Stimulation of inositol 1,4,5-trisphosphate production by peptides corresponding to the effector domain of different Rab3 isoforms and cross-linking of an effector domain peptide target.
ABSTRACT: Rab3 proteins are localized on secretory vesicles and appear to be involved in regulated exocytosis. We have previously shown that a modified peptide corresponding to the effector domain of the small molecular mass GTP-binding protein Rab3A, Rab3AAL, stimulates inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and amylase release in digitonin-permeabilized pancreatic acini. Experiments using monoclonal antibodies reveal that the Rab3-like protein present in pancreatic acini is not the Rab3A isoform. However, since the putative effector domains of the four as yet known Rab3 proteins (A, B, C and D) differ only in the C-terminal four amino acid residues, Rab3A effector domain peptide could mimic the action of the pancreas-specific Rab3 isoform. In the present study we report that peptides corresponding to the different Rab3 isoforms stimulate both Ins(1,4,5)P3 production and amylase secretion with an order of potency Rab3B/D > Rab3AAL > Rab3A = Rab3C. For Rab3A, B/D and C effector domain peptides the concentrations causing half-maximal response (EC50) were 3, 0.2 and 3 nM for Ins(1,4,5)P3 accumulation and 0.3, 0.02 and 0.3 nM for amylase release, respectively. A Rab1A effector domain peptide, Rab1AAL, and a scrambled peptide of Rab3AAL were less potent by several orders of magnitude in eliciting these responses compared with native Rab3 effector domain peptides. None of the peptides influenced Ins(1,4,5)P3 production and amylase release in intact acini. Cross-linking of 125I-Rab3B/D peptide to pancreatic acinar membranes showed a band at 70 to 75 kDa with maximum intensity at 75 kDa. Radiolabelling of the substrates could be displaced by unlabelled Rab3B/D peptide, and to a lesser extend by Rab3A peptide, whereas the scrambled peptide of Rab3AAL had no effect. These data suggest that phospholipase C and exocytosis might be regulated by Rab3B-or Rab3D-like proteins in pancreatic acinar cells. A 75 kDa protein that preferentially cross-linked to 125I-Rab3B/D effector domain peptide is a potential candidate as an effector protein of Rab3 effector domain peptides.
Project description:We have recently shown that synthetic peptides of the effector domain of the low-molecular-mass GTP-binding protein Rab3 stimulate inositol 1,4,5-trisphosphate production in various permeabilized cells. To investigate the mechanism of the peptide-induced activation of phospholipase C (PLC) and to identify the PLC isoenzyme(s) targeted by these peptides, isolated pancreatic acinar membranes and cytosol were preincubated with anti-PLC antibodies before examination of PLC activity in response to the Rab3B/D effector-domain peptide (VSTVGIDFKVKTVYRH, peptide P1). Western blot analysis revealed the presence of PLC-beta1, -beta3, -gamma1 and -delta1 in membrane and cytosolic fractions. P1 stimulated PLC activity in both membrane and cytosolic fractions. Anti-(PLC-beta1) antibody inhibited P1-induced PLC activity in both subcellular fractions almost completely. Moreover, P1-induced amylase release in digitonin-permeabilized pancreatic acini was also inhibited. Other immunoneutralizing anti-PLC antibodies had no effect, suggesting that P1 activates PLC-beta1 but not PLC-beta3, -gamma1 or -delta1. P1 also activated recombinant PLC-beta1, indicating direct activation of PLC-beta1 by Rab3 effector-domain peptides. To investigate further the structure-function relationship of the peptides, truncated peptides of P1 were tested for their ability to activate PLC in isolated pancreatic acinar membranes and to stimulate amylase release from digitonin-permeabilized pancreatic acini. Peptides containing a BXBXXXB(B) motif (where B represents a basic residue and X any residue)[KVKTVYRH (EC50 of 1 nM to stimulate amylase release) approximately TVGIDFKVKTVYRH > TVGIDFKVKTVYR] were potent stimulators of amylase release and PLC activity, whereas deletion of the C-terminus (VSTVGIDF), of the two basic C-terminal amino acid residues (VSTVGIDFKVKTVY and KVKTVY), or destruction of the BXB motif (VKTVYR) resulted in inactive peptides. In conclusion, the results of the present study show that short peptides containing a BXBXXXB motif represent promising pharmacological agents to activate the PLC-beta1 isoenzyme.
