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P. R. Robinson, J. R. Blasic, Jr., S. Hattar, R. L. Brown; Light-Dependent Phosphorylation of Melanopsin in vitro. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5034.
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Melanopsin is the photopigment responsible for light-dependent regulation of many non-image forming functions. Most G-protein coupled receptors (GPCRs), including rod and cone opsins, are regulated by G-protein coupled receptor kinases (GRKs), a class of serine/threonine kinases. The common deactivation mechanism of GPCRs consists of phosphorylation of the carboxy tail of the activated receptor, followed by binding of a member of the arrestin family of proteins. We are investigating the role of phosphorylation in the deactivation and adaptation of light-activated melanopsin.
First, we assayed for light-dependent phosphorylation of melanopsin in a heterologus expression system using an in vitro kinase assay. Second, we identified candidate GRKs that may interact with melanopsin endogenously using RT-PCR on single, isolated melanopsin-expressing ganglion cells (mRGCs). Finally, the functional consequences of phosphorylation were explored using calcium imaging of the light-evoked response of melanopsin expressed in HEK-293 cells.
We have demonstrated light-dependent phosphorylation of melanopsin in vitro, and we also have identified transcripts of both GRK2 and GRK4 in mRGCs. Finally, results from the calcium imaging experiments suggest that phosphorylation is involved in melanopsin inactivation.
In vitro melanopsin is phosphorylated in a light-dependent manner and this post translational modification appears to be involved in the deactivation of light- activated melanopsin.
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