The role of the calmodulin‐binding and calmodulin‐like domains of the epidermal growth factor receptor in tyrosine kinase activation

• Published in: Journal of cellular physiology 2021-07, Vol.236 (7), p.4997-5011
• Main Authors: Abdelli, Faten, Jellali, Karim, Anguita, Estefanía, González‐Muñoz, María, Villalobo, Eduardo, Madroñal, Ivan, Alcalde, Juan, Ben Ali, Mamdouh, Elloumi‐Mseddi, Jihene, Jemel, Ikram, Tebar, Francesc, Enrich, Carlos, Aifa, Sami, Villalobo, Antonio
• Format: Article
• Language: English
• Subjects: Allosteric properties; Asymmetric structures; Binding; Calcium-binding protein; Calmodulin; calmodulin‐binding domain; calmodulin‐like domain; Confocal microscopy; Deactivation; Deletion; Dimers; Domains; Electrostatic properties; Epidermal growth factor; epidermal growth factor receptor; Epidermal growth factor receptors; Flow cytometry; Fluorescence; Green fluorescent protein; Growth factors; Histidine; Inactivation; Internalization; Kinases; Molecular structure; Mutants; Protein-tyrosine kinase; receptor internalization; Receptors; Tyrosine; tyrosine kinase activity; Valine
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.30205

The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)‐binding domain (CaM‐BD) and a CaM‐like domain (CaM‐LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM‐BD (EGFR/CaM‐BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM‐LD (EGFR/CaM‐LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM‐LD with a histidine/valine‐rich peptide (EGFR/InvCaM‐LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR‐green fluorescent protein (GFP)/CaM‐BD∆, the EGFR/CaM‐LD∆, and EGFR/InvCaM‐LD mutants all bind tetramethylrhodamine‐labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild‐type receptor does. However, only the EGFR/CaM‐LD∆ and EGFR/InvCaM‐LD mutants appear to undergo ligand‐dependent internalization, while the EGFR‐GFP/CaM‐BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM‐BD/CaM‐LD electrostatic interaction in the allosteric activation of the EGFR TK. Model of the asymmetric structure of the epidermal growth factor receptor kinase dimer showing that the calmodulin‐binding domain (blue surface) and calmodulin‐like domain (red surface) of apposed monomers are well positioned to establish electrostatic interaction. The model highlights the transmembrane‐juxtamembrane segments and the activator and receiver monomers in relaxed and activated states.