Skip to main content
SpringerLink
Log in

Molecular cloning, sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain

  • Original Article
  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

We have isolated a cDNA clone from rat brain using a human platelet α2-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney α2-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3′-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5′-untranslated region. Southern-blot analysis with a human platelet α2-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain α2-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A+) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the α2-adrenergic antagonist [3H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the α2A subclass. We conclude that the rat brain cDNA encodes a new α2-adrenergic receptor subtype that may be brain-specific.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

G protein:

guanine nucleotide-binding proteins

cA2-47:

α2-adrenergic receptor cDNA from rat brain

SSC:

(1X SSC contains 0.15 M NaCl, 15 mM Na3citrate, pH 7.0)

References

  1. Limbird LE, Sweatt JD: In: PM Conn (ed) The Receptors, Vol II. Academic Press, Orlando, FL, 1985, pp 281–306

  2. Bylund DB: Subtypes of α2-adrenoceptors: Pharmacological and molecular biological evidence converge. Trends Pharmacol Sci 9: 356–361, 1988

    Google Scholar 

  3. Cheung Y-D, Barnett DB, Narhorski SR: Phorbol 12, 13-dibutyrate potentiates a2-adrenoceptor-mediated contraction in the rat saphenous vein. Eur J Pharmacol 84: 79–85, 1982

    Google Scholar 

  4. Dickinson KEJ, McKernan RM, Miles CMM, Leys KS, Sever PD: Heterogeneity of mammalian α2-adrenoceptors delineated by [3H-yohimbine binding. Eur J Pharmacol 120: 285–293, 1986

    Google Scholar 

  5. Graham RM, Lanier SM: α2-Adrenergic receptors: Apparent interaction with multiple effector systems. Chapter 8. In: HM Fozzard, E Haber, R Jennings, A Katz, H Morgan (eds) The Heart and Cardiovascular System, Vol II. Raven Press, New York, pp 1059–1095, 1986

    Google Scholar 

  6. Lanier SM, Homcy CJ, Patenaude C, Graham RM: Identification of structurally distinct α2-adrenergic receptors. J Biol Chem 263: 14491–14496, 1988

    Google Scholar 

  7. Fraser CM, Arakawa S, McCombie WR, Venter JC: Cloning, sequence analysis, and permanent expression of a human α2-receptor in Chinese hamster ovary cells. 264: 11754–11761, 1989

    Google Scholar 

  8. Kobilka BK, Matsu H, Kobilka TS, Yang-Feng TL, Francke U, Caron MG, Lefkowitz RJ, Regan JW: Cloning, sequencing, and expression of the gene coding for the human platelet α2-adrenergic receptor. Science 238: 650–656, 1987

    CAS  PubMed  Google Scholar 

  9. Regan JW, Kobilka TS, Yang-Feng TL, Caron MG, Lefkowitz RJ, Kobilka BK: Cloning and expression of a human kidney cDNA for an α2-receptor subtype. Proc Natl Acad Sci USA 85: 6301–6305, 1988

    CAS  PubMed  Google Scholar 

  10. Jaiswal RK, Marshak DR, Sharma RK: Molecular comparison of α2-adrenergic receptors from rat adrenocortical carcinoma and human blood platelet. Mol Cell Biochem 86: 41–53, 1989

    Google Scholar 

  11. Nambi P, Aiyar NV, Sharma RK: Identification and characterization of ectopic a2-receptors in adrenocortical carcinoma membranes. J Nutrition, Growth and Cancer 1: 77–84, 1983

    Google Scholar 

  12. Benton WD, Davis RW: Screening q gt recombinant clones by hybridization to single plaques in situ. Science 196: 180–182, 1977

    Google Scholar 

  13. Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction fragments to high specific activity. Anal Biochem 132: 6–12, 1984

    Google Scholar 

  14. Short JM, Fernandez JM, Sorge JA, Huse WD: Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucl Acids Res 16: 7583–7600, 1988

    Google Scholar 

  15. Sanger F, Nicklen S, Coulsen AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467, 1977

    CAS  PubMed  Google Scholar 

  16. Henikoff S: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359, 1984

    Google Scholar 

  17. Cullen BR: Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enzymol 152: 684–704, 1987

    Google Scholar 

  18. Maniatis T, Frisch EF, Sambrook J: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, 1982

  19. Kozak M: Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucl Acids Res 12: 857–872, 1984

    Google Scholar 

  20. Kyte J, Doolittle RR: A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105–132, 1982

    CAS  PubMed  Google Scholar 

  21. Dohlman HG, Caron MG, Lefkowitz RJ: A family of receptors coupled to guanine nucleotide regulatory proteins. Biochemistry 26: 2657–2664, 1987

    Google Scholar 

  22. Ovchinnikov YA: Rhodopsin and bacteriorhodopsin: structure-function relationships. FEBS Lett 148: 179–191, 1982

    Google Scholar 

  23. Emini EA, Hughes JV, Perlow DS, Boger J: Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J Virology 55: 836–839, 1985

    Google Scholar 

  24. Jameson BA, Wolf H: The antigenic index: a novel algorithm for predicting antigenic determinants. Computer Applications in the Biosciences 4: 181–186, 1988

    Google Scholar 

  25. Chou PY, Fasman GD: Prediction of the secondary structure of proteins from their amino acid sequence. Advances in Enzymology 47: 45–145, 1978

    Google Scholar 

  26. Garnier J, Osguthorpe DJ, Robson B: Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol 120: 97–120, 1978

    Google Scholar 

  27. Duda T, Chalberg S, Sharma RK: Genetic evidence for α2-adrenergic receptor subtypes in rat brain, heart and adrenal gland. Mol Cell Biochem 92: 69–75, 1990

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Section of Regulatory Biology, Department of Brain and Vascular Research, Cleveland Clinic Research Institute, Cleveland Clinic Foundation, 44195-5068, Cleveland, Ohio, USA

    Stephen C. Chalberg, Teresa Duda, Janet A. Rhine & Rameshwar K. Sharma

Authors
  1. Stephen C. Chalberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Teresa Duda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Janet A. Rhine
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rameshwar K. Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chalberg, S.C., Duda, T., Rhine, J.A. et al. Molecular cloning, sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain. Mol Cell Biochem 97, 161–172 (1990). https://doi.org/10.1007/BF00221058

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00221058

Key words

Search

Navigation