The Role of Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) in the Internalization of Platelet-derived Growth Factor Receptor (PDGFR) in Mouse Embryonic Fibroblast

KREIMANN EL; BRADSHAW BL; GEORGESCU MM

Houston, TX

Simposio; 58th Annual Symposium on Cancer Research; 2005

MD Anderson Cancer Center

EBP50 is an adaptor protein containing two PDZ domains (PDZ1 and PDZ2) that interacts with a wide array of proteins. EBP50 binds to the platelet-derived growth factor receptor (PDGFR) (Maudsley et al., 2000) through the PDZ1 domain. We hypothesize that the interaction between EPB50 and PDGFR is important in PDGFR’s function, stability, and internalization. PDGFR binding to PDGF induces receptor dimerization, autophosphorylation, and subsequent signal transduction.  PDGF is one of the most potent serum mitogens as well as one of the most studied growth factors and is up-regulated in different types of cancers (Heldin and Westermark, 1999) such as medulloblastomas, malignant gliomas, and ovarian tumors (Andrae et al., 2002, Henriksen et al., 1993).             The activation and internalization of the PDGFR was studied in Mouse embryonic fibroblasts (MEFs) immortalized with SV40 t-antigens. The MEFs were generated from the EBP50-deficient mice developed in our laboratory. EBP50 (+/+) and (-/-) MEFs were incubated with a biotinylated PDGFR antibody (PDGFR-Ab-B) followed by incubation with Streptavidin-coated Quantum Dots 565 semiconductor nanocrystals (S-QDOTs) at 4˚C to permit binding. After washing the excess of S-QDOTs, the cells were incubated at 37˚C for T=0 (control), 10, 30 and 60 minutes to allow the internalization of the PDGFR-PDGFR-Ab–B-S-QDOTs complex. In a parallel experiment, after the incubation with the S-QDOTs, the cells were analyzed by immunofluorescence using phospho-PDGFR (T=O), Rab5 (T=10), and LAMP (T=30 and 60) antibodies to visualize the subcellular localization of the S-QDOTs.           The streptavidin-QDOTs nanocrystals were able to bind the PDGFR antibody coupled to biotin. In the control sample lacking the PDGFR antibody, the S-QDOTs incubated directly with the cells were completely washed away and no S-QDOTs were seen on the cell surface. This showed that the binding to the receptor was specific with no nonspecific binding to other membrane structures.  Both EBP50 (+/+) and (-/-) cells internalized the S-QDOTs nanocrystals; however, the internalization was faster in the EBP50 (-/-) cells, as we observed after 10 minutes of incubation with the S-QDOTs at 37˚C. Further studies need to be carried out in order to quantify the differences between the EBP50 (+/+) and (-/-) cells and also to characterize the endosomal traffic and recycling of the PDGFR in the absence of EBP50.  In conclusion, these results showed an increase in the internalization of the PDGFR in EBP50 null cells supporting a role of EBP50 in the stabilization of the PDGFR at the plasma membrane.