Abstract
Previous studies have indicated the presence of a cholera toxin-sensitive phospholipase D (PLD) in cultured RBL-2H3 mast cells that is synergistically activated via calcium, protein kinase C, and another unidentified signal. Here we identify a third potential signal for activation transduced by a pertussis toxin-sensitive trimeric GTP-binding protein, most likely via Gi2 or Gi3. Quercetin-treated RBL-2H3 cells in which expression of Gαi2 and Gαi3 is enhanced more than 7-fold respond to the Gi stimulant compound 48/80 with the activation of PLD, a transient activation of phospholipase C, and enhanced membrane GTPase activity. The activation of PLD was blocked in pertussis toxin-treated cells and, as with other stimulants of PLD, was enhanced in cholera toxin-treated cells. The PLD response to compound 48/80 was only partially inhibited by calcium deprivation and inhibition of protein kinase C to indicate a component of the response that was independent of calcium, protein kinase C, and, presumably, phospholipase C. Based on these and other data, we hypothesized that βγ-subunits, released from Gi2 or Gi3 by compound 48/80 or from Gs by cholera toxin, provide an additional signal for the activation of PLD. Consistent with this hypothesis, recombinant Gβ2γ2 subunits, but not Gαi-3 subunits, at concentrations of 50 to 300 nM markedly synergized PLD activation by compound 48/80 in permeabilized RBL-2H3 cells.
Footnotes
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Send reprint requests to: Dr. Michael A. Beaven, Room 8N109/Bldg. 10, National Institutes of Health, Bethesda, MD 20892-1760. E-mail:beaven{at}helix.nih.gov
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Received for publication May 13, 1999.
- Abbreviations:
- PLD
- phospholipase D
- PLC
- phospholipase C
- G protein
- trimeric GTP-binding protein
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate
- PIPES
- piperazine-N,N′-bis(2-ethanesulfonic acid)
- ARF
- ADP-ribosylation factor
- mARF
- myristoylated ADP-ribosylation factor
- Accepted September 17, 1999.
- U.S. Government
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