Functional analysis of the second extracellular loop of rhodopsin by characterizing split variants.
The second extracellular loop (ECL2) of the family 1 G protein-coupled receptors (GPCR) is known to function in ligand recognition and to show structural diversity in a variety of GPCRs. In rhodopsin, ECL2 forms a rigid structure including a beta-sheet and interacts with the transmembrane region via a disulfide bond, hydrogen bonds, and hydrophobic interactions. It forms the chromophore-binding pocket with transmembrane helices and directly interacts with the 11-cis-retinal chromophore. To clarify the functional role of the rigid ECL2 in bovine rhodopsin, we designed split rhodopsins in which the polypeptide chain of rhodopsin was cleaved at the C-terminal end and/or N-terminal end of ECL2 by genetic engineering. A reconstitution study of pigment from two peptide fragments and 11-cis-retinal showed that fixation of the N-terminus of ECL2 to the transmembrane region is essential for folding into the native rhodopsin structure. Split rhodopsin was resistant to hydroxylamine and activated transducin upon light absorption similarly to wild-type rhodopsin, but was readily disassembled by photobleaching. Analyses of the photobleaching processes of split rhodopsins and rhodopsin mutant lacking disulfide bond showed that the rigid structure of ECL2 is required for facilitating the formation of the active state. These results suggest that ECL2 'mechanically' drives the conformational change of rhodopsin.
Sakai
Kazumi
K
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.
Imamoto
Yasushi
Y
Yamashita
Takahiro
T
Shichida
Yoshinori
Y
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D012243:Rhodopsin; D012172:Retinaldehyde