Low molecular weight GTP-binding proteins in human neutrophil granule membranes

• Published in: The Journal of biological chemistry Vol. 266; no. 2; pp. 1289 - 1298
• Main Authors: Philips, M R, Abramson, S B, Kolasinski, S L, Haines, K A, Weissmann, G, Rosenfeld, M G
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15.01.1991; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Diphosphate Ribose; Affinity Labels; Analytical, structural and metabolic biochemistry; Antibodies, Monoclonal - immunology; Binding and carrier proteins; Biological and medical sciences; Blotting, Western; Botulinum Toxins - metabolism; Cell Fractionation; Cell Membrane - chemistry; Complement C3 - metabolism; Fundamental and applied biological sciences. Psychology; GTP Phosphohydrolases - metabolism; GTP-Binding Proteins - analysis; GTP-Binding Proteins - immunology; Humans; Molecular Weight; Neutrophils - chemistry; Neutrophils - enzymology; Proteins; Human; Binding protein; GTP; Regulation(control); Secretion; Degranulation; Immunoblotting assay; Photoaffinity labelling; Localization; Blood; G protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)35314-0

Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments.