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. 1999 Jan 15;337(Pt 2):281–288.

Interaction sites of the C-terminal region of the cGMP phosphodiesterase inhibitory subunit with the GDP-bound transducin alpha-subunit.

Y Liu 1, V Y Arshavsky 1, A E Ruoho 1
PMCID: PMC1219963  PMID: 9882626

Abstract

In the present report, the region of interaction between the GDP-bound alpha-subunit of transducin (alphat.GTP) and the cGMP phosphodiesterase inhibitory gamma-subunit (Pgamma) has been studied. It is widely accepted that the alphat.GTP is the active form of transducin and that the GDP-bound transducin alpha-subunit (alphat. GDP) is the inactive form. We have reported previously that the binding region of the C-terminal of Pgamma on alphat.GTP is in a region between the exposed face of the alpha3 and alpha4 helices of alphat.GTP [Liu, Arshavsky and Ruoho (1996) J. Biol. Chem. 271, 26900-26907]. We now report that N-[(3-[125I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) ]cysteine ([125I]ACTP)-derivatized Pgamma (at Cys-68) reversibly undergoes a unique disulphide exchange of the radioiodinated moiety N-(3-[125I]iodo-4-azidophenylpropionamido)cysteine ([125I]APC) from Cys-68 of Pgamma to alphat.GDP but not to the guanosine 5'-(gamma-thio)-triphosphate (GTP[S])-bound transducin alpha-subunit (alphat-GTP[S]). The specificity of the interaction was demonstrated by the fact that exchange was protected by the functionally active Cys-68-->Ala Pgamma mutant, and by pretreatment of the alphat.GDP with the betagamma-subunit of transducin. Chemical cleavage and amino acid sequencing demonstrated that the [125I]ACTP-derived Pgamma specifically transferred the [125I]APC group to Cys-250 and Cys-210 of alphat.GDP. These data indicate that the C-terminal region (especially Cys-68-Trp-70) of Pgamma interacts with alphat. GDP on the exposed interface between alpha2/beta4 and alpha3/beta5 of the alpha-subunit of transducin. Disulphide exchange was also observed with the alpha-subunit of holotransducin but this was only approx. 60% of that of pure alphat.GDP. The variation in the binding pattern between alphat.GDP and alphat.GTP with the C-terminal region of Pgamma may contribute to the functional difference between the GDP- and GTP-bound states.

