guanosine-diphosphate and geranylgeranyl-pyrophosphate

Lysophosphatidylcholine (LPC) is known to increase intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells. This study was conducted to investigate the effects of HMG-CoA reductase inhibitors (statins) on the increase in [Ca2+]i and membrane current induced by LPC.. [Ca2+]i was determined in cultured human aortic endothelial cells by fura-2 assay, and membrane current was measured by whole-cell patch clamp. The [Ca2+]i increase induced by LPC was abolished by inhibitors of phospholipase C (PLC). Statins markedly decreased the [Ca2+]i increase caused by LPC. This suppressive effect was quickly reversed by geranylgeranylpyrophosphate (GGPP) and was mimicked by inhibitors of Rho and Rho kinase. LPC induced the translocation of the GTP-bound active form of RhoA into membranes within 1 minute as determined by a pull-down assay and reduced the levels of RhoA in the cytoplasm, indicating that LPC quickly increases the GTP/GDP ratio of RhoA and induces membrane translocation. Statins prevented the GTP/GDP exchange of RhoA and its membrane translocation from the cytoplasm caused by LPC, and these effects of statins were reversed by GGPP. The responses of RhoA activation to statins and GGPP concurred with their effects on Ca2+ mobilization. LPC also induced a nonselective cation current after a lag. Statins prolonged the lag and decreased the current amplitude, and GGPP abolished the inhibitory effect on the current.. LPC induced Ca2+ mobilization and membrane current via a Rho activation-dependent PLC pathway in endothelial cells, and statins blocked these effects by preventing the GGPP-dependent lipid modification of Rho. The present study implicates Rho in LPC stimulation of Ca2+ movement.

Topics: ADP Ribose Transferases; Amides; Aorta; Botulinum Toxins; Calcium; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intracellular Fluid; Intracellular Signaling Peptides and Proteins; Lysophosphatidylcholines; Membrane Potentials; Mevalonic Acid; Patch-Clamp Techniques; Polyisoprenyl Phosphates; Protein Serine-Threonine Kinases; Pyridines; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Type C Phospholipases

GTPases of the Rab family are key components of vesicular transport in eukaryotic cells. Posttranslational attachment of geranylgeranyl moieties is essential for Rab function. Geranylgeranyltransferase type II (GGTase-II) catalyzes the modification of Rab proteins once they are in complex with their escort protein (REP). Upon completion of prenylation, REP and modified Rab leave the enzyme, enabling a new round of catalysis. We have studied the mechanism underlying substrate binding and product release in the geranylgeranylation of Rab proteins. Binding of the Rab7:REP-1 complex to GGTase-II was found to be strongly modulated by geranylgeranyl pyrophosphate (GGpp). The affinity of GGTase-II for the Rab7:REP-1 complex increases from ca. 120 nM to ca. 2 nM in the presence of GGpp. To study the effect of GGpp on interaction of the enzyme with its product, we generated semisynthetic doubly prenylated Rab7 bearing a fluorescent reporter group. Using this novel compound, we demonstrated that the affinity of doubly prenylated Rab7:REP-1 complex for GGTase-II was 2 and 18 nM in the absence and presence of GGpp, respectively. The difference in affinities originates mainly from a difference in the dissociation rates. Thus, binding of the new isoprenoid substrate molecule facilitates the product release by GGTase-II. The affinity of GGpp for the prenylated Rab7:REP-1:GGTase-II was K(d) = 22 nM, with one molecule of GGpp binding per molecule of prenylated ternary complex. We interpreted this finding as an indication that the geranylgeranyl moieties transferred to Rab protein do not occupy the GGpp binding site of the GGTase-II. In summary, these results demonstrate that GGpp acts as an allosteric activator that stabilizes the Rab7:REP-1:GGTase-II complex and triggers product release upon prenylation, preventing product inhibition of the enzyme.

Topics: Adaptor Proteins, Signal Transducing; Alkyl and Aryl Transferases; Allosteric Regulation; Animals; Binding Sites; Fluorescent Dyes; Guanosine Diphosphate; Kinetics; Macromolecular Substances; ortho-Aminobenzoates; Polyisoprenyl Phosphates; Protein Binding; Protein Prenylation; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Rats; Spectrometry, Fluorescence; Substrate Specificity; Titrimetry