Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1997 Sep 15;100(6):1547–1556. doi: 10.1172/JCI119678

Reconstitution of mutant V2 vasopressin receptors by adenovirus-mediated gene transfer. Molecular basis and clinical implication.

T Schöneberg 1, V Sandig 1, J Wess 1, T Gudermann 1, G Schultz 1
PMCID: PMC508336  PMID: 9294123

Abstract

Recent studies with transfected COS-7 cells have shown that functionally inactive mutant V2 vasopressin receptors (occurring in patients with nephrogenic diabetes insipidus) can be functionally rescued by coexpression of a carboxy-terminal V2 receptor fragment (V2-tail) spanning the region where various mutations occur [Schöneberg, T., J. Yun, D. Wenkert, and J. Wess. 1996. EMBO (Eur. Mol. Biol. Organ.) J. 15:1283-1291]. In this study, we set out to characterize the underlying molecular mechanism. Using a coimmunoprecipitation strategy and a newly developed sandwich ELISA system, a direct and highly specific interaction between the mutant V2 vasopressin receptor proteins and the V2-tail polypeptide was demonstrated. To study the potential therapeutic usefulness of these findings, Chinese hamster ovary (CHO) cell lines stably expressing low levels of functionally inactive mutant V2 vasopressin receptors were created and infected with a recombinant adenovirus carrying the V2-tail gene fragment. After adenovirus infection, vasopressin gained the ability to stimulate cAMP formation with high potency and efficacy in all CHO cell clones studied. Moreover, adenovirus-mediated gene transfer also proved to be a highly efficient method for achieving expression of the V2-tail fragment (as well as the wild-type V2 receptor) in Madin-Darby canine kidney tubular cells. Taken together, these studies clarify the molecular mechanisms by which receptor fragments can restore function of mutationally inactivated G protein-coupled receptors and suggest that adenovirus-mediated expression of receptor fragments may lead to novel strategies for the treatment of a variety of human diseases.

Full Text

The Full Text of this article is available as a PDF (353.4 KB).

Selected References

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

  1. Baker E. K., Colley N. J., Zuker C. S. The cyclophilin homolog NinaA functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin. EMBO J. 1994 Oct 17;13(20):4886–4895. doi: 10.1002/j.1460-2075.1994.tb06816.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berkower C., Michaelis S. Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily. EMBO J. 1991 Dec;10(12):3777–3785. doi: 10.1002/j.1460-2075.1991.tb04947.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bett A. J., Haddara W., Prevec L., Graham F. L. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8802–8806. doi: 10.1073/pnas.91.19.8802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bibi E., Kaback H. R. In vivo expression of the lacY gene in two segments leads to functional lac permease. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4325–4329. doi: 10.1073/pnas.87.11.4325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Chang H., Katoh T., Noda M., Kanegae Y., Saito I., Asano S., Kurokawa K. Highly efficient adenovirus-mediated gene transfer into renal cells in culture. Kidney Int. 1995 Jan;47(1):322–326. doi: 10.1038/ki.1995.42. [DOI] [PubMed] [Google Scholar]
  7. Cullen B. R. Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enzymol. 1987;152:684–704. doi: 10.1016/0076-6879(87)52074-2. [DOI] [PubMed] [Google Scholar]
  8. Ferreira P. A., Nakayama T. A., Pak W. L., Travis G. H. Cyclophilin-related protein RanBP2 acts as chaperone for red/green opsin. Nature. 1996 Oct 17;383(6601):637–640. doi: 10.1038/383637a0. [DOI] [PubMed] [Google Scholar]
  9. Gudermann T., Birnbaumer M., Birnbaumer L. Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells. J Biol Chem. 1992 Mar 5;267(7):4479–4488. [PubMed] [Google Scholar]
  10. Hebert T. E., Moffett S., Morello J. P., Loisel T. P., Bichet D. G., Barret C., Bouvier M. A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J Biol Chem. 1996 Jul 5;271(27):16384–16392. doi: 10.1074/jbc.271.27.16384. [DOI] [PubMed] [Google Scholar]
  11. Kanegae Y., Makimura M., Saito I. A simple and efficient method for purification of infectious recombinant adenovirus. Jpn J Med Sci Biol. 1994 Jun;47(3):157–166. doi: 10.7883/yoken1952.47.157. [DOI] [PubMed] [Google Scholar]
  12. Kimura T., Tanizawa O., Mori K., Brownstein M. J., Okayama H. Structure and expression of a human oxytocin receptor. Nature. 1992 Apr 9;356(6369):526–529. doi: 10.1038/356526a0. [DOI] [PubMed] [Google Scholar]
  13. Kolodziej P. A., Young R. A. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. doi: 10.1016/0076-6879(91)94038-e. [DOI] [PubMed] [Google Scholar]
  14. Lieber A., Sandig V., Strauss M. A mutant T7 phage promoter is specifically transcribed by T7-RNA polymerase in mammalian cells. Eur J Biochem. 1993 Oct 1;217(1):387–394. doi: 10.1111/j.1432-1033.1993.tb18257.x. [DOI] [PubMed] [Google Scholar]
  15. Maggio R., Vogel Z., Wess J. Coexpression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular "cross-talk" between G-protein-linked receptors. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):3103–3107. doi: 10.1073/pnas.90.7.3103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McGrory W. J., Bautista D. S., Graham F. L. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1988 Apr;163(2):614–617. doi: 10.1016/0042-6822(88)90302-9. [DOI] [PubMed] [Google Scholar]
  17. Monnot C., Bihoreau C., Conchon S., Curnow K. M., Corvol P., Clauser E. Polar residues in the transmembrane domains of the type 1 angiotensin II receptor are required for binding and coupling. Reconstitution of the binding site by co-expression of two deficient mutants. J Biol Chem. 1996 Jan 19;271(3):1507–1513. doi: 10.1074/jbc.271.3.1507. [DOI] [PubMed] [Google Scholar]
  18. Morel A., O'Carroll A. M., Brownstein M. J., Lolait S. J. Molecular cloning and expression of a rat V1a arginine vasopressin receptor. Nature. 1992 Apr 9;356(6369):523–526. doi: 10.1038/356523a0. [DOI] [PubMed] [Google Scholar]
  19. Moullier P., Friedlander G., Calise D., Ronco P., Perricaudet M., Ferry N. Adenoviral-mediated gene transfer to renal tubular cells in vivo. Kidney Int. 1994 Apr;45(4):1220–1225. doi: 10.1038/ki.1994.162. [DOI] [PubMed] [Google Scholar]
  20. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  21. Parma J., Duprez L., Van Sande J., Cochaux P., Gervy C., Mockel J., Dumont J., Vassart G. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature. 1993 Oct 14;365(6447):649–651. doi: 10.1038/365649a0. [DOI] [PubMed] [Google Scholar]
  22. Ridge K. D., Lee S. S., Yao L. L. In vivo assembly of rhodopsin from expressed polypeptide fragments. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3204–3208. doi: 10.1073/pnas.92.8.3204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rosenfeld M. A., Siegfried W., Yoshimura K., Yoneyama K., Fukayama M., Stier L. E., Päkkö P. K., Gilardi P., Stratford-Perricaudet L. D., Perricaudet M. Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science. 1991 Apr 19;252(5004):431–434. doi: 10.1126/science.2017680. [DOI] [PubMed] [Google Scholar]
  24. Rosenfeld M. A., Yoshimura K., Trapnell B. C., Yoneyama K., Rosenthal E. R., Dalemans W., Fukayama M., Bargon J., Stier L. E., Stratford-Perricaudet L. In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell. 1992 Jan 10;68(1):143–155. doi: 10.1016/0092-8674(92)90213-v. [DOI] [PubMed] [Google Scholar]
  25. Rosenthal W., Seibold A., Antaramian A., Lonergan M., Arthus M. F., Hendy G. N., Birnbaumer M., Bichet D. G. Molecular identification of the gene responsible for congenital nephrogenic diabetes insipidus. Nature. 1992 Sep 17;359(6392):233–235. doi: 10.1038/359233a0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Schoneberg T., Yun J., Wenkert D., Wess J. Functional rescue of mutant V2 vasopressin receptors causing nephrogenic diabetes insipidus by a co-expressed receptor polypeptide. EMBO J. 1996 Mar 15;15(6):1283–1291. [PMC free article] [PubMed] [Google Scholar]
  28. Schöneberg T., Liu J., Wess J. Plasma membrane localization and functional rescue of truncated forms of a G protein-coupled receptor. J Biol Chem. 1995 Jul 28;270(30):18000–18006. doi: 10.1074/jbc.270.30.18000. [DOI] [PubMed] [Google Scholar]
  29. Shenker A., Laue L., Kosugi S., Merendino J. J., Jr, Minegishi T., Cutler G. B., Jr A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature. 1993 Oct 14;365(6447):652–654. doi: 10.1038/365652a0. [DOI] [PubMed] [Google Scholar]
  30. Siebert P. D., Larrick J. W. Competitive PCR. Nature. 1992 Oct 8;359(6395):557–558. doi: 10.1038/359557a0. [DOI] [PubMed] [Google Scholar]
  31. Spiegel A. M. Defects in G protein-coupled signal transduction in human disease. Annu Rev Physiol. 1996;58:143–170. doi: 10.1146/annurev.ph.58.030196.001043. [DOI] [PubMed] [Google Scholar]
  32. Strader C. D., Fong T. M., Tota M. R., Underwood D., Dixon R. A. Structure and function of G protein-coupled receptors. Annu Rev Biochem. 1994;63:101–132. doi: 10.1146/annurev.bi.63.070194.000533. [DOI] [PubMed] [Google Scholar]
  33. Stratford-Perricaudet L. D., Makeh I., Perricaudet M., Briand P. Widespread long-term gene transfer to mouse skeletal muscles and heart. J Clin Invest. 1992 Aug;90(2):626–630. doi: 10.1172/JCI115902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stühmer W., Conti F., Suzuki H., Wang X. D., Noda M., Yahagi N., Kubo H., Numa S. Structural parts involved in activation and inactivation of the sodium channel. Nature. 1989 Jun 22;339(6226):597–603. doi: 10.1038/339597a0. [DOI] [PubMed] [Google Scholar]
  35. Sung C. H., Schneider B. G., Agarwal N., Papermaster D. S., Nathans J. Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8840–8844. doi: 10.1073/pnas.88.19.8840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tang W. J., Stanzel M., Gilman A. G. Truncation and alanine-scanning mutants of type I adenylyl cyclase. Biochemistry. 1995 Nov 7;34(44):14563–14572. doi: 10.1021/bi00044a035. [DOI] [PubMed] [Google Scholar]
  37. Wank S. A., Harkins R., Jensen R. T., Shapira H., de Weerth A., Slattery T. Purification, molecular cloning, and functional expression of the cholecystokinin receptor from rat pancreas. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3125–3129. doi: 10.1073/pnas.89.7.3125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wenkert D., Schoneberg T., Merendino J. J., Jr, Rodriguez Pena M. S., Vinitsky R., Goldsmith P. K., Wess J., Spiegel A. M. Functional characterization of five V2 vasopressin receptor gene mutations. Mol Cell Endocrinol. 1996 Nov 29;124(1-2):43–50. doi: 10.1016/s0303-7207(96)03926-3. [DOI] [PubMed] [Google Scholar]
  39. Wildin R. S., Antush M. J., Bennett R. L., Schoof J. M., Scott C. R. Heterogeneous AVPR2 gene mutations in congenital nephrogenic diabetes insipidus. Am J Hum Genet. 1994 Aug;55(2):266–277. [PMC free article] [PubMed] [Google Scholar]
  40. Zen K. H., McKenna E., Bibi E., Hardy D., Kaback H. R. Expression of lactose permease in contiguous fragments as a probe for membrane-spanning domains. Biochemistry. 1994 Jul 12;33(27):8198–8206. doi: 10.1021/bi00193a005. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES