Phosphorylation of mouse melanopsin by protein kinase A.
The visual pigment melanopsin is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) in the mammalian retina, where it is involved in non-image forming light responses including circadian photoentrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep. It has recently been shown that the melanopsin-based light response in ipRGCs is attenuated by the neurotransmitter dopamine. Here, we use a heterologous expression system to demonstrate that mouse melanopsin can be phosphorylated by protein kinase A, and that phosphorylation can inhibit melanopsin signaling in HEK cells. Site-directed mutagenesis experiments revealed that this inhibitory effect is primarily mediated by phosphorylation of sites T186 and S287 located in the second and third intracellular loops of melanopsin, respectively. Furthermore, we show that this phosphorylation can occur in vivo using an in situ proximity-dependent ligation assay (PLA). Based on these data, we suggest that the attenuation of the melanopsin-based light response by dopamine is mediated by direct PKA phosphorylation of melanopsin, rather than phosphorylation of a downstream component of the signaling cascade.
Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, Maryland, USA.
Joseph R
JR
Blasic
R Lane
RL
Brown
Phyllis R
PR
Robinson
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004298:Dopamine / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects; D057809:HEK293 Cells; D006801:Humans; D008027:Light; D055537:Light Signal Transduction / Q000187:drug effects / Q000528:radiation effects; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects; D017299:Rod Opsins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000099210:Melanopsin
D017868:Cyclic AMP-Dependent Protein Kinases; D004298:Dopamine; D017299:Rod Opsins; D012694:Serine; D013912:Threonine; D000099210:Melanopsin