Absence of G(i) proteins in the Sf9 insect cell. Characterization of the uncoupled recombinant N-formyl peptide receptor.
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.
Quehenberger
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Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
Prossnitz
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Cochrane
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Ye
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D000246:Adenosine Diphosphate Ribose / Q000737:chemistry; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D004247:DNA; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D011233:Precipitin Tests; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D000345:Affinity Labels; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D009240:N-Formylmethionine Leucyl-Phenylalanine; D004247:DNA; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins