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

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.Consejería de Educación, Juventud y Deportes–Comunidad de Madrid,Grant/Award Number: B2017/BMD‐36involving contributions from the EuropeanFunds for Regional Development (EFRD) andthe Social European Fund (SEF); ConsejoSuperior de Investigaciones Científicas, Grant/Award Number: COOPA20053;Secretaría de Estado de Investigación, Desarrollo e Innovación, Grant/Award Number: SAF2014‐52048‐R; Agencia Española de Cooperación Internacional para el Desarrollo, Grant/Award Numbers: A/019018/08,A/5444/06, A/8197/0

Funcionalidad del dominio de unión a calmodulina y del dominio similar a calmodulina del receptor del factor de crecimiento epidérmico

Trabajo Fin de Grado: Grado en Biología Sanitaria.[ES] El receptor del factor de crecimiento epidérmico (EGFR) es un receptor transmembrana con actividad tirosina quinasa. Este trabajo se centra en comprender el funcionamiento de este receptor, más concretamente en cómo se produce su dimerización y activación. El EGFR tiene una secuencia citosólica yuxtamembranal muy básica denominada dominio de unión a calmodulina (CaMBD) y otra secuencia acídica, localizada distalmente del dominio catalítico, que se denomina dominio parecido a calmodulina (CaMLD). Debido a estudios previos se postuló que la dimerización del receptor ocurre de la siguiente forma: tras su unión con el ligando factor de crecimiento epidérmico (EGF), el receptor se dimeriza y al mismo tiempo el complejo Ca2+/calmodulina (Ca2+/CaM) se une al CaMBD, induciendo la separación de este dominio de la membrana celular, a la que está unida electrostáticamente, favoreciendo la activación del receptor. El CaMBD de uno de los monómeros interacciona electrostáticamente con el CaMLD del monómero opuesto, liberando Ca2+/CaM del primero, y estabilizando el dímero. Para intentar validar este modelo, se han realizado experimentos de coinmunoprecipitación y cromatografía de gradiente de masas con la proteína recombinante glutatión S-transferasa (GST) fusionada a la secuencias del CaMBD y del CaMLD, para comprobar si existe dicha interacción. Los resultados de los experimentos, sin embargo, fueron inconcluyentes debido a que la proteína GST sola interacciona consigo misma formando dímeros y posiblemente oligómeros y con otras proteínas.[EN] The epidermal growth factor receptor (EGFR) is a transmembrane receptor with intrinsic tyrosine-kinase activity. This project tries to understand some aspects of the EGFR functionality. More specifically, the mechanism of dimerization and activation of the EGFR. This receptor has a highly basic cytosolic juxtamembrane sequence denoted the calmodulin-binding domain (CaMBD), and another acidic sequence denoted the calmodulin-like domain (CaMLD), located distally of the catalytic domain. Based on previous studies, it was proposed a hypothetical model in which the dimerization and activation of the EGFR happen in the following manner: the epidermal growth factor (EGF) binds to the EGFR inducing its dimerization. At the same time, Ca2+/calmodulin (Ca2+/CaM) binds to the CaMBD inducing the separation of this domain from the inner leaflet of the plasma membrane, where is bound electrostatically. Thereafter, the CaMBD of one monomer interacts electrostatically with the CaMLD of the apposed monomer, releasing the Ca2+/CaM complex, and stabilizing the dimer. Two types of experiments were performed in this project to validate this model. These experiments were coimmunoprecipitation and gel filtration chromatography with the recombinant protein glutathione-S-transferase (GST) fused to the CaMBD and CaMLD sequences. In this manner, the project tries to confirm whether both sequences are able to interact which each other. However, the results of the experiments were inconclusive because the protein GST alone was able of self-association forming dimers, and perhaps oligomers.Peer reviewe

A cell-permeable peptide corresponding to the calmodulin-binding domain of Grb7 inhibits proliferation and migration of A431 cells

Trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018.[Introduction]: Grb7 (growth factor receptor bound 7) is a mammalian adaptor protein that transduces signals from tyrosine kinase receptors. This protein is overexpressed, together with ErbB2, in different human carcinomas. Grb7 is implicated in cell migration, contributing to the invasiveness and metastatic capacity of tumour cells. Also, Grb7 controls cell proliferation and tumour-associated angiogenesis. Our group demonstrated that calmodulin (CaM) binds to the proximal region of the pleckstrin homology (PH) domain of human Grb7 in a Ca2+-dependent manner, comprising the sequence 243RKLWKRFFCFLRRS256. A deletion mutant of Grb7, lacking its CaM-binding domain (CaM-BD), impairs the adhesion and migratory capacity of transfected cells. Moreover, the expression of this deletion mutant in rat C6 glioblastoma cells strongly inhibited its proliferation, the growth of brain tumours derived from stereotaxically implanted tumour cells, and tumour-associated angiogenesis. Therefore, the CaM-BD of Grb7 could be a potential target to inhibit tumour cells invasiveness and metastasis development. [Materials and Methods]: Human carcinoma epidermoid A431 cells were used in all the experiments. Custom-made synthesis of peptides (>99% purity) with the amino acid sequence RKLWKRFFCFLRRS, and a derivative with a myristoyl group at its N-terminus (Myr-RKLWKRFFCFLRRS) to facilitate cell internalization, were manufactured by Wuxi Nordisk Biotech Ltd. (China). Cell migration was measured by artificial wound healing assays and time-lapse microscopy, and cell proliferation was measured by Crystal Violet staining. The assays were done in the absence and presence of 10 nM EGF, and 20-50 µg/ml of the different peptides, or 10-50 µM W-7 and W-12 (CaM antagonists). [Results]: The addition of EGF first induced a 12-18 h lag phase in which the migration of A431 cells was arrested, and a significant retraction of the border of the artificial wound was observed. Thereafter, the migration rate of the cells increased several folds as compared to the rate of migration of cells in the absence of EGF. The RKLWKRFFCFLRRS peptide inhibited the migration of A431 cells both in the absence and presence of EGF. This inhibitory effect was more evident using the Myr-RKLWKRFFCFLRRS peptide. The high affinity CaM antagonist W-7, and in lesser extent its low affinity analogue W-12, incremented the lag phase in which the migration of the cells was arrested upon addition of EGF and decreased its subsequent migration rate. As previously demonstrated, EGF induced a significant inhibition of cell proliferation in A431 cells, and both the RKLWKRFFCFLRRS and Myr-RKLWKRFFCFLRRS peptides further inhibited the proliferative response. [Conclusion]: The mechanism of action of the peptides under study could be due to the capacity to sequester CaM and/or to the prevention of Grb7 dimerization, possibilities that are under study. These peptides, upon selected delivery to tumour cells, could have potential therapeutic effects against cancer and metastasis development.Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2014-52048-R) and Consejería de Educación, Juventud y Deportes - Comunidad de Madrid (B2017/BMD-36).Peer Reviewe