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
. 1988 Apr;85(7):2378–2382. doi: 10.1073/pnas.85.7.2378

Purification and reconstitution of serotonin receptors from bovine brain.

T K Gallaher 1, H H Wang 1
PMCID: PMC279996  PMID: 3353386

Abstract

An affinity-chromatography column was used to isolate and purify 5-hydroxytryptamine (serotonin, 5-HT) receptors from bovine brain frontal cortex. The affinity ligand lysergic acid ethylamidoethylbromide was synthesized and coupled to an agarose matrix via a thioether bond. Receptors in the crude cortical membrane fragments were solubilized using 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), affinity purified, and reconstituted into lipid vesicles. [3H]5-HT binding analysis indicates a single class of high-affinity binding site (Kd, 16.9 nM) that was reconstituted. 5-Methoxytryptamine, a competitor for high-affinity serotonin sites, inhibited this binding and showed a Ki of 27.4 nM. Ketanserin, a high-affinity ligand for 5-HT2 type receptors, was ineffective in displacing [3H]5-HT binding at concentrations up to 4 microM indicating a 5-HT1 receptor as the primary receptor type isolated. The average specific activity of 359 pmol/mg in the reconstituted fractions is an enrichment of 1062-fold over crude membrane fragments. Sodium dodecyl-sulfate electrophoresis showed the presence of four proteins in the reconstituted vesicles with approximate relative Mr values of 63,000, 70,000, 81,000, and 94,000.

