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Absence of G(i) proteins in the Sf9 insect cell. Characterization of the uncoupled recombinant N-formyl peptide receptor

Academic Article
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Overview

authors

  • Quehenberger, O.
  • Prossnitz, E. R.
  • Cochrane, Charles
  • Ye, R. D.

publication date

  • October 1992

journal

  • Journal of Biological Chemistry  Journal

abstract

  • We investigated the interaction of the N-formyl peptide receptor (NFPR) with G proteins in infected Sf9 insect cells expressing the recombinant NFPR. Recombinant receptor expression of up to 27 pmol/mg protein was achieved in these cells. The receptor was recognized by an antiserum raised against an NFPR carboxyl-terminal peptide, and displayed specific and saturable binding of the formyl peptide ligand fMet-Leu-[3H]Phe. Scatchard analysis of the binding data yielded a dissociation constant of approximately 62 nM, a binding affinity of 60- to 120-fold lower than that of the high affinity sites in neutrophils and in transfected mammalian cell lines expressing the NFPR. That this low binding affinity was due to a lack of receptor coupling to G protein was suggested by the failure of guanine nucleotides to regulate receptor affinity and by the lack of formyl peptide-stimulated GTPase activity in these cells. Furthermore, immunoblotting with an anti-G(i) antibody and ADP-ribosylation experiments indicated that the approximately 40-kDa G(i) alpha subunit, which couples to the NFPR in neutrophils, is not present in Sf9 cell membranes. Thus, the current study provides for the first time evidence that a major G protein is absent in the Sf9 insect cells. Potential applications of the Sf9 system for in vitro reconstitution of the NFPR-G protein interaction are discussed.

subject areas

  • Adenosine Diphosphate Ribose
  • Affinity Labels
  • Animals
  • Binding Sites
  • Blotting, Western
  • Cell Line
  • DNA
  • Enzyme Activation
  • GTP Phosphohydrolases
  • GTP-Binding Proteins
  • Humans
  • Insecta
  • N-Formylmethionine Leucyl-Phenylalanine
  • Neutrophils
  • Precipitin Tests
  • Receptors, Formyl Peptide
  • Receptors, Immunologic
  • Recombinant Proteins
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

PubMed ID

  • 1400288
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Additional Document Info

start page

  • 19757

end page

  • 19760

volume

  • 267

issue

  • 28

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