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Selected References

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  1. Angleson J. K., Wensel T. G. A GTPase-accelerating factor for transducin, distinct from its effector cGMP phosphodiesterase, in rod outer segment membranes. Neuron. 1993 Nov;11(5):939–949. doi: 10.1016/0896-6273(93)90123-9. [DOI] [PubMed] [Google Scholar]
  2. Arshavsky VYu, Antoch M. P., Lukjanov K. A., Philippov P. P. Transducin GTPase provides for rapid quenching of the cGMP cascade in rod outer segments. FEBS Lett. 1989 Jul 3;250(2):353–356. doi: 10.1016/0014-5793(89)80754-9. [DOI] [PubMed] [Google Scholar]
  3. Arshavsky VYu, Bownds M. D. Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP. Nature. 1992 Jun 4;357(6377):416–417. doi: 10.1038/357416a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arshavsky V. Y., Dumke C. L., Zhu Y., Artemyev N. O., Skiba N. P., Hamm H. E., Bownds M. D. Regulation of transducin GTPase activity in bovine rod outer segments. J Biol Chem. 1994 Aug 5;269(31):19882–19887. [PubMed] [Google Scholar]
  5. Artemyev N. O., Mills J. S., Thornburg K. R., Knapp D. R., Schey K. L., Hamm H. E. A site on transducin alpha-subunit of interaction with the polycationic region of cGMP phosphodiesterase inhibitory subunit. J Biol Chem. 1993 Nov 5;268(31):23611–23615. [PubMed] [Google Scholar]
  6. Artemyev N. O., Rarick H. M., Mills J. S., Skiba N. P., Hamm H. E. Sites of interaction between rod G-protein alpha-subunit and cGMP-phosphodiesterase gamma-subunit. Implications for the phosphodiesterase activation mechanism. J Biol Chem. 1992 Dec 15;267(35):25067–25072. [PubMed] [Google Scholar]
  7. Bennett N., Clerc A. Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin). Biochemistry. 1989 Sep 5;28(18):7418–7424. doi: 10.1021/bi00444a040. [DOI] [PubMed] [Google Scholar]
  8. Bennett N., Dupont Y. The G-protein of retinal rod outer segments (transducin). Mechanism of interaction with rhodopsin and nucleotides. J Biol Chem. 1985 Apr 10;260(7):4156–4168. [PubMed] [Google Scholar]
  9. Bornstein P., Balian G. Cleavage at Asn-Gly bonds with hydroxylamine. Methods Enzymol. 1977;47:132–145. doi: 10.1016/0076-6879(77)47016-2. [DOI] [PubMed] [Google Scholar]
  10. Bourne H. R., Stryer L. G proteins. The target sets the tempo. Nature. 1992 Aug 13;358(6387):541–543. doi: 10.1038/358541a0. [DOI] [PubMed] [Google Scholar]
  11. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  12. Brown R. L. Functional regions of the inhibitory subunit of retinal rod cGMP phosphodiesterase identified by site-specific mutagenesis and fluorescence spectroscopy. Biochemistry. 1992 Jun 30;31(25):5918–5925. doi: 10.1021/bi00140a031. [DOI] [PubMed] [Google Scholar]
  13. Brown R. L., Stryer L. Expression in bacteria of functional inhibitory subunit of retinal rod cGMP phosphodiesterase. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4922–4926. doi: 10.1073/pnas.86.13.4922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chabre M., Deterre P. Molecular mechanism of visual transduction. Eur J Biochem. 1989 Feb 1;179(2):255–266. doi: 10.1111/j.1432-1033.1989.tb14549.x. [DOI] [PubMed] [Google Scholar]
  15. Clerc A., Bennett N. Activated cGMP phosphodiesterase of retinal rods. A complex with transducin alpha subunit. J Biol Chem. 1992 Apr 5;267(10):6620–6627. [PubMed] [Google Scholar]
  16. Clerc A., Catty P., Bennett N. Interaction between cGMP-phosphodiesterase and transducin alpha-subunit in retinal rods. A cross-linking study. J Biol Chem. 1992 Oct 5;267(28):19948–19953. [PubMed] [Google Scholar]
  17. Cowan C. W., Fariss R. N., Sokal I., Palczewski K., Wensel T. G. High expression levels in cones of RGS9, the predominant GTPase accelerating protein of rods. Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5351–5356. doi: 10.1073/pnas.95.9.5351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Crimmins D. L., McCourt D. W., Thoma R. S., Scott M. G., Macke K., Schwartz B. D. In situ chemical cleavage of proteins immobilized to glass-fiber and polyvinylidenedifluoride membranes: cleavage at tryptophan residues with 2-(2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine to obtain internal amino acid sequence. Anal Biochem. 1990 May 15;187(1):27–38. doi: 10.1016/0003-2697(90)90412-3. [DOI] [PubMed] [Google Scholar]