Full text

PDF
2378

Images in this article

2381Image
on p.2381
2381Image
on p.2381

Selected References

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

  1. Barbaccia M. L., Brunello N., Chuang D. M., Costa E. Serotonin-elicited amplification of adenylate cyclase activity in hippocampal membranes from adult rat. J Neurochem. 1983 Jun;40(6):1671–1679. doi: 10.1111/j.1471-4159.1983.tb08141.x. [DOI] [PubMed] [Google Scholar]
  2. Berry-Kravis E., Dawson G. Evidence for [D-Ala2,D-Leu5]enkephalin-induced supersensitivity to 5-hydroxytryptamine in a neurotumor x brain hybrid cell line (NCB-20). J Neurochem. 1985 Dec;45(6):1731–1738. doi: 10.1111/j.1471-4159.1985.tb10528.x. [DOI] [PubMed] [Google Scholar]
  3. Berry-Kravis E., Dawson G. Possible role of gangliosides in regulating an adenylate cyclase-linked 5-hydroxytryptamine (5-HT1) receptor. J Neurochem. 1985 Dec;45(6):1739–1747. doi: 10.1111/j.1471-4159.1985.tb10529.x. [DOI] [PubMed] [Google Scholar]
  4. Blurton P. A., Wood M. D. Identification of multiple binding sites for [3H]5-hydroxytryptamine in the rat CNS. J Neurochem. 1986 May;46(5):1392–1398. doi: 10.1111/j.1471-4159.1986.tb01753.x. [DOI] [PubMed] [Google Scholar]
  5. Cheng S. H., Shih J. C. Photoaffinity labeling of serotonin-binding proteins. Life Sci. 1979 Dec 24;25(26):2197–2203. doi: 10.1016/0024-3205(79)90092-4. [DOI] [PubMed] [Google Scholar]
  6. Conn P. J., Sanders-Bush E. Serotonin-stimulated phosphoinositide turnover: mediation by the S2 binding site in rat cerebral cortex but not in subcortical regions. J Pharmacol Exp Ther. 1985 Jul;234(1):195–203. [PubMed] [Google Scholar]
  7. Dixon R. A., Sigal I. S., Rands E., Register R. B., Candelore M. R., Blake A. D., Strader C. D. Ligand binding to the beta-adrenergic receptor involves its rhodopsin-like core. Nature. 1987 Mar 5;326(6108):73–77. doi: 10.1038/326073a0. [DOI] [PubMed] [Google Scholar]
  8. Emerit M. B., el Mestikawy S., Gozlan H., Cossery J. M., Besselievre R., Marquet A., Hamon M. Identification of the 5-HT1A receptor binding subunit in rat brain membranes using the photoaffinity probe [3H]8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin. J Neurochem. 1987 Aug;49(2):373–380. doi: 10.1111/j.1471-4159.1987.tb02875.x. [DOI] [PubMed] [Google Scholar]
  9. Enjalbert A., Bourgoin S., Hamon M., Adrien J., Bockaert J. Postsynaptic serotonin-sensitive adenylate cyclase in the central nervous system. I. Development and distribution of serotonin and dopamine-sensitive adenylate cyclases in rat and guinea pig brain. Mol Pharmacol. 1978 Jan;14(1):2–10. [PubMed] [Google Scholar]
  10. Enjalbert A., Hamon M., Bourgoin S., Bockaert J. Postsynaptic serotonin-sensitive adenylate cyclase in the central nervous system. II. Comparison with dopamine- and isoproterenol-sensitive adenylate cyclases in rat brain. Mol Pharmacol. 1978 Jan;14(1):11–23. [PubMed] [Google Scholar]
  11. Haga K., Haga T. Affinity chromatography of the muscarinic acetylcholine receptor. J Biol Chem. 1983 Nov 25;258(22):13575–13579. [PubMed] [Google Scholar]
  12. Kendall D. A., Nahorski S. R. 5-Hydroxytryptamine-stimulated inositol phospholipid hydrolysis in rat cerebral cortex slices: pharmacological characterization and effects of antidepressants. J Pharmacol Exp Ther. 1985 May;233(2):473–479. [PubMed] [Google Scholar]
  13. Kubo T., Fukuda K., Mikami A., Maeda A., Takahashi H., Mishina M., Haga T., Haga K., Ichiyama A., Kangawa K. Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature. 1986 Oct 2;323(6087):411–416. doi: 10.1038/323411a0. [DOI] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Leysen J. E., Niemegeers C. J., Van Nueten J. M., Laduron P. M. [3H]Ketanserin (R 41 468), a selective 3H-ligand for serotonin2 receptor binding sites. Binding properties, brain distribution, and functional role. Mol Pharmacol. 1982 Mar;21(2):301–314. [PubMed] [Google Scholar]
  16. MacDermot J., Higashida H., Wilson S. P., Matsuzawa H., Minna J., Nirenberg M. Adenylate cyclase and acetylcholine release regulated by separate serotonin receptors of somatic cell hybrids. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1135–1139. doi: 10.1073/pnas.76.3.1135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mehl E., Rüther E., Redemann J. Endogenous ligands of a putative LSD-serotonin receptor in the cerebrospinal fluid: higher level of LSD-displacing factors (LDF) in unmedicated psychotic patients. Psychopharmacology (Berl) 1977 Aug 31;54(1):9–16. doi: 10.1007/BF00426533. [DOI] [PubMed] [Google Scholar]
  18. Pedigo N. W., Yamamura H. I., Nelson D. L. Discrimination of multiple [3H]5-hydroxytryptamine binding sites by the neuroleptic spiperone in rat brain. J Neurochem. 1981 Jan;36(1):220–226. doi: 10.1111/j.1471-4159.1981.tb02397.x. [DOI] [PubMed] [Google Scholar]
  19. Peroutka S. J., Lebovitz R. M., Snyder S. H. Serotonin receptor binding sites affected differentially by guanine nucleotides. Mol Pharmacol. 1979 Nov;16(3):700–708. [PubMed] [Google Scholar]
  20. Peroutka S. J., Lebovitz R. M., Snyder S. H. Two distinct central serotonin receptors with different physiological functions. Science. 1981 May 15;212(4496):827–829. doi: 10.1126/science.7221567. [DOI] [PubMed] [Google Scholar]
  21. Peroutka S. J. Pharmacological differentiation and characterization of 5-HT1A, 5-HT1B, and 5-HT1C binding sites in rat frontal cortex. J Neurochem. 1986 Aug;47(2):529–540. doi: 10.1111/j.1471-4159.1986.tb04532.x. [DOI] [PubMed] [Google Scholar]
  22. Peroutka S. J., Snyder S. H. Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid diethylamide and [3H]spiroperidol. Mol Pharmacol. 1979 Nov;16(3):687–699. [PubMed] [Google Scholar]
  23. Saitoh T., Shih J. C. Photoaffinity labeling of adenylate cyclase-linked serotonin receptors in Aplysia neurons. J Neurochem. 1987 Nov;49(5):1361–1366. doi: 10.1111/j.1471-4159.1987.tb01000.x. [DOI] [PubMed] [Google Scholar]
  24. Schlegel J. R., Peroutka S. J. Nucleotide interactions with 5-HT1A binding sites directly labeled by [3H]-8-hydroxy-2-(DI-n-propylamino)tetralin ([3H]-8-OH-DPAT). Biochem Pharmacol. 1986 Jun 15;35(12):1943–1949. doi: 10.1016/0006-2952(86)90725-2. [DOI] [PubMed] [Google Scholar]
  25. Shorr R. G., Lefkowitz R. J., Caron M. G. Purification of the beta-adrenergic receptor. Identification of the hormone binding subunit. J Biol Chem. 1981 Jun 10;256(11):5820–5826. [PubMed] [Google Scholar]
  26. Todd R. D., Ciaranello R. D. Demonstration of inter- and intraspecies differences in serotonin binding sites by antibodies from an autistic child. Proc Natl Acad Sci U S A. 1985 Jan;82(2):612–616. doi: 10.1073/pnas.82.2.612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. VandenBerg S. R., Allgren R. L., Todd R. D., Ciaranello R. D. Solubilization and characterization of high-affinity [3H]serotonin binding sites from bovine cortical membranes. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3508–3512. doi: 10.1073/pnas.80.11.3508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wouters W., Van Dun J., Laduron P. M. Identification of the serotonin-S2 receptor ligand binding site by photoaffinity labelling with 7-azido-8-[125I]ketanserin ([125I]AZIK). FEBS Lett. 1987 Mar 23;213(2):359–364. doi: 10.1016/0014-5793(87)81522-3. [DOI] [PubMed] [Google Scholar]
  29. von Hungen K., Roberts S., Hill D. F. Serotonin-sensitive adenylate cyclase activity of immature rat brain. Brain Res. 1975 Feb 7;84(2):257–267. doi: 10.1016/0006-8993(75)90980-4. [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