Role of the intradiscal domain in rhodopsin assembly and function.

Abstract

The role of the intradiscal polypeptide loops in bovine rhodopsin has been investigated by deletions in the N-terminal tail and in loops B-C, D-E, and E-F as well as by single amino acid substitutions in the D-E loop. Mutants with three types of phenotypes were observed. Type I mutants showed a rhodopsin-like chromophore and glycosylation. Type II mutants did not regenerate the chromophore and showed abnormal glycosylation. Type mI mutants showed poor chromophore regeneration and abnormal glycosylation. Reduced transducin activation was shown by some type I and Ill mutants. Single amino acid substitutions in the D-E loop gave mostly type I mutants. Deletions in loops B-C, D-E, and F-G gave type II mutants, whereas deletions in the N-terminal tail produced type HI mutants. Systematic deletions of two adjacent amino acids in loop D-E indicated that the amino acid sequences 171-182 and 189-192 were essential to rhodopsin structure. Immunofluorescence double-staining and transmission electron microscopy of one type II mutant (with residues 189 and 190 deleted) showed that it was mostly in the endoplasmic reticulum, whereas the wild-type protein was in the plasma membrane. We conclude that the first step in the assembly of the rhodopsin molecule is the formation of a three-dimensional structure in the intradiscal domain involving the bulk of the out-of-the-membrane polypeptide segments followed by the linkage of Cys-110 and Cys-187 through a disulfide bond.Rhodopsin, the photoreceptor in the vertebrate rod cells (1), consists of a single polypeptide chain of 348 amino acids. It is believed to contain seven transmembrane helical segments (2) (Fig. 1), with roughly half of the protein embedded in the membrane and the other half distributed between the cytoplasmic and intradiscal domains, which contain peptide loops of various sizes and the N-and C-terminal tails. Light activation of rhodopsin prepares the molecule for interaction with transducin, the GTP-binding regulatory protein, and allows its phosphorylation by rhodopsin kinase and subsequent interaction with the 48-kDa protein, S antigen (3). These interactions all occur on the cytoplasmic face of rhodopsin. In contrast, the role of the intradiscal domain in rhodopsin structure and/or function has remained unclear. Recently, systematic replacements of cysteine residues in rhodopsin were carried out (4). This study showed that the intradiscal Cys-110 and Cys-187 ( Fig. 1) are essential for the formation of functional rhodopsin and that these two cysteines form a disulfide bond between them. To clarify further the role of the intradiscal domain in rhodopsin function, we have now performed deletion mutagenesis in the N-terminal tail and the three intradiscal loops as well as single amino acid substitutions in loop D-E (Fig. 1). The results show that relatively short deletions in any one of the loops or in the N-terminal tail affect the assembly of functional rhodopsin.We conclude that the role of the intradiscal domain in rhodopsin is structural. Very likely ...

Retinoids Assist the Cellular Folding of the Autosomal Dominant Retinitis Pigmentosa Opsin Mutant P23H

“…Previous studies (7,8,15,25,27,28) of these proteins led to the conclusion that the mutant opsins were misfolded based on low pigment yield, intracellular accumulation, altered glycosylation, or non-native disulfide bonds but did not provide direct evidence for defects in protein thermal stability or folding kinetics. However, several mutant rhodopsins associated with ADRP were recently shown to exhibit decreased thermal stability (16,29,30); our studies of P23H rhodopsin provide another example.…”

Coupling of retinal isomerization to the activation of rhodopsin

“…The lack of changes in the chemical shifts of Tyr-178 and Tyr-268 is consistent with rigid body motion of H6 that occurs on the cytoplasmic side of Pro-267 (3). Mutational data indicate that EL2 is important for the stability of the retinal binding site in rhodopsin and meta II (24,25). Recent computational studies suggest that the Cys-110-Cys-187 disulfide bridge along with several residues on EL2 and at the ends of H3 and H4 are part of a stable folding core in rhodopsin (26).…”

Molecular evolution of color vision in vertebrates

“…Studies on the structure and function of rhodopsins by Doi et al (1990) and the subsequent series of papers by H.G. Khorana and his colleagues have dramatically improved our understanding of the roles of key amino acids in visual pigments (see also Sakmar et al, 1989;Zhukovsky and Oprian, 1989;Nathans, 1990a,b;Weitz and Nathans, 1993).…”