Recruitment of the Adaptor Protein Grb2 to EGFR Tetramers

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• 作者：Noga Kozer ; Dipak Barua ; Christine Henderson ; Edouard C. Nice ; Antony W. Burgess ; William S. Hlavacek ; Andrew H. A. Clayton
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：April 29, 2014
• 年：2014
• 卷：53
• 期：16
• 页码：2594-2604
• 全文大小：483K
• 年卷期：v.53,no.16(April 29, 2014)
• ISSN：1520-4995

文摘

Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected F枚rster resonance energy transfer (also known as FLIM-based FRET or FLIM鈥揊RET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2鈥搈RFP) and EGFR (EGFR鈥揺GFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2鈥搈RFP with EGFR鈥揺GFP clusters, which contained, on average, 4 卤 1 copies of EGFR鈥揺GFP per cluster. In contrast, the pool of EGFR鈥揺GFP without Grb2鈥搈RFP had an average cluster size of 1 卤 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR鈥揺GFP and Grb2鈥搈RFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit.