Biochemical analysis of a rhodopsin photoactivatable GFP fusion as a model of G-protein coupled receptor transport.
Biochemical analysis of a rhodopsin photoactivatable GFP fusion as a model of G-protein coupled receptor transport.
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DOI：
10.1016/j.visres.2013.10.008
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发表时间：
2013-12-18
期刊：
影响因子：
1.8
通讯作者：
Gross, Alecia K.
中科院分区：
文献类型：
--
作者：
Sammons, Joshua D.;Gross, Alecia K.
视紫红质以高保真度被运输到脊椎动物视杆细胞的视杆外节。当视紫红质的运输被破坏时，视网膜光感受器就会产生葡萄糖，导致致盲性疾病常染色体显性视网膜色素变性。在此，我们引入了视紫红质光活化GFP-1D 4（rhodopsin-paGFP-1D 4），用于监测活细胞中视紫红质的转运。视紫红质-paGFP-1D 4含有与视紫红质的C-末端融合的可光活化GFP（paGFP）和附加到paGFP的C-末端的视紫红质（1D 4）的最后8个氨基酸。该融合蛋白结合发色团11-顺式视黄醛，并在光激活时类似于视紫红质发生光异构化。它以与视紫红质相似的动力学激活G蛋白转导蛋白。视紫红质-paGFP-1D 4在体外和体内定位于与视紫红质相同的隔室，即培养的IMCD细胞中的初级纤毛和视杆细胞的外节。这使其能够用作视紫红质转运的模型，并详细说明了游离视紫红质C-末端在视杆细胞定位和健康中的重要性。
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.
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DOI：
10.1083/jcb.151.7.1369
发表时间：
2000-12-25
期刊：
The Journal of cell biology
影响因子：
--
作者：
Tam BM;Moritz OL;Hurd LB;Papermaster DS
通讯作者：
Papermaster DS
影响因子：
1.8
作者：
Concepcion, F;Mendez, A;Chen, J
通讯作者：
Chen, J
影响因子：
--
作者：
Millar, Robert P.;Newton, Claire L.
通讯作者：
Newton, Claire L.
影响因子：
5.3
作者：
Lodowski, Kerrie H.;Lee, Richard;Imanishi, Yoshikazu
通讯作者：
Imanishi, Yoshikazu
影响因子：
1.8
作者：
Wensel, TG;Gross, AK;Wilson, JH
通讯作者：
Wilson, JH