  19. Cunnick J., Twamley C., Udovichenko I., Gonzalez K., Takemoto D. J. Identification of a binding site on retinal transducin alpha for the phosphodiesterase inhibitory gamma subunit. Biochem J. 1994 Jan 1;297(Pt 1):87–91. doi: 10.1042/bj2970087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Dhanasekaran N., Wessling-Resnick M., Kelleher D. J., Johnson G. L., Ruoho A. E. Mapping of the carboxyl terminus within the tertiary structure of transducin's alpha subunit using the heterobifunctional cross-linking reagent, 125I-N-(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) cysteine. J Biol Chem. 1988 Dec 5;263(34):17942–17950. [PubMed] [Google Scholar]
  21. He W., Cowan C. W., Wensel T. G. RGS9, a GTPase accelerator for phototransduction. Neuron. 1998 Jan;20(1):95–102. doi: 10.1016/s0896-6273(00)80437-7. [DOI] [PubMed] [Google Scholar]
  22. Hingorani V. N., Tobias D. T., Henderson J. T., Ho Y. K. Chemical cross-linking of bovine retinal transducin and cGMP phosphodiesterase. J Biol Chem. 1988 May 15;263(14):6916–6926. [PubMed] [Google Scholar]
  23. Ho Y. K., Fung B. K. Characterization of transducin from bovine retinal rod outer segments. The role of sulfhydryl groups. J Biol Chem. 1984 May 25;259(10):6694–6699. [PubMed] [Google Scholar]
  24. Johnson G. L., Dhanasekaran N., Gupta S. K., Lowndes J. M., Vaillancourt R. R., Ruoho A. E. Genetic and structural analysis of G protein alpha subunit regulatory domains. J Cell Biochem. 1991 Oct;47(2):136–146. doi: 10.1002/jcb.240470207. [DOI] [PubMed] [Google Scholar]
  25. Koutalos Y., Yau K. W. Regulation of sensitivity in vertebrate rod photoreceptors by calcium. Trends Neurosci. 1996 Feb;19(2):73–81. doi: 10.1016/0166-2236(96)89624-x. [DOI] [PubMed] [Google Scholar]
  26. Kroll S., Phillips W. J., Cerione R. A. The regulation of the cyclic GMP phosphodiesterase by the GDP-bound form of the alpha subunit of transducin. J Biol Chem. 1989 Mar 15;264(8):4490–4497. [PubMed] [Google Scholar]
  27. Kutuzov M., Pfister C. Activation of the retinal cGMP-specific phosphodiesterase by the GDP-loaded alpha-subunit of transducin. Eur J Biochem. 1994 Mar 15;220(3):963–971. doi: 10.1111/j.1432-1033.1994.tb18700.x. [DOI] [PubMed] [Google Scholar]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. Lagnado L., Baylor D. Signal flow in visual transduction. Neuron. 1992 Jun;8(6):995–1002. doi: 10.1016/0896-6273(92)90122-t. [DOI] [PubMed] [Google Scholar]
  30. Lambright D. G., Noel J. P., Hamm H. E., Sigler P. B. Structural determinants for activation of the alpha-subunit of a heterotrimeric G protein. Nature. 1994 Jun 23;369(6482):621–628. doi: 10.1038/369621a0. [DOI] [PubMed] [Google Scholar]
  31. Lambright D. G., Sondek J., Bohm A., Skiba N. P., Hamm H. E., Sigler P. B. The 2.0 A crystal structure of a heterotrimeric G protein. Nature. 1996 Jan 25;379(6563):311–319. doi: 10.1038/379311a0. [DOI] [PubMed] [Google Scholar]
  32. Lipkin V. M., Bondarenko V. A., Zagranichny V. E., Dobrynina L. N., Muradov K. G., Natochin MYu Site-directed mutagenesis of the cGMP phosphodiesterase gamma subunit from bovine rod outer segments: role of separate amino acid residues in the interaction with catalytic subunits and transducin alpha subunit. Biochim Biophys Acta. 1993 Apr 16;1176(3):250–256. doi: 10.1016/0167-4889(93)90052-q. [DOI] [PubMed] [Google Scholar]
  33. Lipkin V. M., Dumler I. L., Muradov K. G., Artemyev N. O., Etingof R. N. Active sites of the cyclic GMP phosphodiesterase gamma-subunit of retinal rod outer segments. FEBS Lett. 1988 Jul 18;234(2):287–290. doi: 10.1016/0014-5793(88)80100-5. [DOI] [PubMed] [Google Scholar]
  34. Liu Y., Arshavsky V. Y., Ruoho A. E. Interaction sites of the COOH-terminal region of the gamma subunit of cGMP phosphodiesterase with the GTP-bound alpha subunit of transducin. J Biol Chem. 1996 Oct 25;271(43):26900–26907. doi: 10.1074/jbc.271.43.26900. [DOI] [PubMed] [Google Scholar]
  35. Mixon M. B., Lee E., Coleman D. E., Berghuis A. M., Gilman A. G., Sprang S. R. Tertiary and quaternary structural changes in Gi alpha 1 induced by GTP hydrolysis. Science. 1995 Nov 10;270(5238):954–960. doi: 10.1126/science.270.5238.954. [DOI] [PubMed] [Google Scholar]
  36. Morrison D. F., Cunnick J. M., Oppert B., Takemoto D. J. Interaction of the gamma-subunit of retinal rod outer segment phosphodiesterase with transducin. Use of synthetic peptides as functional probes. J Biol Chem. 1989 Jul 15;264(20):11671–11681. [PubMed] [Google Scholar]