Project description:In this study we examined the biochemical properties and subcellular localization of Rab3A, Rab3B and Rab3C in bovine adrenal chromaffin cells. The Kd for guanosine 5'-[gamma-thio]triphosphate (GTP[S]) of the three Rab3 proteins was 15, 2700 and 204 nM for Rab3A, Rab3B and Rab3C respectively. The intrinsic GTPase activity of the three Rab3 proteins seemed similar and was increased approx. 3-fold by bovine chromaffin cell lysate. Truncation of the C-terminal 31 amino acid residues decreased the binding affinity for GTP[S] of the three Rab3 proteins. When the C-terminus of Rab3C was replaced with that of Rab3A, the binding affinity of Rab3C for GTP[S] was decreased, but the replacement did not affect the affinity of Rab3B for GTP[S]. Immunostaining experiments showed that Rab3A, Rab3B and Rab3C are localized separately within chromaffin cells. Anti-Rab3A and anti-Rab3C antibodies stained vesicle-like structures, whereas anti-Rab3B antibody distinctly stained the plasma membrane. In summary, bovine chromaffin cells express the three Rab3 proteins but the subcellular localization and biochemical properties of the three Rab3 proteins are distinct.
Project description:Rab3B, similar to other Rab3 isoforms, is a synaptic vesicle protein that interacts with the Rab3-interacting molecule (RIM) isoforms RIM1? and RIM2? as effector proteins in a GTP-dependent manner. Previous studies showed that at excitatory synapses, Rab3A and RIM1? are essential for presynaptically expressed long-term potentiation (LTP), whereas at inhibitory synapses RIM1? is required for endocannabinoid-dependent long-term depression (referred to as "i-LTD"). However, it remained unknown whether i-LTD also involves a Rab3 isoform and whether i-LTD, similar to other forms of long-term plasticity, is important for learning and memory. Here we show that Rab3B is highly enriched in inhibitory synapses in the CA1 region of the hippocampus. Using electrophysiological recordings in acute slices, we demonstrate that knockout (KO) of Rab3B does not alter the strength or short-term plasticity of excitatory or inhibitory synapses but does impair i-LTD significantly without changing classical NMDA receptor-dependent LTP. Behaviorally, we found that Rab3B KO mice exhibit no detectable changes in all basic parameters tested, including the initial phase of learning and memory. However, Rab3B KO mice did display a selective enhancement in reversal learning, as measured using Morris water-maze and fear-conditioning assays. Our data support the notion that presynaptic forms of long-term plasticity at excitatory and inhibitory synapses generally are mediated by a common Rab3/RIM-dependent pathway, with various types of synapses using distinct Rab3 isoforms. Moreover, our results suggest that i-LTD contributes to learning and memory, presumably by stabilizing circuits established in previous learning processes.
Project description:Rab3A, Rab3B, Rab3C, and Rab3D are closely related GTP-binding proteins of synaptic vesicles that may function in neurotransmitter release. We have produced knock-out (KO) mice for Rab3B and Rab3C and crossed them with previously generated Rab3A and 3D knock-out mice to generate double, triple, and quadruple Rab3 knock-out mice. We have found that all single and double Rab3 knock-out mice are viable and fertile. Most triple Rab3 knock-out mice perish whenever Rab3A is one of the three deleted proteins, whereas all triple knock-out mice that express Rab3A are viable and fertile. Finally, all quadruple knock-out mice die shortly after birth. Quadruple Rab3 KO mice initially develop normally and are born alive but succumb to respiratory failure. Rab3-deficient mice display no apparent changes in synapse structure or brain composition except for a loss of rabphilin, a Rab3-binding protein. Analysis of cultured hippocampal neurons from quadruple knock-out mice uncovered no significant change in spontaneous or sucrose-evoked release but an approximately 30% decrease in evoked responses. This decrease was caused by a decline in the synaptic and the vesicular release probabilities, suggesting that Rab3 proteins are essential for the normal regulation of Ca2+-triggered synaptic vesicle exocytosis but not for synaptic vesicle exocytosis as such. Our data show that Rab3 is required for survival in mice and that the four Rab3 proteins are functionally redundant in this role. Furthermore, our data demonstrate that Rab3 is not in itself essential for synaptic membrane traffic but functions to modulate the basic release machinery.
