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GTP‐dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein
Author(s) -
Rosales Jesusa L.,
Ernst Joel D.
Publication year - 2000
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/1097-4644(20010101)80:1<37::aid-jcb40>3.0.co;2-i
Subject(s) - gtp' , secretion , cytosol , guanosine , gtp binding protein regulators , guanosine triphosphate , g protein , gtpase , biochemistry , microbiology and biotechnology , biology , signal transduction , enzyme
Guanosine triphosphate (GTP) has been implicated in the regulation of Ca 2+ ‐mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found that, in the presence of GTP, 1.0 μM free Ca 2+ causes maximum secretion—equivalent to that achieved with 100 μM free Ca 2+ —whereas GTPγS inhibits Ca 2+ ‐stimulated secretion. Interestingly, GTP by itself stimulates secretion. These results indicate the existence of a GTP‐regulated mechanism of secretion in neutrophils that requires GTP hydrolysis to stimulate secretion in the presence and absence of Ca 2+ . The stimulatory effect of GTP is only observed when GTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, gives no stimulatory response, implying that the GTP‐dependent secretory apparatus requires at least one cytosolic protein. GTP‐dependent secretion can be reconstituted with crude HL‐60 and bovine liver cytosol. The reconstituting activity binds to GTP‐agarose, suggesting that the cytosolic factor is a GTP‐binding protein or forms a complex with a GTP‐binding protein. However, it is not a member of the rho or rac families of GTPases. By gel filtration chromatography, the secretion‐reconstituting activity eluted at 870 and 200 kDa, but in the presence of GTP, eluted at 120 kDa, indicating that it is part of a high‐molecular‐weight complex that dissociates in the presence of GTP. Retention of adenosine diphosphate‐ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activity to brefeldin A led to our speculation that ARF6 may be the GTPase involved in GTP‐dependent secretion, and that activity from a BFA‐insensitive ARF6 guanine nucleotide exchange factor reconstitutes secretion. J. Cell. Biochem. 80:37–45, 2000. © 2000 Wiley‐Liss, Inc.