Functional implication of the calmodulin-binding domain of the adaptor protein Grb7

Resumen

The growth factor receptor bound protein 7 (Grb7) gives name to a mammalian adaptor protein family that also includes Grb10 and Grb14. As an adaptor protein, Grb7 mediates signal transduction events initiated by tyrosine kinase receptors present in the plasma membrane or other cytosolic kinases through its Src homology 2 (SH2) domain. Our laboratory previously described the presence of a calmodulin (CaM)-binding domain (CaM-BD) located in the proximal region of the pleckstrin homology (PH) domain of Grb7 and described that Grb7 was able to bind CaM in a Ca2+-dependent manner both in vitro and in living cells. In this Thesis, we first described that the other Grb7 family members, Grb10 and Grb14, are also CaM-binding proteins (CaM-BP). To investigate the role that CaM plays in Grb7 functionality, we generated a deletion mutant lacking the CaM-BD named Grb7delta. We report the differential behavior in vitro and in vivo of cells expressing Grb7delta, as compared to those expressing either its wild type counterpart or cells lacking expression of this adaptor protein. Our results showed that deletion of the CaM-BD resulted in a lower cell migration rate, impaired cell adhesion ability to the extracellular matrix, and a lower proliferation rate. Additionally, we demonstrated that Grb7delta was unable to localize in the nuclear fraction and that inhibition of CaM favored the nuclear localization of wild type Grb7. Therefore, we proposed that the CaM-BD of Grb7 overlapped its nuclear localization sequence (NLS). Besides, we performed in vivo experiments to study the role of the CaM-BD of Grb7 in tumor growth and tumor-associated angiogenesis by implanting glioma C6 cells stably expressing EYFP, EYFP-Grb7 or EYFP-Grb7delta in the brain of rats and monitoring the tumor growth and its associated angiogenesis using Magnetic Resonance Imaging (MRI). We found that tumors generated by EYFP-Grb7delta-expressing cells were smaller and less vascularized than those grown from cells expressing its wild type EYFP-Grb7 counterpart or EYFP-expressing control cells, suggesting an anti-angiogenic effect of the mutant protein. Finally, using co-immunoprecipitation and Mass Spectrometry-based proteomics, we described novel Grb7 and/or Grb7delta binding partners. We validated by Western blot Hsc70 (heat shock cognate 70 protein) and caprin-1 (cytoplasmic activation/proliferation-associated protein-1) as Grb7 and Grb7delta associated partners, and Nedd4 (neural precursor cell expressed developmentally down-regulated protein 4) as a Grb7 binding partner.