The adaptors Grb10 and Grb14 are calmodulin‐binding proteins

We identified the Grb7 family members, Grb10 and Grb14, as Ca2+‐dependent CaM‐binding proteins using Ca2+‐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 t...

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Bibliographic Details
Published in:FEBS letters 2017-04, Vol.591 (8), p.1176-1186
Main Authors: García‐Palmero, Irene, Pompas‐Veganzones, Noemí, Villalobo, Eduardo, Gioria, Sophie, Haiech, Jacques, Villalobo, Antonio
Format: Article
Language:English
Subjects:
adaptor proteins
Adaptor Proteins, Signal Transducing - chemistry
Adaptor Proteins, Signal Transducing - genetics
Adaptor Proteins, Signal Transducing - metabolism
Amino Acid Sequence
Binding Sites
Calcium Signaling
calmodulin
Calmodulin - chemistry
Calmodulin - metabolism
calmodulin‐binding sites
Chromatography, Affinity
Conserved Sequence
Gene Deletion
Grb10
GRB10 Adaptor Protein - chemistry
GRB10 Adaptor Protein - genetics
GRB10 Adaptor Protein - metabolism
Grb14
Grb7
GRB7 Adaptor Protein - chemistry
GRB7 Adaptor Protein - genetics
GRB7 Adaptor Protein - metabolism
HEK293 Cells
Humans
Kinetics
Mutagenesis, Site-Directed
Peptide Fragments - chemistry
Peptide Fragments - genetics
Peptide Fragments - metabolism
Protein Conformation
Protein Interaction Domains and Motifs
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - metabolism
Sequence Homology, Amino Acid
Structural Homology, Protein
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Summary:We identified the Grb7 family members, Grb10 and Grb14, as Ca2+‐dependent CaM‐binding proteins using Ca2+‐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. We identify Grb7, Grb10 and Grb14 as calmodulin (CaM)‐binding proteins. The interaction with CaM is Ca2+‐dependent. We identify the potential CaM‐binding domains of these adaptor proteins.
ISSN:0014-5793
1873-3468
DOI:10.1002/1873-3468.12623