Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Oct 25;91(22):10521–10525. doi: 10.1073/pnas.91.22.10521

Topological analysis of the human beta 2-adrenergic receptor expressed in Escherichia coli.

R M Lacatena 1, A Cellini 1, F Scavizzi 1, G P Tocchini-Valentini 1
PMCID: PMC45053  PMID: 7937986

Abstract

We have investigated the topology of the human beta 2-adrenergic receptor expressed in Escherichia coli, using the genetic method described by Beckwith and coworkers. We found that fusions with alkaline phosphatase beyond a certain point on the human beta 2-adrenergic receptor sequence were assembled into the bacterial membrane with the same topology as the human beta 2-adrenergic receptor in the mammalian membrane. The pattern that might have been expected on the basis of the topology of the human beta 2-adrenergic receptor in mammalian membranes was not reflected in the levels of alkaline phosphatase activity of the fusions occurring between the N-terminal region and positions close to the second external domain. Our data suggest that the correct positioning of the N terminus of the receptor depends on the presence of its C-terminal portions.

Full text

PDF
10521

Images in this article

10522Image
on p.10522
10522Image
on p.10522
10523Image
on p.10523
10523Image
on p.10523
10523Image
on p.10523

Selected References

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

  1. Boyd D., Beckwith J. The role of charged amino acids in the localization of secreted and membrane proteins. Cell. 1990 Sep 21;62(6):1031–1033. doi: 10.1016/0092-8674(90)90378-r. [DOI] [PubMed] [Google Scholar]
  2. Boyd D., Traxler B., Beckwith J. Analysis of the topology of a membrane protein by using a minimum number of alkaline phosphatase fusions. J Bacteriol. 1993 Jan;175(2):553–556. doi: 10.1128/jb.175.2.553-556.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradbury A., Persic L., Werge T., Cattaneo A. Use of living columns to select specific phage antibodies. Biotechnology (N Y) 1993 Dec;11(13):1565–1569. doi: 10.1038/nbt1293-1565. [DOI] [PubMed] [Google Scholar]
  4. Breyer R. M., Strosberg A. D., Guillet J. G. Mutational analysis of ligand binding activity of beta 2 adrenergic receptor expressed in Escherichia coli. EMBO J. 1990 Sep;9(9):2679–2684. doi: 10.1002/j.1460-2075.1990.tb07453.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burkholder A. C., Hartwell L. H. The yeast alpha-factor receptor: structural properties deduced from the sequence of the STE2 gene. Nucleic Acids Res. 1985 Dec 9;13(23):8463–8475. doi: 10.1093/nar/13.23.8463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Calamia J., Manoil C. Membrane protein spanning segments as export signals. J Mol Biol. 1992 Apr 5;224(3):539–543. doi: 10.1016/0022-2836(92)90542-r. [DOI] [PubMed] [Google Scholar]
  7. Calamia J., Manoil C. lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4937–4941. doi: 10.1073/pnas.87.13.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Charbit A., Ronco J., Michel V., Werts C., Hofnung M. Permissive sites and topology of an outer membrane protein with a reporter epitope. J Bacteriol. 1991 Jan;173(1):262–275. doi: 10.1128/jb.173.1.262-275.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dalbey R. E. Positively charged residues are important determinants of membrane protein topology. Trends Biochem Sci. 1990 Jul;15(7):253–257. doi: 10.1016/0968-0004(90)90047-f. [DOI] [PubMed] [Google Scholar]
  10. Derman A. I., Beckwith J. Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm. J Bacteriol. 1991 Dec;173(23):7719–7722. doi: 10.1128/jb.173.23.7719-7722.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Derman A. I., Beckwith J. Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm. J Bacteriol. 1991 Dec;173(23):7719–7722. doi: 10.1128/jb.173.23.7719-7722.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dohlman H. G., Bouvier M., Benovic J. L., Caron M. G., Lefkowitz R. J. The multiple membrane spanning topography of the beta 2-adrenergic receptor. Localization of the sites of binding, glycosylation, and regulatory phosphorylation by limited proteolysis. J Biol Chem. 1987 Oct 15;262(29):14282–14288. [PubMed] [Google Scholar]
  13. Dohlman H. G., Caron M. G., Lefkowitz R. J. A family of receptors coupled to guanine nucleotide regulatory proteins. Biochemistry. 1987 May 19;26(10):2657–2664. doi: 10.1021/bi00384a001. [DOI] [PubMed] [Google Scholar]
