Abstract
Grb7 is a signalling adapter protein that engages activated receptor tyrosine kinases at cellular membranes to effect downstream pathways of cell migration, proliferation and survival. Grb7’s cellular location was shown to be regulated by the small calcium binding protein calmodulin (CaM). While evidence for a Grb7/CaM interaction is compelling, a direct interaction between CaM and purified Grb7 has not been demonstrated and quantitated. In this study we sought to determine this, and prepared pure full-length Grb7, as well as its RA-PH and SH2 subdomains, and tested for CaM binding using surface plasmon resonance. We report a direct interaction between full-length Grb7 and CaM that occurs in a calcium dependent manner. While no binding was observed to the SH2 domain alone, we observed a high micromolar affinity interaction between the Grb7 RA-PH domain and CaM, suggesting that the Grb7/CaM interaction is mediated through this region of Grb7. Together, our data support the model of a CaM interaction with Grb7 via its RA-PH domain.
Highlights
Growth receptor bound 7 (Grb7) protein is a cytoplasmic adapter protein that couples activated tyrosine kinase receptors to a nexus of downstream proliferative, migratory and survival signalling pathways [1,2]
Full-length Grb7, Grb7-RA-Pleckstrin homology (PH) and Grb7-Src-homology 2 (SH2) were prepared as GST-tagged constructs to enable efficient capture on biosensor chips via anti GST-antibodies (Figure 3A)
We investigated the relationship between Grb7 and the calcium sensor protein CaM
Summary
Growth receptor bound 7 (Grb7) protein is a cytoplasmic adapter protein that couples activated tyrosine kinase receptors to a nexus of downstream proliferative, migratory and survival signalling pathways [1,2]. Mig-10 (the Grb and Mig region, GM) and a C-terminal Src-homology 2 (SH2) domain [3]. The GM domain, in turn, is made up of Ras-associating (RA) and Pleckstrin homology (PH) domains and a BPS (between PH and SH2) domain (Figure 1A). It is through the C-terminal SH2 domain that. The RA domain can influence proliferative signalling pathways by interacting with activated GTP bound
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