Light-Controlled PI3K Activation Mimics TRPV1 Potentiation by NGF

Abstract

Membrane receptor signaling is extensively studied, yet unclear. For example, activation of TrkA by nerve growth factor (NGF) triggers many downstream signaling pathways, including: Phosphoinositide 3-kinase (PI3K), PLC and MAPK. One target of TrkA signaling is the pain-transducing ion channel TRPV1, an essential player in inflammatory hyperalgesia. Although regulation of TRPV1 by NGF has been studied for over a decade, the relative contributions of PI3K, PLC, and MAPK are still controversial.Innovative approaches have recently been developed to bypass cell-surface receptors and activate specific signaling pathways using light-dependent dimerization. One new system (Levskaya et al, Nature, 2009) involves dimerization of phytochrome B (PhyB) and phytochrome interaction factor (PIF) by 650 nm light and their dissociation by 750 nm. By expressing plasma membrane anchored PhyB and PIF fused to a lipid-active enzyme, 650 nm light recruits the PIF-enzyme to the plasma membrane, “activating” the enzyme by colocalizing it with its substrate. This interaction is fully reversible by 750 nm light over tens of seconds.We applied this light-controlled dimerization system to dissect the signaling cascade leading to TRPV1 potentiation by NGF. Our goal was to bypass TrkA, activating only the PI3K branch, to determine if activation of PI3K with light mimics potentiation of capsaicin-induced calcium responses to the TRPV1 agonist capsaicin. The PhyB/PIF system was successful in reversibly activating PI3K as measured by levels of its product, PI(3,4,5)P3. Importantly, potentiation of capsaicin responses by light and NGF were similar. Our interpretation is that PI3K activation is sufficient to mimic potentiation of TRPV1 by NGF, at least on the acute time scale of our experiments. Although inflammatory hyperalgesia is a complex process likely involving numerous components, control of PI3K with light allowed us to identify a major player definitively.