No abstract

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor ,B (TGF-Pi)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57-to 62-kDa) and type II (75-to 82-kDa) receptor components for TGF-, and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-,B and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP4 binding and the appearance of a major affinity-labeled product of -64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs.The transforming growth factor ,B (TGF-,B) superfamily contains a large number of growth, differentiation, and morphogenetic cytokines that are active as homo-or heterodimers.This superfamily includes the TGF-1 family, the activin family, the Mullerian inhibiting substance (MIS), and the bone morphogenetic protein (BMP)/Vg family, which composes the largest group (reviewed in reference 39). Human BMP-2 and BMP-4 and their Drosophila homolog dpp are highly related (74 to 76% sequence identity [72]), as are BMP-5, BMP-6, and BMP-7 and their Drosophila homolog 60A (69 to 73% sequence identity [21]). Given the high degree of structural similarity among these family members, it is expected that their receptors will also form a family of related molecules. activin (2, 40) have been cloned. It has recently been determined that the product of the daf-4 gene from Caenorhabditis elegans, which is involved in both inhibition of dauer larva formation and exit from the dauer stage, is a type II receptor for BMP-2 and BMP-4 (24). Each of these receptors is predicted to be a transmembrane serine/threonine kinase. Ligand binding to each of these type II receptors in a variety of cell types does not appear to require coexpression of additional receptor components, indicating that type II receptors are capable of...

The 78,000-dalton glucose-regulated protein (GRP78) and the immunoglobulin heavy-chain-binding protein (BiP) were shown to be the same protein by NH2-terminal sequence comparison. Immunoprecipitation of GRP78-BiP induced by glucose starvation and a temperature-sensitive mutation in a hamster fibroblast cell line demonstrated the association of GRP78-BiP with other cellular proteins. In both fibroblasts and lymphoid cells, GRP78-BiP was found to label with 32Pi and [3H]adenosine. Phosphoamino acid analysis demonstrated that GRP78-BiP is phosphorylated on serine and threonine residues. Conditions which induce increased production of GRP78-BiP resulted in decreased incorporation of 32p; and [3H]adenosine into GRP78-BiP. Furthermore, we report here that the phosphorylated form of BiP resides in the endoplasmic reticulum and that BiP which is associated with heavy chains is not phosphorylated or labeled with [3H]adenosine, whereas free BiP is. This suggests that posttranslational modifications may be important in regulating the synthesis and binding of BiP.

The isolation and characterization of a human functional GRP78 gene and a processed pseudogene are described. We present the complete primary structure of the human GRP78 gene, which spans over 5 kb and consists of eight exons. Sequence comparisons reveal that the GRP78 gene shares unusual homology among the human, rat, and hamster in the protein-coding and 3' untranslated regions. In addition, short domains highly conserved with HSP70 isolated from human, Drosophila, Xenopus, yeast, and E. coli DNA are identified within the hydrophobic regions of GRP78. The intronless pseudogene resembles that of a processed gene. It is flanked by a short direct repeat and is embedded within an AT-rich genomic region. The highly active promoter from the functional human GRP78 gene contains a TATA box, five CCAAT sequences, and two potential binding sites for the transcriptional factor Sp1. It consists of a distal domain that enhances basal level expression and a proximal domain essential for responses to calcium ionophore and for a temperature-sensitive mutation which induce the GRP78 gene. Both domains are highly conserved between the rat and the human GRP78 promoters.

Abstract. The calcium ionophore A23187 can reversibly induce the expression of two glucose-regulated genes, p3C5 and p4A3. This induction requires a continuous presence of the ionophore for over 2 h. Although extracellular Ca 2+ is important for the optimal effect of A23187, it is not necessary for the induction, since a similar response with a lower magnitude can be triggered in cells cultured in low Ca 2+ medium buffered with EGTA. Both the basal and induced levels of p3C5 and p4A3 transcripts can be modulated by the calmodulin antagonist W-7, indicating the involvement of CaE+/calmodulin-associated pathways. In addition, the sensitivity of the A23187 induction to cycloheximide suggests that the induction process is dependent on de novo protein synthesis.