No abstract

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The type 1 angiotensin II (AngII) receptor (AT 1 R) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the AT 1 R was able to transactivate HER2, but not HER3. Mechanistically, AT 1 R-mediated ERK1/2 activation was dependent on G q/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of G q/11 and only partially dependent on EGFR tyrosine kinase activity. This G q/11 independence of EGFR transactivation was confirmed using AT 1 R mutants and in CRISPR cell lines lacking G q/11. EGFR transactivation was also apparently independent of βarrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII-and EGF-stimulation promoted AT 1 R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT 1 R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT 1 R and EGFR.

The traditional medicinal mushroom Hericium erinaceus is known for enhancing peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. Here, we purified and identified biologically new active compounds from H. erinaceus, based on their ability to promote neurite outgrowth in hippocampal neurons. N‐de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom, together with its hydrophobic derivative hericene A, were highly potent in promoting extensive axon outgrowth and neurite branching in cultured hippocampal neurons even in the absence of serum, demonstrating potent neurotrophic activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA‐12 only partly prevented the NDPIH‐induced neurotrophic activity, suggesting a potential link with BDNF signaling. However, we found that NDPIH activated ERK1/2 signaling in the absence of TrkB in HEK‐293T cells, an effect that was not sensitive to ANA‐12 in the presence of TrkB. Our results demonstrate that NDPIH acts via a complementary neurotrophic pathway independent of TrkB with converging downstream ERK1/2 activation. Mice fed with H. erinaceus crude extract and hericene A also exhibited increased neurotrophin expression and downstream signaling, resulting in significantly enhanced hippocampal memory. Hericene A therefore acts through a novel pan‐neurotrophic signaling pathway, leading to improved cognitive performance.

No abstract