title: Biochemical analysis of a rhodopsin photoactivatable GFP fusion as a model of G-protein coupled receptor transport.
abstract: Rhodopsin is trafficked to the rod outer segment of vertebrate rod cells with high fidelity. When rhodopsin transport is disrupted retinal photoreceptors apoptose, resulting in the blinding disease autosomal dominant retinitis pigmentosa. Herein, we introduce rhodopsin-photoactivatable GFP-1D4 (rhodopsin-paGFP-1D4) for the purposes of monitoring rhodopsin transport in living cells. Rhodopsin-paGFP-1D4 contains photoactivatable GFP (paGFP) fused to rhodopsin's C-terminus and the last eight amino acids of rhodopsin (1D4) appended to the C-terminus of paGFP. The fusion protein binds the chromophore 11-cis retinal and photoisomerizes upon light activation similarly to rhodopsin. It activates the G-protein transducin with similar kinetics as does rhodopsin. Rhodopsin-paGFP-1D4 localizes to the same compartments, the primary cilium in cultured IMCD cells and the outer segment of rod cells, as rhodopsin in vitro and in vivo. This enables its use as a model of rhodopsin transport and details the importance of a free rhodopsin C-terminus in rod cell localization and health.
authors.affiliation: Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Sammons
authors.forename: Alecia K
authors.initials: AK
authors.lastname: Gross
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology; D019556:COS Cells; D002522:Chlorocebus aethiops; D002923:Cilia / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000502:physiology*; D017510:Protein Folding; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells / Q000502:physiology*; D012243:Rhodopsin / Q000737:chemistry / Q000502:physiology*; D015293:Transducin / Q000378:metabolism
chemical_list: D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D012243:Rhodopsin; D015293:Transducin
keywords: C-terminus; G-protein coupled receptor; GPCR; IMCD; MDCK; Madin–Darby Canine Kidney; OCT; Photoactivatable GFP; Photoreceptors; ROS; Rhodopsin; Trafficking; XOP; Xenopus opsin promoter; carboxy-terminus; i3 loop; inner medullary collecting duct; intracellular loop 3; optimal cutting temperature medium; paGFP; photoactivatable GFP; rod outer segment