  14. Ehrmann M., Boyd D., Beckwith J. Genetic analysis of membrane protein topology by a sandwich gene fusion approach. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7574–7578. doi: 10.1073/pnas.87.19.7574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Freissmuth M., Selzer E., Marullo S., Schütz W., Strosberg A. D. Expression of two human beta-adrenergic receptors in Escherichia coli: functional interaction with two forms of the stimulatory G protein. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8548–8552. doi: 10.1073/pnas.88.19.8548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Galli G., Matteoni R., Bianchi E., Testa L., Marazziti D., Rossi N., Tocchini-Valentini G. Replica filter assay of human beta-adrenergic receptors expressed in E.coli. Biochem Biophys Res Commun. 1990 Dec 14;173(2):680–688. doi: 10.1016/s0006-291x(05)80089-0. [DOI] [PubMed] [Google Scholar]
  18. Gutierrez C., Devedjian J. C. A plasmid facilitating in vitro construction of phoA gene fusions in Escherichia coli. Nucleic Acids Res. 1989 May 25;17(10):3999–3999. doi: 10.1093/nar/17.10.3999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Henderson R., Baldwin J. M., Ceska T. A., Zemlin F., Beckmann E., Downing K. H. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol. 1990 Jun 20;213(4):899–929. doi: 10.1016/S0022-2836(05)80271-2. [DOI] [PubMed] [Google Scholar]
  20. High S., Flint N., Dobberstein B. Requirements for the membrane insertion of signal-anchor type proteins. J Cell Biol. 1991 Apr;113(1):25–34. doi: 10.1083/jcb.113.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ito K., Bassford P. J., Jr, Beckwith J. Protein localization in E. coli: is there a common step in the secretion of periplasmic and outer-membrane proteins? Cell. 1981 Jun;24(3):707–717. doi: 10.1016/0092-8674(81)90097-0. [DOI] [PubMed] [Google Scholar]
  22. Kobilka B. K., Dixon R. A., Frielle T., Dohlman H. G., Bolanowski M. A., Sigal I. S., Yang-Feng T. L., Francke U., Caron M. G., Lefkowitz R. J. cDNA for the human beta 2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proc Natl Acad Sci U S A. 1987 Jan;84(1):46–50. doi: 10.1073/pnas.84.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  24. Lee J. I., Kuhn A., Dalbey R. E. Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion. J Biol Chem. 1992 Jan 15;267(2):938–943. [PubMed] [Google Scholar]
  25. Manoil C., Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 1986 Sep 26;233(4771):1403–1408. doi: 10.1126/science.3529391. [DOI] [PubMed] [Google Scholar]
  26. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Manoil C., Mekalanos J. J., Beckwith J. Alkaline phosphatase fusions: sensors of subcellular location. J Bacteriol. 1990 Feb;172(2):515–518. doi: 10.1128/jb.172.2.515-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Marullo S., Delavier-Klutchko C., Eshdat Y., Strosberg A. D., Emorine L. Human beta 2-adrenergic receptors expressed in Escherichia coli membranes retain their pharmacological properties. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7551–7555. doi: 10.1073/pnas.85.20.7551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McGovern K., Ehrmann M., Beckwith J. Decoding signals for membrane protein assembly using alkaline phosphatase fusions. EMBO J. 1991 Oct;10(10):2773–2782. doi: 10.1002/j.1460-2075.1991.tb07826.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Michaelis S., Inouye H., Oliver D., Beckwith J. Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. J Bacteriol. 1983 Apr;154(1):366–374. doi: 10.1128/jb.154.1.366-374.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nilsson I., von Heijne G. Fine-tuning the topology of a polytopic membrane protein: role of positively and negatively charged amino acids. Cell. 1990 Sep 21;62(6):1135–1141. doi: 10.1016/0092-8674(90)90390-z. [DOI] [PubMed] [Google Scholar]
  32. San Millan J. L., Boyd D., Dalbey R., Wickner W., Beckwith J. Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase. J Bacteriol. 1989 Oct;171(10):5536–5541. doi: 10.1128/jb.171.10.5536-5541.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Traxler B., Lee C., Boyd D., Beckwith J. The dynamics of assembly of a cytoplasmic membrane protein in Escherichia coli. J Biol Chem. 1992 Mar 15;267(8):5339–5345. [PubMed] [Google Scholar]
  34. Wang H., Lipfert L., Malbon C. C., Bahouth S. Site-directed anti-peptide antibodies define the topography of the beta-adrenergic receptor. J Biol Chem. 1989 Aug 25;264(24):14424–14431. [PubMed] [Google Scholar]
  35. von Heijne G. Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol. 1992 May 20;225(2):487–494. doi: 10.1016/0022-2836(92)90934-c. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES