DOI QR코드

DOI QR Code

Chromophore 형성과 rhodopsin kinase 활성을 이용한 항활성 로돕신 mutant의 분석

Chromophore formation and phosphorylation analysis of constitutively active rhodopsin mutants

  • 발행 : 2007.06.25

초록

G protein-coupled receptor, (GPCR)는 세포외부의 신호를 인식 시 G 단백질을 활성화시켜 신호를 전달하며 kinase에 의한 인산화를 통하여 지속적인 신호전달을 억제한다. 외부 신호물질이 없는 조건에서도 활성을 나타내는 항활성 돌연변이종(CAM)은 GPCR의 신호전달 이상에 기인한 질병 치료나 활성화 구조변화의 좋은 연구대상이다. 희미한 빛을 인식하는 시각수용체인 로돕신의 CAM으로는 salt bridge에 직접적인 영향을 미치는 돌연변이인 G90D, El13Q, 그리고 K296E와, 직접적인 영향이 없는 돌연변이인 E134q와 M25Y등 두 가지 계통의 종류가 알려져 있다. 본 연구에서는 각각의 돌연변이가 복합된 mutant를 구성하여 agonist와 inverse agonist에 대한친화도와 로돕신 kinase에 대한 활성을 조사하여 각 종에서의 구조변화의 차이를 분석하였다. 로돕신 mutant의constitutive activity는 all-trans-retinal에 대한 친화도에 비례하며 11-cis-retinal에 대한 친화도와는 역상관 관계를 보여준다. 같은 계통에 속하는 돌연변이가 합쳐진 복합 mutant는 단일 mutant에 비하여 미약한 정도의 로돕신 kinase 항활성화 증가를 보여주나, 다른 계통에 속하는 두 가지 돌연변이가 합쳐진 mutant는 항활성화가 크게 증가되었음을 보여주었다. 이 결과는 다른 계통에 속하는 mutant에서는 상이한 구조변화가 일어나며 로돕신이완전한 활성화에 이르기 위해서는 최소한 두 가지 종류의 돌연변이에 의하여 생기는 구조변화들이 함께 일어나야함을 의 미 한다. G protein 활성화와 유사한 항활성화 분석 결과는 rhodopsin kinase가 인식하는 로돕신의 활성화상태 구조가 G protein이 인식하는 구조와 유사함을 의미한다. 특히 가장 강한 활성을 나타내는 El13Q/E134Q/M257Y는 활성화상태 GPCR 단백질의 결정 시도에 이용 될 수 있을 것이다.

G protein coupled receptors (GPCRs) transmit various extracellular signals into the cells. Upon binding of the ligands, conformational changes in the extracellular and/or transmembrane (TM) domains of CPCRs were propagated into the cytoplasmic (CP) domain of the molecule leading to the activation of their cognate heterotrimeric C proteins and kinases. Constitutively active GPCR mutants causing the activation of C Protein signaling even in the absence of ligand binding are of interest for the study of activation mechanism of GPCRs. Two classes of constitutively active mutations, categorized by their effects on the salt bridge between Ell3 and K296, were found in the TM domain of rhodopsin. Opsin mutants containing combinations of the mutations were constructed to study the conformational changes required for the activation of rhodopsin. Rhodopsin chromophore regenerated with 11-cis-retinal showed a thermal stability inversely correlated with its constitutive activity. In contrast, rhodopsin mutants exhibited a binding affinity to an agonist, all-trans-retinal, in a constitutive activity-dependent manner. In order to test whether the conformational changes responsible for the activation of trans-ducin (Gt) are the same as the conformation required for the recognition of rhodopsin kinase, analysis of the mutants were carried out with phosphorylation by rhodopsin kinase. Rhodopsin mutants containing combinations of different classes of the mutations showed a strong synergistic effect on the phosphorylation of the mutants in the dark as similar to that of Gt activation. The results suggest that at least two or three kinds of segmental and independent conformational changes are required for the activation of rhodopsin and the conformational changes responsible for activating rhodopsin kinase and Gt are similar to each other.

키워드

참고문헌

  1. Bennett, N., M. Michel-Villaz and H. Kuhn. 1982. Lightinduced interaction between rhodopsin and GTP-binding protein. Metarhodopsin II is the major product involved. Eur. J. Biochem. 127, 97-103 https://doi.org/10.1111/j.1432-1033.1982.tb06842.x
  2. Brown, N. G., C. Fowles, R. Sharma and M. Akhtar. 1992. Mechanistic studies on rhodopsin kinase. Light-dependent phosphorylation of C-terminal peptides of rhodopsin. Eur. J. Biochem. 208, 659-667 https://doi.org/10.1111/j.1432-1033.1992.tb17232.x
  3. Bruel, C., K. Cha, L. Niu, P. J. Reeves and H. G. Khorana. 2000. Rhodopsin kinase : Two mAbs binding near the carboxy terminus cause time-dependent inactivation. Proc. Natl. Acad. Sci. USA 97, 3010-3015 https://doi.org/10.1073/pnas.97.7.3010
  4. Cohen, G. B., T. Yang, P. R. Robinson and D. D. Oprian. 1993. Constitutive activation of opsin : Influence of charge at position 134 and size at position 296. Biochemistry 32, 6111-6115 https://doi.org/10.1021/bi00074a024
  5. Franke, R. R., T. P. Sakmar, R. M. Graham and H. G. Khorana. 1992. Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin. J. Biol. Chem. 267, 14767-14774
  6. Gether, U. 2000. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocrine Rev. 21, 90-113 https://doi.org/10.1210/er.21.1.90
  7. Han, M., S. O. Smith and T. P. Sakmar. 1998. Constitutive activation of opsin by mutation of methionine 257 on transmembrane helix 6. Biochemistry 37, 8253-8261 https://doi.org/10.1021/bi980147r
  8. Karnik, S. S., C. Gognea, S. Patil, Y. Saad and T. Takezako. 2003. Activation of G-protein-coupled receptors: a common molecular mechanism. Trends Endocrinol. Metabol. 14, 431-437 https://doi.org/10.1016/j.tem.2003.09.007
  9. Khorana, H. G., P. J. Reeves and J.-M. Kim. 2002. Structure and mechanism in G protein-coupled receptors. Pharmaceut. Rev. 9, 287-294
  10. Khorana, H. G. 2000. Molecular Biology of light transduction by the mammalian photoreceptor, rhodopsin. J. Biomol. Struct. Dyn. 11. 1-6
  11. Kim, J. M., C. Altenbach, R. L. Thurmond, H. G. Khorana and W. L. Hubbell. 1997. Structure and function in rhodopsin: rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state. Proc. Natl. Acad. Sci. USA 94, 14273-14278 https://doi.org/10.1073/pnas.94.26.14273
  12. Kim, J. M., C. Altenbach, M. Kono, D. D. Oprian, W. L. Hubbell and H. G. Khorana. 2004. Structural origins of constitutive activation in rhodopsin : Role of the K296/ E113 salt bridge. Proc. Natl. Acad. Sci. USA 101, 12508-12513 https://doi.org/10.1073/pnas.0404519101
  13. Knowles, A. and A. Priestley. 1978. The preparation of 11-cis-retinal. Vision Res. 18, 115-116 https://doi.org/10.1016/0042-6989(78)90086-X
  14. Kuhn, H. and W. J. Dreyer. 1972. Light-dependent phosphorylation by ATP. FEBS. Lett. 20, 1-6 https://doi.org/10.1016/0014-5793(72)80002-4
  15. Lefkowitz, R. J. 2000. The superfamily of heptahelical receptors. Nature Cell. Biol. 2, 133-136 https://doi.org/10.1038/35017152
  16. Meng, E. C. and H. R. Bourne. 2001. Receptor activation: what does the rhodopsin structure tell us? Trends Pharmacol. Sci. 22, 587-593 https://doi.org/10.1016/S0165-6147(00)01825-3
  17. Molday, R. S. and D. MacKenzie. 1983. Monoclonal antibodies to rhodopsin: characterization, cross-reactivity and application as structural probes. Biochemistry 22, 653-660 https://doi.org/10.1021/bi00272a020
  18. Oprian, D. D., R. S. Molday, R. J. Kaufman and H. G. Khorana. 1987. Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc. Natl. Acad. Sci. USA 84, 8874-8878 https://doi.org/10.1073/pnas.84.24.8874
  19. Palczewski, K., T. Kumasaka, T. Hori, C. A. Behnke, H. Motoshima, B. A. Fox, I. Le Trong, D. C. Teller, T. Okada, R. E. Stenkamp, M. Yamamoto and M. Miyano. 2000. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739-745 https://doi.org/10.1126/science.289.5480.739
  20. Papermaster, D. S. 1982. Preparation of antibodies to rhodopsin and the large protein of rod outer segments. Methods Enzymol. 81, 240-246 https://doi.org/10.1016/S0076-6879(82)81037-9
  21. Parnot, C., S. Miserey-Lenkei, S. Bardin, P. Corvol and E. Clauser. 2002. Lessons from constitutively active mutants of G protein-coupled receptors. Trends Endocrinol. Metabol. 13, 336-343 https://doi.org/10.1016/S1043-2760(02)00628-8
  22. Rao, V. R. and D. D. Oprian. 1996. Activating mutations of rhodopsin and other G protein-coupled receptors. Ann. Rev. Biophys. Biomol. Str. 25. 287-314 https://doi.org/10.1146/annurev.bb.25.060196.001443
  23. Rao, V. R., G. B. Cohen and D. D. Oprian. 1994. Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness. Nature 367, 639-642 https://doi.org/10.1038/367639a0
  24. Ridge, K., Z. Lu, X. Liu and H. G. Khorana. 1995. Structure and function in rhodopsin. Separation and characterization of the correctly folded and misfolded opsins produced on expression of an opsin mutant gene containing only the native intradiscal cysteine codons. Biochemistry 34, 3261-3267 https://doi.org/10.1021/bi00010a016
  25. Robinson, P. R., G. B. Cohen, E. A. Zhukovsky and D. D. Oprian. 1992. Constitutively active mutants of rhodopsin. Neuron 9, 719-725 https://doi.org/10.1016/0896-6273(92)90034-B
  26. Sakmar, T. P., R. R. Franke and H. G. Khorana. 1989. Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Proc. Natl. Acad. Sci. USA 86, 8309-8313 https://doi.org/10.1073/pnas.86.21.8309
  27. Sambrook, J. and D. W. Russell. 2001. Molecular Cloning : A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press. Plainview, New York.
  28. Teller, D. C., T. Okada, C. A. Behnke, K. Palczewski and R. E. Stenkamp. 2001. Advances in determination of a high-resolution three-dimensional structure of rhodopsin, a model of G-protein-coupled receptors (GPCRs). Biochemistry 40, 7761-7772 https://doi.org/10.1021/bi0155091
  29. Thurmond, R. L., C. Creuzenet, P. J. Reeves and H. G. Khorana. 1997. Structure and function in rhodopsin : Peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase. Proc. Natl. Acad. Sci. USA. 94, 1715-1720 https://doi.org/10.1073/pnas.94.5.1715