Optical Activation of TrkA Signaling

Abstract

Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. As TrkA signaling is highly dynamic, mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.

Optical activation of TrkB receptors

“…Particularly, we found that CRY2-based Opto-iTrkB + CIBN-CAAX is the most effective strategy to activate TrkB receptors by quantifying the degrees of PC12 neurite outgrowth and comparing the levels of phosphorylated ERK and Akt. The result agrees with our previous report that the same design for TrkA receptors, Opto-iTrkA + CIBN-CAAX, can activate TrkA signaling better than other methods exploiting full-length receptors or membrane-bound intracellular domain of the receptors [24]. To further improve the activation efficiency in the future, CRY2 variants with enhanced homo-interacting abilities, such as CRY2high [41], CRY2clust [42] or CRY2oligo [43], can be incorporated into the Opto-iTrkB + CIBN-CAAX construct.…”

“…Lyn-iTrkB-mCh-CRY2: The intracellular domain of TrkB (a.a. 455-822) was inserted into our previous construct Lyn-iTrkA-mCh-CRY2 [24] at SacI and BsrGI sites using In-Fusion (Clontech).…”

“…CRY2-mCh-iTrkB: iTrkB was inserted into our previous construct CRY2-mCh-iTrkA [24] at BsrGI and MunI sites using In-Fusion (Clontech).…”

“…However, the full-length receptor fused to photosensory proteins is still responsive to ligands via its extracellular domain. In addition, whether the membrane-bound light-responsive TrkB possesses the best optical activation efficacy is unclear, as we previously found that light-induced membrane recruitment and homo-interaction of the intracellular domain of tropomyosin receptor kinase A (iTrkA) is more effective to activate TrkA signaling than the optical homo-interaction of membrane-bound iTrkA [24]. Here in this report, we develop and compare different optical approaches to regulate TrkB signaling (OptoTrkB) and inspect their efficiency.…”

Construction of light-activated neurotrophin receptors using the improved Light-Induced Dimerizer (iLID)

“…Our group and others have successfully leveraged the Arabidopsis thaliana photoreceptor CRY2 3 to make light-inducible constructs of several RTKs, including FGFR 4,5 , EphB2 6 , and the neurotrophin receptors TrkA, TrkB, and TrkC [7][8][9] . The blue light-induced clustering of CRY2 lends itself especially well to reproducing the conditions for RTK activation.…”