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Integrin alpha 2 beta 1-dependent EGF receptor activation at cell-cell contact sites
1Institute of Life Science and Research Center for Innovative Cancer Therapy, Kurume University, Kurume, Fukuoka 839-0861, Japan.
Insights
Integrin alpha 2 beta 1 physically associates with epidermal growth factor receptor (EGFR) at cell junctions. This interaction drives EGFR phosphorylation independently of serum, highlighting integrins
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Integrins are cell surface receptors involved in cell-cell and cell-extracellular matrix interactions.
- Epidermal Growth Factor Receptor (EGFR) is a key regulator of cell growth, proliferation, and survival.
- The localization and function of integrins and EGFR at intercellular sites suggest potential crosstalk.
Purpose of the Study:
- To investigate the physical and functional association between integrin alpha 2 beta 1 and EGFR at intercellular adhesion sites.
- To determine the role of integrin alpha 2 beta 1 in regulating EGFR activity at cell-cell contacts.
Main Methods:
- Co-immunoprecipitation assays to detect protein-protein interactions.
- Chemical crosslinking to confirm direct association.
- Double immunofluorescence staining for colocalization studies.
- Western blotting using antibodies specific to activated EGFR.
- Functional blocking antibodies to assess the role of integrins in EGFR phosphorylation.
Main Results:
- Integrin alpha 2 beta 1 physically associates with EGFR at intercellular adhesion sites in A431 cells.
- EGFR at cell-cell contact sites is phosphorylated independently of serum, mediated by integrin alpha 2 beta 1.
- Cell-cell adhesion is crucial for this serum-independent EGFR phosphorylation.
Conclusions:
- Integrin alpha 2 beta 1 forms a physical complex with EGFR at cell-cell contact sites.
- Integrin alpha 2 beta 1 plays a critical role in the activation of EGFR at intercellular junctions, independent of EGF stimulation.
- This study reveals a novel mechanism for integrin-mediated regulation of receptor tyrosine kinase signaling in cell-cell interactions.
Abstract:
Certain integrins including alpha 2 beta 1 and alpha 3 beta 1 localize to intercellular binding sites, and thus may participate in cell-cell interactions. We demonstrated here the physical and functional associations of integrin alpha 2 beta 1 with epidermal growth factor receptor (EGFR) at intercellular adhesion sites. Immunoprecipitation with anti-integrin alpha 2 antibodies or anti-integrin beta 1 antibody resulted in preferential coprecipitation of EGFR from A431 cell lysates, while anti-EGFR antibody coprecipitated integrin alpha 2 beta 1. Chemical crosslinking confirmed the association of integrin alpha 2 beta 1 and EGFR. Colocalization of integrin alpha 2 beta 1 and EGFR at cell-cell contact sites was observed by double immunofluorescence staining of A431 cells. EGF-induced EGFR stimulation did not affect the association of integrin alpha 2 beta 1 and EGFR. However, immunostaining with the antibody specific to activated-EGFR revealed that EGFR localized at cell-cell contact sites are phosphorylated even in serum-depleted conditions, while EGFR localized to other sites is totally dephosphorylated in the same conditions. The EGFR phosphorylation in cell-cell contact sites observed in a serum-depleted culture was abrogated with a function-blocking antibody of integrin alpha 2, but not with a non-function-blocking alpha 2 antibody or function-blocking alpha 3 antibody. Moreover, the EGFR phosphorylation in serum-depleted conditions was not observed in suspended cells, or largely abrogated in sparse cells, indicating that cell-cell adhesion is required for EGFR phosphorylation. These results indicate that integrin alpha 2 beta 1 not only physically associates with EGFR but also functions in serum-independent EGFR activation at cell-cell contact sites. The present results shed a new light on the role of intercellular integrins in cell-cell interactions.