Modulation of Ins(2,4,5)P3-stimulated Ca2+ mobilization by Ins(1,3,4,5)P4:: enhancement by activated G-proteins, and evidence for the involvement of a GAP1 protein, a putative Ins(1,3,4,5)P4 receptor

We have previously shown that addition of Ins(1,3,4,5)P-4 to permeabilized L1210 cells increases the amount of Ca2+ mobilized by a submaximal concentration of Ins(2,4,5)P-3, and we suggested that, in doing this, Ins(1,3,4,5)P, is not working via an InsP(3) receptor but indirectly via an InsP(4) receptor [Loomis-Husselbee, Cullen, Dreikhausen, Irvine and Dawson (1996) Biochem. J. 314, 811-816]. Here we have investigated whether this effect might be mediated by GAP1(IP4BP), recently identified as a putative receptor for Ins(1,3,4,5)P-4. GAP1(IP4BP) is a protein that interacts with one or more monomeric G-proteins, so we sought evidence for involvement of monomeric G-proteins in the effects of Ins(1,3 4,5)P-4 in permeabilized L1210 cells. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) enhanced the effect of Ins(1,3,4,5)P-4 on Ins(2,4,5)(3)-stimulated Ca2+ mobilization, but had no effect on the action of Ins(2,4,5)P-3 alone. A specific enhancement of only the action of Ins(1,3,4,5)P-4 was also seen with GTP[S]-loaded R-Ras or Rap1a (two G-proteins known to interact with GAP1(IP4BP)), whereas H-Ras was inactive at similar concentrations. Guanosine 5'-[P-thio]diphosphate (GDP[S]) did not alter the action of either Ins(2,4,5)P-3 or Ins(1,3,4,5)P-4. Finally, the addition of exogenous GAP1(IP4BP), purified from platelets, markedly enhanced the effect of Ins(1,3,4,5)P-4, and again, the amount of Ca2+ mobilized by Ins(2,4,5)P-3 alone was unaltered. We conclude that the increase in Ins(2,4,5)P-3-stimulated Ca2+ mobilization by Ins(1,3,4,5)P-4 may be mediated by GAP1(IP4BP) or a closely related protein (such as GAP1(m)), and if so, the action of the GAP1 is not solely to regulate GTP loading of a G-protein, but rather it acts with a G-protein to cause its effect.