Low molecular weight GTP-binding proteins in human neutrophil granule membranes.
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.
Philips
M R
MR
Department of Medicine, New York University School of Medicine, New York 10016.
Abramson
S B
SB
Kolasinski
S L
SL
Haines
K A
KA
Weissmann
G
G
Rosenfeld
M G
MG
D000246:Adenosine Diphosphate Ribose; D000345:Affinity Labels; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000378:metabolism; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D003176:Complement C3 / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000276:immunology; D006801:Humans; D008970:Molecular Weight; D009504:Neutrophils / Q000737:chemistry* / Q000201:enzymology
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D003176:Complement C3; D000246:Adenosine Diphosphate Ribose; D001905:Botulinum Toxins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins