Expression and localization of an exogenous G protein-coupled receptor fused with the rhodopsin C-terminal sequence in the retinal rod cells of knockin mice.
Vertebrate rod cell outer segments are highly differentiated compartments consisting of closely packed disk membranes, in which the photoreceptor rhodopsin is embedded at high density. To explore the unusually efficient mechanism of rhodopsin biosynthesis, folding and transport, we challenged it with the ectopic expression in rod cells of human endothelin receptor subtype B (hET(B)R) fused with the C-terminal 10 residues of rhodopsin, under the control of the mouse opsin promoter/enhancer, by gene targeted replacement (knockin), because the C-terminal eight residues are essential to target rhodopsin to the outer segment. The hET(B)R, a type-I G protein-coupled receptor, was successfully expressed and folded in a functional structure in the rod cells of knockin mice. However, while the mRNA level of hET(B)R was one tenth of that of rhodopsin, the hET(B)R protein level was approximately one-thousandth of the rhodopsin level in heterozygous mice, suggesting an intrinsically distinct efficiency in the production of functional receptor protein. In addition, a substantial fraction of the hET(B)R was successfully transported to the outer segment, suggesting that the addition of the C-terminal sequence of rhodopsin enabled hET(B)R to be translocated to the outer segment.
Kodama
Takashi
T
Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
Imai
Hiroo
H
Doi
Tomoko
T
Chisaka
Osamu
O
Shichida
Yoshinori
Y
Fujiyoshi
Yoshinori
Y
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000032:analysis; D044023:Receptor, Endothelin B / Q000032:analysis* / Q000235:genetics; D012160:Retina / Q000033:anatomy & histology; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012243:Rhodopsin / Q000032:analysis / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000737:chemistry; D017299:Rod Opsins / Q000235:genetics; D014178:Translocation, Genetic / Q000235:genetics
D012333:RNA, Messenger; D044023:Receptor, Endothelin B; D017299:Rod Opsins; D012243:Rhodopsin