Subcellular distribution and characterization of GTP-binding proteins in human neutrophils

Leili Khachatrian, Jeffrey B. Rubins, Eric C. Manning, David Dexter, Alfred I. Tauber, Burton F. Dickey

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

The subcellular distribution of GTP binding proteins in human neutrophils and their functional coupling to the N-formylmethionylleucylphenylalanine (FMLP) receptor was characterized to provide insight into mechanisms of cellular activation. Human neutrophils were nitrogen cavitated and fractionated on discontinuous Percoll gradients. Four subcellular fractions were obtained: cytosol, light membranes enriched for plasma membranes, specific granules and azurophilic granules. ADP-ribosylation catalyzed by pertussis toxin (PT) revealed a major substrate of 40 kDa only in plasma membrane and cytosol, and antiserum specific for Giα confirmed the presence of neutrophil Giα in plasma membrane and cytosol and its absence from specific granules. The cytosolic PT substrate was shown to be mostly in monomeric form by molecular sieve chromatography. The rate of the ribosyltransferase reaction was several-fold lower in cytosol compared to plasma membranes, and the extent of ADP-ribosylation was greatly augmented by supplementation with βγ subunits in cytosol. ADP-ribosylation catalyzed by cholera toxin (CT) revealed substrates of 52, 43 and 40 kDa in plasma membrane alone. FMLP receptors in plasma membrane were shown to be coupled to the 40 kDa substrate for CT by ligand-modulation of ADP-ribosylation, while FMLP added to specific granules did not induce ribosylation of this substrate even though FMLP receptors were found in high density in this compartment. Both 24 and 26 kDa [32P]GTP binding proteins were found to codistribute with FMLP receptors in specific granules and plasma membranes. Functional evidence for the coupling of GTP binding proteins to the FMLP receptor in specific granules was obtained by modulating [3H]FMLP binding with GTPγS, and by accelerating [35S]GTPγS binding with FMLP.

Original languageEnglish (US)
Pages (from-to)237-245
Number of pages9
JournalBBA - Molecular Cell Research
Volume1054
Issue number2
DOIs
StatePublished - Sep 1 1990
Externally publishedYes

Bibliographical note

Funding Information:
We wish to thank Nellie Pavlotsky for assistance in preparation of neutrophil subcellular fractions, Joseph Troll for scanning our autoradiograms, and Hanna Bloomfield Rubins for assistance with the computerized statistical analyses. This work was supported by NIH grants GM39608 (L.K.), HL07864 (J.B.R.), HL33565 and AI20064 (A.I.T.), and HL05503 and an American Lung Association Research Grant (B.F.D.).

Keywords

  • ADP-ribosylation
  • G-protein
  • GTP binding protein
  • Neutrophil

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