Functional expression, targeting and Ca2+ signaling of a mouse melanopsin-eYFP fusion protein in a retinal pigment epithelium cell line.
Melanopsin, first discovered in Xenopus melanophores, is now established as a functional sensory photopigment of the intrinsically photosensitive retinal ganglion cells. These ganglion cells drive circadian rhythm and pupillary adjustments through projection to the brain. Melanopsin shares structural similarities with all known opsins. Comprehensive characterization of melanopsin with respect to its spectral properties, photochemical cascade and signaling partners requires a suitable recombinant system and high expression levels. This combination has not yet been described. To address this issue, we have expressed recombinant mouse melanopsin in several cell lines. Using enhanced yellow fluorescent protein (eYFP) as a visualization tag, expression was observed in all cell lines. Confocal microscopy revealed that melanopsin was properly routed to the plasma membrane only in retinal pigment epithelium (RPE)-derived D407 cells and in human embryonic kidney (HEK) cells. Further, we performed intracellular calcium measurements in order to probe the melanopsin signaling activity of this fusion protein. Transfected cells were loaded with the calcium indicator Fura2-AM. Upon illumination, an immediate but transient calcium response was observed in HEK as well as in D407 cells, while mock-transfected cells showed no calcium response under identical conditions. Supplementation with 11-cis retinal or all-trans retinal enhanced the response. After prolonged illumination the cells became desensitized. Thus, RPE-derived cells expressing recombinant melanopsin may constitute a suitable system for the study of the structural and functional characteristics of melanopsin.
Giesbers
Maikel E
ME
Department of Biochemistry, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.
Shirzad-Wasei
Nazhat
N
Bosman
Giel J C G M
GJ
de Grip
Willem J
WJ
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D004247:DNA / Q000235:genetics; D017931:DNA Primers; D006801:Humans; D051379:Mice; D010857:Pigment Epithelium of Eye / Q000502:physiology*; D017299:Rod Opsins / Q000235:genetics* / Q000502:physiology*; D015398:Signal Transduction; D014981:Xenopus; D000099210:Melanopsin
D002118:Calcium; D004247:DNA; D017931:DNA Primers; D017299:Rod Opsins; D000099210:Melanopsin