Identification of a novel 135-kDa Grb2-binding protein in osteoclasts.
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.
Sahni
M
M
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Zhou
X M
XM
Bakiri
L
L
Schlessinger
J
J
Baron
R
R
Levy
J B
JB
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D051380:GRB2 Adaptor Protein; D016173:Macrophage Colony-Stimulating Factor / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010010:Osteoclasts / Q000737:chemistry*; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine; D055232:Proline-Rich Protein Domains; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry; D050721:Proto-Oncogene Proteins c-cbl; D015398:Signal Transduction; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D010455:Peptides; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D016173:Macrophage Colony-Stimulating Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C493849:Cbl protein, mouse