This component will continue elucidating the molecular mechanisms underlying 'ontogenesis' of kappaopioid receptor (KOR), i.e. production of KOR protein during developmental stages and differentiatingneurons. Studies in the previous funding cycles have delineated several regulatory pathways for the controlof KOR mRNA production during developmental stages, principally transcriptional control. This concludesthe first phase of studies focusing on the regulation of KOR mRNA synthesis, a hall markd of KORneurogenesis involving signaling pathways of retinoic acid (vitamin A) and nitric oxide and requireschromatin remodeling of KOR gene regulatory regions, as well as more recent findings in epigenetic control.Importantly, ontogenesis of KOR appears to be controlled, most crucially, by post-transcriptionalmechanisms such as mRNA stability, transport and translation. This renewal component will focus ontranslational mechanism by extending from our preliminary studies that have identified Netrin-1 as atranslational stimulator for KOR, and Grb7 as a translational represser of KOR.Two specfiic aims are:1. To elucidate the mechanism that activates KOR translation via Netrin-1/Grb7 pathway. We will focus on i)regulation of translational initiation of KOR by Grb7, including studies of its molecular and biochemicalfeatures and funcitonal domains, and KOR RNA sequences bound by Grb7 which can be activated byNetrin-1, and ii) specific translational initiation step that is targeted by Grb7 including initiation factors andsubcellular distribution and possible circularization of KOR mRNA.2. Pharmacological and physiological relevance of Grb7/Netrin-1 signaling to KOR ontogenesis. We will i)perform gain- and loss-of-function studies to validate the relevance of Netrin-1/Grb7 in KOR ontogenesisusing stem cells and primary neurons, and ii) examine the physiological relevance of KOR synthesis inneuronal activity such as in a specific pain circiitry and during nerve injury or as a stress response.
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