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Functional expression of the human serotonin 5-ht1a receptor in escherichia-coli - ligand-binding properties and interaction with recombinant g-protein alpha-subunits

Academic Article
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Overview

authors

  • Bertin, B.
  • Freissmuth, M.
  • Breyer, R. M.
  • Schutz, W.
  • Strosberg, Donny
  • Marullo, S.

publication date

  • April 1992

journal

  • Journal of Biological Chemistry  Journal

abstract

  • Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r5-HT1A) and G alpha-subunits (rG alpha) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E. coli inner membranes. Binding of the selective agonist [3H] +/- 8-hydroxy-(2-N-dipropylamine)tetralin ([3H]8-OH-DPAT) to intact bacteria or E. coli membranes was saturable with a KD of approximately 8 nM and an average of 120 sites/bacterium. Binding properties of several serotoninergic ligands to r5-HT1A receptors were comparable with those measured in mammalian cells. Incubation of rG alpha.beta gamma with E. coli membranes resulted in high affinity agonist [3H]8-OH-DPAT binding (KD = 0.7 nM) and titration with a panel of rG alpha subtypes showed the order of potency: rGi alpha-3 greater than rGi alpha-2 greater than rGi alpha-1 much greater than rGo alpha, while rGs alpha appeared incapable of interacting with 5-HT1A receptors. Moreover, agonist-mediated enhancement of [35S]guanosine 5'-O-(3-thiotriphosphate) binding to rGi alpha confirmed the achievement of the functional interaction between receptor and G proteins. Our findings are in agreement with the in vivo ability of 5-HT1A receptors to activate Gi alpha related to adenylyl cyclase inhibition or K+ channel activation, but do not support previously reported adenylyl cyclase stimulation through interaction with Gs alpha.

subject areas

  • 8-Hydroxy-2-(di-n-propylamino)tetralin
  • Amino Acid Sequence
  • Binding, Competitive
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli
  • GTP-Binding Proteins
  • Gene Expression
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Humans
  • Ligands
  • Molecular Sequence Data
  • Plasmids
  • Receptors, Serotonin
  • Recombinant Proteins
  • Signal Transduction
  • Tetrahydronaphthalenes
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

PubMed ID

  • 1533220
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Additional Document Info

start page

  • 8200

end page

  • 8206

volume

  • 267

issue

  • 12

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