high levels of cell surface expression of IFN alpha R1 in human cells and in mouse cells transfected with IFN alpha R1 cDNA, whereas no cross-reactivity was observed in control mouse L929 cells expressing only the endogenous mouse receptor. The subunit was rapidly down-regulated by IFN alpha (80% decrease within 2 hr) and degraded upon internalization. The IFN alpha R1 chain appeared to be constitutively associated with the 115-kDa subunit of the IFN alpha/beta receptor, since the mAbs coprecipitated this protein. IFN alpha/beta treatment induced tyrosine phosphorylation of IFN alpha R1 within 1 min, with kinetics paralleling that of the IFN-activated protein-tyrosine kinases Jak1 and Tyk2. Ligand-induced tyrosine phosphorylation of IFN alpha R1 was blocked by the kinase inhibitors genistein or staurosporine. Although IFN alpha R1 cDNA-transfected mouse cells expressed high levels of this subunit when compared with empty vector-transfected cells the number of binding sites for human IFN alpha (50-75 sites per cell) was not increased. Human IFN alpha induced the expression of a mouse IFN alpha/beta-responsive gene (the 204 gene) in mouse L929 cells transfected with the IFN alpha R1 cDNA, but not in mock-transfected cells. These results suggest that the IFN alpha R1 subunit acts as a species-specific signal transduction component of the IFN alpha/beta receptor complex.

The leukocyte common antigen-related protein tyrosine phosphatase (LRP) is a widely expressed transmembrane glycoprotein thought to be involved in cell growth and differentiation. Similar to most other transmembrane protein tyrosine phosphatases, LRP contains two tandem cytoplasmic phosphatase domains. To understand further the regulation and evolution of LRP, we have isolated and characterized mouse λ genomic clones. Thirteen genomic clones could be divided into two non-overlapping clusters. The first cluster contained the transcription initiation site and the exon encoding most of the 5′ untranslated region. The second cluster contained the remaining exons encoding the protein and the 3′ untranslated region. The gene consists of 22 exons spanning over 75 kb. The distance between exon 1 and exon 2 is at least 25 kb. Characterization of the 5′ ends of LRP mRNA by S1 nuclease protection identifies putative initiation start sites within a G/C-rich region. The upstream region does not contain a TATA box. Comparison of the LRP gene structure to the mammalian protein tyrosine phosphatase gene, CD45, shows striking similarities in size and genomic organization.