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Mutational analysis of ligand-binding activity of beta-2 adrenergic-receptor expressed in escherichia-coli

Academic Article
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Overview

authors

  • Breyer, R. M.
  • Strosberg, Donny
  • Guillet, J. G.

publication date

  • 1990

journal

  • EMBO Journal  Journal

abstract

  • A novel in situ screening procedure was used to identify neutral mutations in the human beta 2 adrenergic receptor (beta 2AR). The coding region of the human beta 2AR gene was subcloned under transcriptional control of an inducible T7 promoter and used to transform Escherichia coli. Colonies expressing the beta 2AR bound the radiolabeled antagonist [125I]iodocyanopindolol and could be identified by autoradiography after transfer to nitrocellulose filters. The region of the beta 2AR between residues 76 and 83, in the second transmembrane helix, was mutagenized by a saturation mutagenesis technique, so that virtually all of the beta 2AR genes contained at least one mutation. Colonies retaining ligand binding activity were isolated using the in situ screen. Sequence analysis of the active mutant receptor genes allowed the identification of individual amino acid side chains which are essential for either ligand binding or structural integrity of the beta 2AR receptor.

subject areas

  • Amino Acid Sequence
  • Escherichia coli
  • Humans
  • Iodocyanopindolol
  • Kinetics
  • Ligands
  • Molecular Sequence Data
  • Mutation
  • Oligonucleotide Probes
  • Pindolol
  • Plasmids
  • Receptors, Adrenergic, beta
  • Recombinant Proteins
  • Restriction Mapping
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Identity

PubMed Central ID

  • PMC551972

International Standard Serial Number (ISSN)

  • 0261-4189

PubMed ID

  • 2167830
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Additional Document Info

start page

  • 2679

end page

  • 2684

volume

  • 9

issue

  • 9

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