title: Growth factor receptor binding protein 2-mediated recruitment of the RING domain of Cbl to the epidermal growth factor receptor is essential and sufficient to support receptor endocytosis.
abstract: Knockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR). To gain insights into the function of Grb2 in EGFR endocytosis, we have generated cell lines in which endogenous Grb2 was replaced by yellow fluorescent protein (YFP)-tagged Grb2 expressed at the physiological level. In these cells, Grb2-YFP fully reversed the inhibitory effect of Grb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis. Overexpression of Grb2-binding protein c-Cbl did not restore endocytosis in Grb2-depleted cells. However, EGFR endocytosis was rescued in Grb2-depleted cells by chimeric proteins consisting of the Src homology (SH) 2 domain of Grb2 fused to c-Cbl. The "knockdown and rescue" analysis revealed that the expression of Cbl-Grb2/SH2 fusions containing RING finger domain of Cbl restores normal ubiquitylation and internalization of the EGFR in the absence of Grb2, consistent with the important role of the RING domain in EGFR endocytosis. In contrast, the carboxy-terminal domain of Cbl, when attached to Grb2 SH2 domain, had 4 times smaller endocytosis-rescue effect compared with the RING-containing chimeras. Together, the data suggest that the interaction of Cbl carboxy terminus with CIN85 has a minor and a redundant role in EGFR internalization. We concluded that Grb2-mediated recruitment of the functional RING domain of Cbl to the EGFR is essential and sufficient to support receptor endocytosis.
authors.affiliation: Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, CO 80045, USA.
authors.forename: Fangtian
authors.initials: F
authors.lastname: Huang
authors.forename: Alexander
authors.initials: A
authors.lastname: Sorkin
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C433810:SH3KBP1 protein, human; D025801:Ubiquitin; C054562:yellow fluorescent protein, Bacteria; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; C067419:CBL protein, human