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. 1990 Jul;87(13):4991–4995. doi: 10.1073/pnas.87.13.4991

Role of the intradiscal domain in rhodopsin assembly and function.

T Doi 1, R S Molday 1, H G Khorana 1
PMCID: PMC54247  PMID: 2367520

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 III mutants showed poor chromophore regeneration and abnormal glycosylation. Reduced transducin activation was shown by some type I and III 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 III 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Applebury M. L., Hargrave P. A. Molecular biology of the visual pigments. Vision Res. 1986;26(12):1881–1895. doi: 10.1016/0042-6989(86)90115-x. [DOI] [PubMed] [Google Scholar]
  2. Baehr W., Morita E. A., Swanson R. J., Applebury M. L. Characterization of bovine rod outer segment G-protein. J Biol Chem. 1982 Jun 10;257(11):6452–6460. [PubMed] [Google Scholar]
  3. Copeland C. S., Doms R. W., Bolzau E. M., Webster R. G., Helenius A. Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J Cell Biol. 1986 Oct;103(4):1179–1191. doi: 10.1083/jcb.103.4.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dixon R. A., Sigal I. S., Candelore M. R., Register R. B., Scattergood W., Rands E., Strader C. D. Structural features required for ligand binding to the beta-adrenergic receptor. EMBO J. 1987 Nov;6(11):3269–3275. doi: 10.1002/j.1460-2075.1987.tb02645.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dryja T. P., McGee T. L., Reichel E., Hahn L. B., Cowley G. S., Yandell D. W., Sandberg M. A., Berson E. L. A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature. 1990 Jan 25;343(6256):364–366. doi: 10.1038/343364a0. [DOI] [PubMed] [Google Scholar]
  6. Ferretti L., Karnik S. S., Khorana H. G., Nassal M., Oprian D. D. Total synthesis of a gene for bovine rhodopsin. Proc Natl Acad Sci U S A. 1986 Feb;83(3):599–603. doi: 10.1073/pnas.83.3.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Franke R. R., Sakmar T. P., Oprian D. D., Khorana H. G. A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin. J Biol Chem. 1988 Feb 15;263(5):2119–2122. [PubMed] [Google Scholar]
  8. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  9. Hong K., Hubbell W. L. Lipid requirements for Rhodopsin regenerability. Biochemistry. 1973 Oct 23;12(22):4517–4523. doi: 10.1021/bi00746a033. [DOI] [PubMed] [Google Scholar]
  10. Karnik S. S., Sakmar T. P., Chen H. B., Khorana H. G. Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8459–8463. doi: 10.1073/pnas.85.22.8459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Khorana H. G., Knox B. E., Nasi E., Swanson R., Thompson D. A. Expression of a bovine rhodopsin gene in Xenopus oocytes: demonstration of light-dependent ionic currents. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7917–7921. doi: 10.1073/pnas.85.21.7917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kozutsumi Y., Segal M., Normington K., Gething M. J., Sambrook J. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature. 1988 Mar 31;332(6163):462–464. doi: 10.1038/332462a0. [DOI] [PubMed] [Google Scholar]
  13. Kreis T. E., Lodish H. F. Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. Cell. 1986 Sep 12;46(6):929–937. doi: 10.1016/0092-8674(86)90075-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Liao C. F., Themmen A. P., Joho R., Barberis C., Birnbaumer M., Birnbaumer L. Molecular cloning and expression of a fifth muscarinic acetylcholine receptor. J Biol Chem. 1989 May 5;264(13):7328–7337. [PubMed] [Google Scholar]
  15. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nathans J. Molecular biology of visual pigments. Annu Rev Neurosci. 1987;10:163–194. doi: 10.1146/annurev.ne.10.030187.001115. [DOI] [PubMed] [Google Scholar]
  17. Oprian D. D., Molday R. S., Kaufman R. J., Khorana H. G. Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8874–8878. doi: 10.1073/pnas.84.24.8874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Papermaster D. S., Dreyer W. J. Rhodopsin content in the outer segment membranes of bovine and frog retinal rods. Biochemistry. 1974 May 21;13(11):2438–2444. doi: 10.1021/bi00708a031. [DOI] [PubMed] [Google Scholar]
  19. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Stryer L. Cyclic GMP cascade of vision. Annu Rev Neurosci. 1986;9:87–119. doi: 10.1146/annurev.ne.09.030186.000511. [DOI] [PubMed] [Google Scholar]
  21. VanAken T., Foxall-VanAken S., Castleman S., Ferguson-Miller S. Alkyl glycoside detergents: synthesis and applications to the study of membrane proteins. Methods Enzymol. 1986;125:27–35. doi: 10.1016/s0076-6879(86)25005-3. [DOI] [PubMed] [Google Scholar]
  22. Wald G. The molecular basis of visual excitation. Nature. 1968 Aug 24;219(5156):800–807. doi: 10.1038/219800a0. [DOI] [PubMed] [Google Scholar]
  23. Wessling-Resnick M., Johnson G. L. Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange. J Biol Chem. 1987 Mar 15;262(8):3697–3705. [PubMed] [Google Scholar]

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