Project description:We compared the time course of increases in isomers of inositol trisphosphate [Ins(1,4,5)P3] and Ins(1,3,4)P3] and the tetrakisphosphate [Ins(1,3,4,5)P4] with changes in cytosolic free Ca2+ [( Ca2+]i) in dispersed pancreatic acini of the rat. There were rapid (5s) increases in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in response to carbachol, caerulein and secretin, whereas Ins(1,3,4)P3 increased more slowly. All three secretagogues induced increases in [Ca2+]i, which reached a peak at 15-20 s. Our results indicate that the very rapid formation of Ins(1,4,5)P3 is compatible with its second-messenger role in the initial elevation of [Ca2+]i.
Project description:Electrically permeabilized rat pancreatic acini were used to evaluate the contributions of GTP and Ins(1,4,5) P3 to hormone-stimulated Ca2+ uptake and release from intracellular pools. Treatment of permeabilized acini with Ca2+-mobilizing hormones, GTP or GTP[S] resulted in stimulation of an ATP-dependent, VO4(2-)-sensitive Ca2+ uptake into a non-mitochondrial intracellular pool. GTP and GTP[S] also augmented the hormone-mediated stimulation of Ca2+ uptake. Including oxalate in the uptake medium increased Ca2+ uptake into this pool but did not modify the stimulation of Ca2+ uptake induced by hormones or GTP. Ins(1,4,5)P3 released all the extra Ca2+ accumulated as a result of hormone, GTP or GTP[S] stimulation. Hence, these stimuli activated the Ca2+ pump localized in the membrane of the hormone and Ins(1,4,5)P3-sensitive Ca2+ pool. Including 2,3-diphosphoglyceric acid (PGA) [an inhibitor of Ins(1,4,5)P3 hydrolysis] in the incubation medium blunted the GTP and GTP[S]-stimulated Ca2+ uptake. In the presence of PGA, the hormones inhibited Ca2+ accumulation, and GTP and GTP[S] augmented this effect. Accordingly, PGA stabilized the Ins(1,4,5)P3-evoked Ca2+ release from intracellular pools. Only in the presence of PGA was it possible to demonstrate hormonally-evoked Ca2+ release from permeabilized cells. GTP, and more importantly GTP[S], augmented the hormone-evoked Ca2+ release. Hormones and Ins(1,4,5)P3 in the presence or absence of GTP or GTP[S] released Ca2+ from the same intracellular pool. The extent of Ca2+ release caused by the combination of hormones and GTP or GTP[S] was similar to that evoked by Ins(1,4,5)P3 alone. Taken together, these results suggest that GTP or GTP[S] facilitates stimulation of phospholipase C by hormones. Such stimulation results in stimulation of protein kinase C and increased levels of Ins(1,4,5)P3 and is sufficient to explain the effects of GTP and GTP[S] on Ca2+ uptake and release from pancreatic acinar cells.
Project description:We have studied the effects of monoclonal antibodies that recognize different epitopes of the cerebellar Ins(1,4,5)P3 receptor on Ins(1,4,5)P3-induced Ca(2+)-release activity. Ins(1,4,5)P3 stimulated Ca2+ flux from cerebellar microsomes, and half-maximal Ca2+ release occurred at 112 +/- 8 nM-Ins(1,4,5)P3 [concentration causing half-maximal effect (EC50) = 112.8 nM]. The minimum concentration of Ins(1,4,5)P3 necessary to initiate Ca2+ release (threshold concentration) was 20 +/- 5 nM. A monoclonal antibody (mAb) 18A10 (50 micrograms/ml), which recognizes the C-terminal region of the Ins(1,4,5)P3 receptor, suppressed Ins(1,4,5)P3-induced Ca2+ release: the EC50 and threshold concentration shifted to 460 +/- 56 nM and 61 +/- 6 nM respectively. On the other hand, the antibody at the same concentration raised the affinity of the receptor for binding to Ins(1,4,5)P3, and the Kd value decreased from 43 +/- 12 nM to 25 +/- 4 nM without a change in the number of Ins(1,4,5)P3-binding sites. However, mAbs that recognize the N-terminal domain affected neither Ca2+ release nor Ins(1,4,5)P3 binding. Among the various synthetic peptides, only the 12-residue-long peptide from the most C-terminal portion of the receptor (amino acid residues 2736-2747) reacted strongly with mAb18A10. From these findings, combined with the Immunogold localization of the cerebellar Ins(1,4,5)P3 receptor [Otsu, Yamamoto, Maeda, Mikoshiba & Tashiro (1990) Cell Struct. Funct. 15, 163-173], we concluded that the C-terminus of the Ins(1,4,5)P3 receptor is exposed to the cytoplasmic side of the smooth endoplasmic reticulum and plays an important role in the regulation of both Ins(1,4,5)P3-binding affinity and channel gating.
