Abstract
The tyrosine kinase receptor for macrophage colony-stimulating factor and the non-receptor tyrosine kinase c-Src play critical roles in osteoclast differentiation and function. Since the ubiquitously expressed adaptor protein Grb2 plays an important role in several tyrosine kinase signal transduction pathways, we used a filter binding assay to identify osteoclast proteins that bind to Grb2. In osteoclasts, there were three major Grb2-binding proteins, two of which, mSos and c-Cbl (p120), have been previously identified as Grb2-binding proteins in many cell types. The third protein, p135, had a restricted pattern of expression and was present at high levels in authentic osteoclasts and osteoclast-like cells formed in an in vitro co-culture system. In addition to binding Grb2 in the filter binding assay, p135 was isolated in complexes with endogenous Grb2 from osteoclast cell extracts. The association of p135 and Grb2 was dependent on an intact Src homology 3 domain and furthermore, was shown to preferentially interact with the N-terminal Src homology 3 domain of Grb2, which is similar to the interaction of mSos and Grb2 in other cell types. p135 was not recognized by antibodies against several known Grb2-binding proteins and thus may be a novel Grb2-binding protein.
Highlights
Signaling events mediated by tyrosine kinases play an important role in regulating cell growth, differentiation, and transformation
GST-growth factor receptor binding protein 2 (Grb2) Fusion Protein Binds to a 135-kDa Protein That Is Highly Expressed in Osteoclasts—To compare the pattern of Grb2-binding proteins in osteoclasts relative to other cell types, cellular lysates were probed with a GST fusion protein containing the entire coding sequence of Grb2
This assay detected a 135-kDa Grb2-binding protein (p135) that is highly enriched in osteoclast-like cells (OCLs) as well as in rabbit osteoclasts (ROCs) relative to other tissue and cell types (Fig. 1, A and B)
Summary
Signaling events mediated by tyrosine kinases play an important role in regulating cell growth, differentiation, and transformation. One protein that mediates the interaction of tyrosine kinases with their targets is the ubiquitously expressed growth factor receptor binding protein 2 (Grb). The SH2 domain of Grb facilitates binding to specific phosphorylated tyrosine residues such as the phosphorylated sites found in ligand-activated growth factor receptors, while the SH3 domains of Grb interact with proline-rich sequences found in a variety of cytosolic proteins. The Grb21⁄7Sos complex aids in transmitting signals from activated tyrosine kinase receptors to the Ras pathway. Another Grb2-binding protein is Shc, whose interaction with the SH2 domain of Grb depends upon the tyrosine phosphorylation of Shc by activated growth factor receptors or by oncogenic tyrosine kinases such as v-src and v-fps [18, 19]. This protein binds to Grb in an in vitro assay and physically associates with Grb in osteoclast lysates
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