Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

Kim Williamson, Burton F. Dickey, Hae Yung Pyun, Javier Navarro

Research output: Contribution to journalArticle

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Abstract

We describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [3H] fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [3H] fMet-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, we also demonstrated fMet-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils.

Original languageEnglish (US)
Pages (from-to)5371-5377
Number of pages7
JournalBiochemistry
Volume27
Issue number14
StatePublished - 1988
Externally publishedYes

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N-Formylmethionine Leucyl-Phenylalanine
GTP-Binding Proteins
Guanine Nucleotides
Wheat Germ Agglutinins
Brain
Fractionation
Chromatography
Guanosine Triphosphate
Sepharose
Gel Chromatography
Hydrolysis
Phospholipids
Neutrophils
Gels
Chemical activation
Binding Sites
Ligands

ASJC Scopus subject areas

  • Biochemistry

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Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein. / Williamson, Kim; Dickey, Burton F.; Pyun, Hae Yung; Navarro, Javier.

In: Biochemistry, Vol. 27, No. 14, 1988, p. 5371-5377.

Research output: Contribution to journalArticle

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