Project description:Chemical modification by phenylglyoxal, an arginine-specific reagent, of both native and recombinant rat brain inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase A was accompanied by irreversible inhibition of enzyme activity. This effect was prevented in the presence of the substrate ATP but not Ins(1,4,5)P3. The modification reaction obeyed pseudo-first-order rate kinetics. Complete inhibition of activity corresponded to incorporation of 1.2 mol of phenylglyoxal per mol of protein. A single [14C]phenylglyoxal-modified peptide was isolated following alpha-chymotrypsin digestion of the radiolabelled Ins(1,4,5)P3 3-kinase and reverse-phase HPLC. ATP prevented the incorporation of radioactivity to this peptide. The peptide sequence (i.e. QWREGISSSTTL) corresponded to amino acids 315 to 326 of rat brain Ins(1,4,5)P3 3-kinase A. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivative showed the modified amino acid to be Arg-317. The data directly identify a reactive arginine residue as part of the ATP-binding site. Arg-317 is located within a sequence segment which is conserved among the catalytic domain of Ins(1,4,5)P3 3-kinase isoenzymes A and B in human and rat species.
Project description:An explanation of the complex effects of hormones on intracellular Ca2+ requires that the intracellular actions of Ins(1,4,5)P3 and the relationships between intracellular Ca2+ stores are fully understood. We have examined the kinetics of 45Ca2+ efflux from pre-loaded intracellular stores after stimulation with Ins(1,4,5)P3 or the stable phosphorothioate analogue, Ins(1,4,5)P3[S]3, by simultaneous addition of one of them with glucose/hexokinase to rapidly deplete the medium of ATP. Under these conditions, a maximal concentration of either Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 evoked rapid efflux of about half of the accumulated 45Ca2+, and thereafter the efflux was the same as occurred under control conditions. Submaximal concentrations of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 caused a smaller rapid initial efflux of 45Ca2+, after which the efflux was similar whatever the concentration of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 present. The failure of submaximal concentrations of Ins(1,4,5)P3 and Ins(1,4,5)P3[S]3 to mobilize fully the Ins(1,4,5)P3-sensitive Ca2+ stores despite prolonged incubation was not due either to inactivation of Ins(1,4,5)P3 or to desensitization of the Ins(1,4,5)P3 receptor. The results suggest that the size of the Ins(1,4,5)P3 sensitive Ca2+ stores depends upon the concentration of Ins(1,4,5)P3.
Project description:To elucidate the functional difference between type 1 and type 3 Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 respectively] we studied the effect of Ca2+ on the ligand-binding properties of both Ins(1,4,5)P3R types. We expressed full-length human Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 from cDNA species in insect ovary Sf9 cells, and the membrane fractions were used for Ins(1,4,5)P3-binding assays. The binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 was differentially regulated by Ca2+. With increasing concentrations of free Ca2+ ([Ca2+]), Ins(1,4,5)P3 binding to Ins(1,4,5)P2R1 decreased, whereas that to Ins(1,4,5)P3R3 increased. Alteration of Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 was observed at [Ca2+] ranging from less than 1 nM to more than 10 microM. The EC50 of Ins(1,4,5)P3 binding was 100 nM Ca2+ for Ins(1,4,5)P3R1. In contrast, Ins(1,4,5)P3 binding to Ins(1,4,5)P3R3 was changed at high [Ca2+] with an EC50 value of 872 nM, and steeply between 100 nM and 10 microM. These Ca2+-dependent alterations of Ins(1,4,5)P3 binding to both Ins(1,4,5)P3R types were reversible. Scatchard analyses revealed that Ca2+ changed the affinity of both Ins(1,4,5)P3R types but not the total number of Ins(1,4,5)P3-binding sites. The Kd values of Ins(1,4,5)P3R1 for Ins(1,4,5)P3 were 78.5 nM with 3 nM free Ca2+, and 312 nM with 1.4 microM free Ca2+. In contrast, Ins(1,4,5)P3R3 exhibited an affinity for Ins(1,4,5)P3 with Kd values of 116 nM with 3 nM free Ca2+, and 62.2 nM with 1.4 microM free Ca2+. These results indicate that (1) both Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 have at least two affinity states, (2) Ca2+ regulates interconversions between these states, and (3) Ca2+ regulates the binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 in opposite manners.