  37. Morrison D. F., Rider M. A., Takemoto D. J. Modulation of retinal transducin and phosphodiesterase activities by synthetic peptides of the phosphodiesterase gamma-subunit. FEBS Lett. 1987 Oct 5;222(2):266–270. doi: 10.1016/0014-5793(87)80383-6. [DOI] [PubMed] [Google Scholar]
  38. Noel J. P., Hamm H. E., Sigler P. B. The 2.2 A crystal structure of transducin-alpha complexed with GTP gamma S. Nature. 1993 Dec 16;366(6456):654–663. doi: 10.1038/366654a0. [DOI] [PubMed] [Google Scholar]
  39. Otto-Bruc A., Antonny B., Vuong T. M., Chardin P., Chabre M. Interaction between the retinal cyclic GMP phosphodiesterase inhibitor and transducin. Kinetics and affinity studies. Biochemistry. 1993 Aug 24;32(33):8636–8645. doi: 10.1021/bi00084a035. [DOI] [PubMed] [Google Scholar]
  40. Pagès F., Deterre P., Pfister C. Enhanced GTPase activity of transducin when bound to cGMP phosphodiesterase in bovine retinal rods. J Biol Chem. 1992 Nov 5;267(31):22018–22021. [PubMed] [Google Scholar]
  41. Pugh E. N., Jr, Lamb T. D. Cyclic GMP and calcium: the internal messengers of excitation and adaptation in vertebrate photoreceptors. Vision Res. 1990;30(12):1923–1948. doi: 10.1016/0042-6989(90)90013-b. [DOI] [PubMed] [Google Scholar]
  42. Rarick H. M., Artemyev N. O., Hamm H. E. A site on rod G protein alpha subunit that mediates effector activation. Science. 1992 May 15;256(5059):1031–1033. doi: 10.1126/science.1317058. [DOI] [PubMed] [Google Scholar]
  43. Schlegel W., Kempner E. S., Rodbell M. Activation of adenylate cyclase in hepatic membranes involves interactions of the catalytic unit with multimeric complexes of regulatory proteins. J Biol Chem. 1979 Jun 25;254(12):5168–5176. [PubMed] [Google Scholar]
  44. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  45. Spickofsky N., Robichon A., Danho W., Fry D., Greeley D., Graves B., Madison V., Margolskee R. F. Biochemical analysis of the transducin-phosphodiesterase interaction. Nat Struct Biol. 1994 Nov;1(11):771–781. doi: 10.1038/nsb1194-771. [DOI] [PubMed] [Google Scholar]
  46. Steinman H. M., Naik V. R., Abernethy J. L., Hill R. L. Bovine erythrocyte superoxide dismutase. Complete amino acid sequence. J Biol Chem. 1974 Nov 25;249(22):7326–7338. [PubMed] [Google Scholar]
  47. Stryer L. Visual excitation and recovery. J Biol Chem. 1991 Jun 15;266(17):10711–10714. [PubMed] [Google Scholar]
  48. Tsang S. H., Burns M. E., Calvert P. D., Gouras P., Baylor D. A., Goff S. P., Arshavsky V. Y. Role for the target enzyme in deactivation of photoreceptor G protein in vivo. Science. 1998 Oct 2;282(5386):117–121. doi: 10.1126/science.282.5386.117. [DOI] [PubMed] [Google Scholar]
  49. Vaillancourt R. R., Dhanasekaran N., Ruoho A. E. The photoactivatable NAD+ analogue [32P]2-azido-NAD+ defines intra- and inter-molecular interactions of the C-terminal domain of the G-protein G alpha t. Biochem J. 1995 Nov 1;311(Pt 3):987–993. doi: 10.1042/bj3110987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wall M. A., Coleman D. E., Lee E., Iñiguez-Lluhi J. A., Posner B. A., Gilman A. G., Sprang S. R. The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2. Cell. 1995 Dec 15;83(6):1047–1058. doi: 10.1016/0092-8674(95)90220-1. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Winslow J. W., Bradley J. D., Smith J. A., Neer E. J. Reactive sulfhydryl groups of alpha 39, a guanine nucleotide-binding protein from brain. Location and function. J Biol Chem. 1987 Apr 5;262(10):4501–4507. [PubMed] [Google Scholar]
  53. Yamazaki A., Hayashi F., Tatsumi M., Bitensky M. W., George J. S. Interactions between the subunits of transducin and cyclic GMP phosphodiesterase in Rana catesbiana rod photoreceptors. J Biol Chem. 1990 Jul 15;265(20):11539–11548. [PubMed] [Google Scholar]
  54. Yamazaki A., Yamazaki M., Tsuboi S., Kishigami A., Umbarger K. O., Hutson L. D., Madland W. T., Hayashi F. Regulation of G protein function by an effector in GTP-dependent signal transduction. An inhibitory subunit of cGMP phosphodiesterase inhibits GTP hydrolysis by transducin in vertebrate rod photoreceptors. J Biol Chem. 1993 Apr 25;268(12):8899–8907. [PubMed] [Google Scholar]

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