The adaptors Grb10 and Grb14 are calmodulin‐binding proteins

Abstract

We identified the Grb7 family members, Grb10 and Grb14, as Ca -dependent CaM-binding proteins using Ca -dependent CaM-affinity chromatography as we previously did with Grb7. The potential CaM-binding sites were identified and experimentally tested using fluorescent-labeled peptides corresponding to these sites. The apparent affinity constant of these peptides for CaM, and the minimum number of calcium ions bound to CaM that are required for effective binding to these peptides were also determined. We prepared deletion mutants of the three adaptor proteins lacking the identified sites and determined that they lost or strongly diminished their CaM-binding capacity following the sequence Grb7 > > Grb14 > Grb10. More than one CaM-binding site and/or accessory CaM-binding sites appear to exist in Grb10 and Grb14, as compared to a single one present in Grb7.

A coding and non-coding transcriptomic perspective on the genomics of human metabolic disease

“…In the present study, we have established the first robust gene expression signature for IS and utilized three strategies to validate our findings and explore the functional implications of our observations: systematic literature review, quantification of responses to clinical interventions; and application of systems biology methods. We independently identified many known IS regulators, including GRB14 , a receptor tyrosine kinase adaptor protein (113) which inhibits insulin signaling (105,114), located at a GWAS significant metabolic disease loci, while providing the first detailed map of the relationship between lncRNAs and IS. We may have expected a greater number of GWAS loci related specifically to insulin, however as noted above, linear modeling does not fully capture the complex interactions between genes, insulin, obesity and risk of development of T2DM.…”

The Role of Calmodulin in Tumor Cell Migration, Invasiveness, and Metastasis

“…The adaptor protein Grb7 binds CaM in a Ca 2+ -dependent manner [284], as this is also the case for the Grb7 family members 10 and 14 [285], and plays an important role in cell migration and tumor cell invasiveness (reviewed in [21,286,287]). Deletion of the CaM-binding site of human Grb7, located in the proximal region of its pleckstrin homology domain, had an inhibitory effect on cell attachment to the substrate, impaired the migratory capacity of the cells and the development of brain tumors in vivo shown by implantation of rat glioblastoma cells transfected with a Grb7-mutant lacking the CaM-binding site [288,289].…”

Direct Interaction between Calmodulin and the Grb7 RA-PH Domain

“…Due to the high level of homology between Grb7 and family members Grb10 and Grb14, it could be predicted that CaM may also interact with these proteins in a similar manner. Indeed, initial work has revealed that Grb10 and Grb14 are also bound by CaM and that the interaction site may be similar to that proposed for Grb7 [13]. Whether these interactions occur via direct interaction or with significant affinity, and the cellular implications, awaits further investigation.…”

“…Typically, calcium activated CaM binds to its target proteins with affinities in the range of 100 nM to 10 pm [25]. Futhermore, the Grb7 peptide, representing the CaM binding site out of the context of surrounding secondary and tertiary structure, was reported to have an affinity for CaM of 2.5 nM [13]. With the affinity measurement observed here > 1000-fold weaker than other reported interactions, it is postulated that additional cellular factors, such as post-translational modifications, and/or conformational rearrangements are likely to play a role to enhance the affinity of the Grb7/CaM interaction.…”

“…For Grb7, the CaM binding site was mapped to the proximal region of the PH domain (Grb7 residues 243-256). A peptide representing this region was shown to have high affinity for CaM [13].…”