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Analysis of Grb7 recruitment by heregulin-activated erbB receptors reveals a novel target selectivity for erbB3
R J Fiddes1, D H Campbell, P W Janes
1Cancer Research Program, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales 2010, Australia.
Abstract:
Heregulin-mediated activation of particular erbB receptor combinations was used as a model system to investigate the interaction of erbB3 and erbB4 with the adaptor protein growth factor receptor-bound (Grb)7. In human breast cancer cell lines, co-immunoprecipitation of Grb7 with both receptors was detected upon heregulin stimulation. This association was direct and mediated by the Grb7 Src homology (SH)2 domain. Co-expression of erbB2 with erbB3 point mutants was used to map Grb7 binding sites. This demonstrated that tyrosine 1180 and 1243 represent the major and minor sites of Grb7 interaction, respectively. Although these recognition sequences possess an Asn residue at +2 relative to the phosphotyrosine and therefore represent potential Grb2 binding sites, phosphopeptide competition and "pull-down" experiments demonstrated that they interact preferentially with the Grb7 versus the Grb2 SH2 domain. Substitution analysis indicated that an Arg residue at +3 could act as a selectivity determinant, but the effect was context-dependent. Consequently, the Grb2 and Grb7 SH2 domains possess overlapping, but distinct, specificities. These studies therefore identify Grb7 as an in vivo target of erbB3 and erbB4 and provide an underlying mechanism for the ability of erbB3 to recruit Grb7 and not Grb2, a property unique among erbB receptors.
Insights
Growth factor receptor-bound 7 (Grb7) directly binds erbB3 and erbB4 receptors, crucial for breast cancer cell signaling. This interaction is specific, distinguishing Grb7 from Grb2 binding sites.
Area of Science:
- Cellular signaling
- Molecular biology
- Cancer research
Background:
- The erbB receptor family plays a critical role in cell growth and differentiation.
- Understanding adaptor protein interactions with erbB receptors is key to deciphering signaling pathways in cancer.
Purpose of the Study:
- To investigate the interaction between erbB3/erbB4 receptors and the adaptor protein growth factor receptor-bound 7 (Grb7).
- To elucidate the molecular mechanisms underlying Grb7 binding to erbB receptors and its specificity compared to Grb2.
Main Methods:
- Co-immunoprecipitation assays in human breast cancer cell lines.
- Site-directed mutagenesis to map Grb7 binding sites on erbB3.
- Phosphopeptide competition and pull-down assays to assess SH2 domain binding specificity.
Main Results:
- Heregulin stimulation induced direct association between Grb7 and both erbB3 and erbB4 receptors.
- Tyrosine residues 1180 (major) and 1243 (minor) on erbB3 were identified as Grb7 binding sites.
- Grb7 preferentially binds to these sites over Grb2, despite sequence similarities, due to specific SH2 domain interactions.
Conclusions:
- Grb7 is identified as an in vivo binding target of erbB3 and erbB4 receptors.
- The findings provide a mechanism for erbB3's unique ability to recruit Grb7 over Grb2.
- This specificity is critical for understanding distinct signaling outcomes mediated by different erbB receptor combinations.