Subcellular localization of G(iα) in human neutrophils

D. Rotrosen, J. I. Gallin, A. M. Spiegel, H. L. Malech

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73 Scopus citations


Subcellular fractions were prepared from human neutrophils by sucrose density gradient centrifugation and analyzed for G(i)-like proteins by pertussis toxin-catalyzed [32P]ADP-ribosylation and by immunoblotting with rabbit antiserum AS/6 which recognizes purified transducin and G(i), but not G(s) or G(o) α-subunits. In resting cells, ≃ 60% of pertussis toxin substrate retrieved from the sucrose density gradient localized to the plasma membrane-enriched fraction, ≃ 35% to the specific granule-enriched fraction, and ≃ 5% to cytosol. The azurophil granule-enriched fraction did not contain pertussis toxin substrate. In contrast to plasma membrane, the specific granule-enriched fraction demonstrated increased AS/6 immunoreactivity of a ≃ 41-kDa protein relative to a ≃ 40-kDa protein. Within the specific granule-enriched fraction, the peak of pertussis toxin substrate detected immunochemically or by [32P]ADP-ribosylation sedimented at a lighter density (ρ = 1.6 g/ml) than did lactoferrin (ρ = 1.19 g/ml), suggesting that the intracellular compartment bearing pertussis toxin substrate may not be the lactoferrin containing specific granule, per se. Furthermore, in neutrophils exposed to 10-8 M N-formylmethionylleucylphenylalanine, a weak degranulating stimulus (7% lactoferrin degranulation), there was a 31-42% decline in pertussis toxin-catalyzed [32P]ADP-ribosylation of ≃ 40-41-kDa proteins in the specific granule-enriched fraction accompanied by a near-quantitative increase in labeling of plasma membrane. The pool of intracellular formyl peptide receptors localized to the specific granule-enriched fraction appeared functionally coupled to a cosedimenting G-protein in experiments demonstrating modulation of high affinity N-formylmethionylleucyl[3H]phenylalanine binding by guanosine 5'-(3-O-thio)triphosphate or pertussis toxin. The data indicate that neutrophils contain a surface translocatable pool of intracellular G-protein sedimenting in the specific granule-enriched fraction and support the view that mobilization of intracellular G-protein represents a mechanism by which cells can regulate receptor activity.

Original languageEnglish (US)
Pages (from-to)10958-10964
Number of pages7
JournalJournal of Biological Chemistry
Issue number22
StatePublished - 1988
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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