BMS 378806 gp120/CD4 inhibitor tated the equivalent residue in the EGFRvIII

tated the equivalent residue in the EGFRvIII, we prevented the ubiquitination and downregulation of this receptor by N1/2 Cbl b. However, the mutation of this residue does not appear to have as significant an effect upon the interaction between the EGFRvIII and WT Cbl b. As the proline rich region of the Cbl proteins can indirectly bind to the WT EGFR via Grb2, this BMS 378806 gp120/CD4 inhibitor is likely also the case with the EGFRvIII. The EGFRvIII has been shown to bind to Grb2 in NIH 3T3 fibroblasts. Interestingly, stable clones of NIH 3T3 cells expressing high levels of the EGFRvIII have decreased levels of Grb2 protein. This is consistent with the ability of the Cbl proteins to downregulate the EGFR signaling complex, including Grb2. In contrast to the present study, Schmidt et al.
reported that the EGFRvIII does not Camptothecin 7689-03-4 interact with either Cbl or Cbl b. In their investigation, HEK 293 cells were transfected with EGFRvIII and either Cbl or Cbl b. Then the EGFRvIII was precipitated with an anti EGFRvIIIspecific antibody. Although they observed the co precipitation of both Cbl and Cbl b with the EGFRvIII, the WT EGFR was also precipitated in their experiments. They concluded that the anti EGFRvIII antibody was crossreacting with the WT receptor, so in subsequent experiments they precleared the lysate with an anti EGFR antibody before the precipitation of the EGFRvIII. Following preclearing of the lysates, they failed to observe either Cbl or Cbl b when the EGFRvIII was precipitated. In addition, they were also unable to observe any ubiquitination of the EGFRvIII following this preclearing.
As the EGFRvIII and the WT EGFR are capable of heterodimerizing, it is possible that this preclearing step removed any of the EGFRvIII that is bound to the WT EGFR. As this heterodimerized protein may be the active pool of the EGFRvIII, this could account for any differences between the two studies. Our experiments in CHO cells, which do not express the WT EGFR, allowed us to investigate the interaction between the EGFRvIII and the Cbl proteins in the absence of the WT receptor. In addition, we used a mutant of Cbl b deficient in E3 activity to test an interaction between the EGFRvIII and Cbl b in CHO cells. As this mutant cannot target the complex of Cbl b and the EGFRvIII for lysosomal degradation, the amount of active EGFRvIII bound to Cbl b should be increased relative to cells transfected with WT Cbl b.
Therefore, any association between these proteins should be detected with a greater sensitivity than if WT Cbl b was used. Only a small fraction of the EGFRvIII protein is active at any given time compared to the WT EGFR that has been stimulated by EGF. Thus, it is possible that the interaction between the EGFRvIII Davies et al. Page 7 Oncogene. Author manuscript, available in PMC 2008 March 25. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript and the Cbl proteins was below the level of sensitivity of the immunoprecipitation and immunoblotting procedure used by Schmidt et al.. The constitutive TK activity of the EGFRvIII results in the malignant transformation of cells. In this study, we found that the EGFRvIII is regulated by the Cbl proteins in an identical manner to the WT EGFR. This is unsurprising given that the activity and phosphorylation pattern of the dimerized EGFRvIII is similar to that of the WT EGFR following EGF stimulation. Indeed, we we