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Analysis of the native murine bone morphogenetic protein serine threonine kinase type I receptor (ALK-3)
1Immunobiology and Cancer Program, Oklahoma Medical Research Foundation Oklahoma City 73104, USA.
Abstract:
The bone morphogenetic proteins, members of the transforming growth factor-beta cytokine family, induce the osteoblast phenotype and promote osteogenesis in the bone marrow stroma. Simultaneously, these cytokines inhibit other mesodermal differentiation pathways, such as adipogenesis and myogenesis. The receptors for the bone morphogenetic proteins belong to a family of transmembrane serine/ threonine kinase TGF beta type I and type II receptor proteins. In man, these include the activin receptor like kinase-3 (ALK-3), a type I receptor protein. We have used a polyclonal antibody to examine the expression of the native murine ALK-3 protein in murine tissues and bone morphogenetic protein-responsive cell lines. On Western blot analyses, we found that the native 85 kDa native ALK-3 protein was expressed in a number of murine tissues; protein and mRNA levels did not necessarily correlate. Two bone morphogenetic protein-responsive cell lines, BMS2 bone marrow stromal cells and C2C12 myoblasts, expressed the ALK-3 protein constitutively. Cell differentiation was accompanied by modest changes in ALK-3 protein levels. Immunoprecipitation of the ALK-3 protein cross linked to [125I] BMP-4 revealed two major receptor complexes of approximately 90 kDa and 170 kDa in size. Biotin surface-labeling experiments revealed that the 85 kDa ALK-3 protein was constitutively associated with a novel 140 kDa surface glycoprotein. Deglycosylation reduced the protein's size to 116 kDa, comparable in size to that of the recently described BMP type II receptor. These findings support the current model that BMP interacts with a pre-existing complex consisting of a type I and type II receptor protein.
Insights
Bone morphogenetic proteins (BMPs) are key to bone formation. This study investigates the activin receptor like kinase-3 (ALK-3) protein, a BMP receptor, revealing its expression and interactions in bone marrow and muscle cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Bone morphogenetic proteins (BMPs), part of the transforming growth factor-beta superfamily, are crucial for inducing osteogenesis and inhibiting other mesodermal differentiation pathways like adipogenesis and myogenesis.
- BMPs signal through a family of transmembrane serine/threonine kinase receptors, including type I and type II receptors. Activin receptor like kinase-3 (ALK-3) is identified as a key type I receptor in humans.
Purpose of the Study:
- To investigate the expression and characteristics of the native murine activin receptor like kinase-3 (ALK-3) protein in various murine tissues and cell lines responsive to bone morphogenetic proteins.
- To elucidate the protein complexes and interactions involving ALK-3 during bone morphogenetic protein signaling.
Main Methods:
- Utilized a polyclonal antibody to detect native murine ALK-3 protein via Western blot analysis in tissue extracts and cell lines.
- Employed immunoprecipitation with [125I] BMP-4 and biotin surface-labeling techniques to identify ALK-3 associated proteins and complexes.
- Performed deglycosylation experiments to analyze the protein structure and compare it to known BMP receptors.
Main Results:
- Western blot analysis confirmed the expression of the 85 kDa ALK-3 protein in multiple murine tissues, with varying correlations between protein and mRNA levels.
- Constitutive expression of ALK-3 protein was observed in BMS2 bone marrow stromal cells and C2C12 myoblasts, with minor changes during cell differentiation.
- Immunoprecipitation revealed ALK-3 associated with 90 kDa and 170 kDa complexes, and surface-labeling identified a novel 140 kDa glycoprotein, which upon deglycosylation, matched the size of the BMP type II receptor.
Conclusions:
- The 85 kDa ALK-3 protein is widely expressed in murine tissues and cell lines, indicating its broad role in BMP signaling.
- ALK-3 forms pre-existing complexes with other receptor proteins, including a novel 140 kDa glycoprotein identified as the BMP type II receptor.
- These findings support the model that BMPs bind to a pre-formed complex of type I and type II receptors to initiate signaling pathways.