Association of ARF and Rabs with complement receptor type-1 storage vesicles in human neutrophils.
During neutrophil activation, the properties of the cell are rapidly altered by increases in the surface expression of functionally important receptors and adherence molecules. At the same time, endocytic and phagocytic activities increase. These alterations require precise regulation of membrane and protein movement, which is achieved, at least in part, by bidirectional movement of small transport vesicles. GTP-binding proteins, including Rabs and ADP-ribosylation factors (ARFs), play critical roles in regulating vesicle trafficking in other types of cells. The ability to immunoisolate the "secretory" vesicle subpopulation in which complement receptor type 1 (CR1) is stored allowed us to determine which types of low-molecular-weight GTP-binding proteins interact with these vesicles and under what conditions. CR1-containing vesicles from resting human neutrophils constitutively copurify with Rabs 3a, 4, and 5a, and reversibly bind an ARF, likely ARF1. ARF binding is dependent on free Mg(2+) and is enhanced by GTPgammaS. Mg(2+) at 0.4 microM is necessary for half-maximal binding of ARFs to CR1 storage vesicles. Artificial phospholipid vesicles and primary and secondary granules from human neutrophils do not bind ARFs themselves and do not compete for recruitment of ARFs to CR1 vesicles, suggesting that specific membrane environments and/or proteins on these vesicles stabilize the ARF-GTP-Mg(2+) complex. Free Ca(2+) at 300 nM does not inhibit ARF binding to CR1 storage vesicles, but 10 mM Ca(2+) does reduce such binding. These findings suggest that ARF-GTP specifically and reversibly interacts with CR1 storage vesicles in human polymorphonuclear leukocytes and may play a role in regulating their transport.
Chaudhuri
S
S
Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, OH, USA.
Kumar
A
A
Berger
M
M
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D000906:Antibodies / Q000276:immunology; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D018375:Neutrophil Activation; D009504:Neutrophils / Q000276:immunology*; D011951:Receptors, Complement / Q000276:immunology / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D000906:Antibodies; D011951:Receptors, Complement; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020823:ADP-Ribosylation Factor 1; D020691:rab GTP-Binding Proteins; D008274:Magnesium; D002118:Calcium