Abstract
The steroid hormone estrogen has wide spread physiological actions on many cell-types including neurons and neuroendocrine cells. The signalling mechanisms are mediated by a number of distinct receptor subtypes including estrogen receptor alpha, estrogen receptor beta, and G protein-coupled estrogen receptor 1. This signalling can be classed into two different categories, classical, slow acting actions, or rapid, membrane-initiated actions. The mechanisms by which classical estrogen actions occur have been well defined, while the rapid effects are currently not fully understood. It has been proposed that estrogens may modulate the nerve growth factor receptor TrkA and influence its intracellular signalling downstream. The current study aimed to determine if estrogen modifies TrkA signalling on PC12 cells, which have high TrkA receptor expression. The current study also demonstrated the presence of estrogen receptor beta and G protein-coupled estrogen receptor 1 on these cells. Single molecule tracking data using the total internal reflection fluorescence microscope showed isolated PC12 cells treated with nerve growth factor (100 nM) demonstrated an interaction with antibody-labelled TrkA receptors resulting in increased periods of receptor immobility. Such periods of immobility have been proposed to coincide with enhanced intracellular signalling activity. Low concentrations of 17-β estradiol (100 pM) also resulted in increased TrkA receptor immobility. Further experiments examined the effect of 17-β estradiol on TrkA-mediated activation of the downstream signalling molecule mitogen-activated protein kinase 1/2. While nerve growth factor rapidly increased the activity of this pathway, as indicated by a rise in its phosphorylated form, 17-β estradiol was without effect, either on its own, or in combination with nerve growth factor. Thus this study shows that while 17-β estradiol interacts with the TrkA receptor to increase its period of immobility, this does not change the activity of its downstream signalling molecule mitogen-activated protein kinase 1/2. Since receptor immobility is believed to be associated with increased signalling, future studies should be directed to examining 17-β estradiol effects on alternative nerve growth factor-activated pathways. These studies are important since both nerve growth factor and 17-β estradiol have shown to have neuroprotective effects, which in combination may aid in the treatment of degenerative conditions such as Alzheimer’s disease.