Identification of a candidate integrin-fraction associated with the activated form of the PDGF-receptor.
The activated PDGF-receptor has been shown to coimmunoprecipitate together with alpha v beta 3 integrin out of the 15,000g soluble supernatant of non-ionic detergent cell lysates. I have now further characterized this complex by ultracentrifugation analysis. The ultracentrifugation-conditions were chosen so that the phosphorylated form of the PDGF-receptor was pelleted out of the 15,000g soluble supernatant. Together with the tyrosine-phosphorylated PDGF-receptor small amounts of integrins, cytoskeletal- and extracellular matrix proteins were recovered in the pelleted material. The results show that (i) the candidate-fraction of integrins interacting with the activated PDGF-receptor is small compared to the overall integrin population in the cell lysate, (ii) several proteins known to be present in focal adhesions such as FAK, talin, and vinculin are absent from the integrin-growth factor receptor complexes, while on the other hand (iii) a tyrosine-phosphorylated protein migrating at 120 kDa was highly enriched in the ultracentrifugation-pellet, and finally (iv) non-ionic detergent cell lysates appear to contain quantitatively small fractions of complexed proteins that are qualitatively distinct from their total cellular population. Thus, the separation of protein-complexes from the total cellular proteom may be instrumental for the investigation of cellular protein complexes in general.
Schneller
M
M
Department of Cell and Molecular Biology, Lund University, SE-221 00 Lund, Sweden. max.schneller@medkem.lu.se
D002458:Cell Fractionation; D002474:Cell-Free System / Q000737:chemistry; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000302:isolation & purification / Q000378:metabolism*; D017830:Octoxynol; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011233:Precipitin Tests; D011485:Protein Binding; D017479:Receptors, Platelet-Derived Growth Factor / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D014443:Tyrosine / Q000378:metabolism; D014461:Ultracentrifugation
D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D016023:Integrins; D010750:Phosphoproteins; D011092:Polyethylene Glycols; D014443:Tyrosine; D017830:Octoxynol; C010615:Nonidet P-40; D017479:Receptors, Platelet-Derived Growth Factor