Panoply™ Human FGFR2 Over-expressing Stable Cell Line

Autism spectrum disorders are neurodevelopmental conditions with diverse aetiologies, all characterized by common core symptoms such as impaired social skills and communication, as well as repetitive behaviour. Cell adhesion molecules, receptor tyrosine kinases, and their downstream signaling pathways are all closely related to neurodevelopment and autism spectrum disorders. Here, researchers found that downregulation of the cell adhesion molecule NEGR1 or the receptor tyrosine kinase fibroblast growth factor receptor 2 (FGFR2) during cortical development in mice affects neuronal migration and dendritic spine density, leading to core behavioral impairments associated with autism spectrum disorders. Mechanistically, NEGR1 physically interacts with FGFR2 and modulates FGFR2-dependent extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways by reducing FGFR2 degradation from the plasma membrane. Therefore, overexpression of FGFR2 can rescue all defects caused by Negr1 knockdown in vivo. Negr1 knockout mice exhibit a similar phenotype to mice with downregulated Negr1 expression. These data suggest that NEGR1 and FGFR2 synergistically regulate cortical development and indicate that defects in the NEGR1-FGFR2 complex and the fusion of downstream ERK and AKT signaling pathways may play a role in autism spectrum disorders.

NEGR1 is susceptible to cleavage by proteases and exists in a soluble form. Therefore, researchers investigated whether soluble NEGR1 added to the culture medium could bind to FGFR2 expressed on the cell membrane. They incubated wild-type and FGFR2-overexpressing HEK293 cells with recombinant FLAG-NEGR1 protein and performed anti-FLAG immunocytochemical staining. FLAG-NEGR1 bound only to FGFR2-overexpressing cells, indicating that NEGR1 and FGFR2 interact in their native state (Figure 1). Interestingly, after treatment with the pan-FGFR agonist FGFb (which induces FGFR endocytosis), FLAG-positive aggregates were observed in the perinuclear region (Figure 1), suggesting that NEGR1 binding to FGFR2 is followed by co-internalization into endocytic vesicles upon stimulation.

Figure 1. Representative fluorescence images of FLAG and DAPI immunostainings in wild-type or FGFR2-expressing HEK293 cells exposed to purified soluble FLAG-NEGR1 (sFLAG-Negr1) or first to sFLAG-Negr1 and subsequently to the pan-FGFRs activator FGFb. (Szczurkowska J, et al., 2018)