Interaction of Rab31 and OCRL-1 in oligodendrocytes: its role in transport of mannose 6-phosphate receptors.

Rab31, a protein that we cloned from an oligodendrocyte cDNA library, is required for transport of mannose 6-phosphate receptors (MPRs) from the trans-Golgi network (TGN) to endosomes and for Golgi/TGN organization. Here we extend the knowledge of the mechanism of action of Rab31 by demonstrating its interaction with OCRL-1, a phosphatidylinositol 4,5-diphosphate 5-phosphatase (PI(4,5)P(2) 5-phosphatase) that regulates the levels of PI(4,5)P(2) and PI(4)P, molecules involved in transport and Golgi/TGN organization. We show that Rab31 interacts with OCRL-1 in a yeast two-hybrid system, GST-Rab31 pull-down experiments, and coimmunoprecipitation of OCRL-1 using oligodendrocyte culture lysates. Rab31 and OCRL-1 colocalize in the TGN, post-TGN carriers, and endosomes. Cation-dependent MPR (CD-MPR) is sorted to OCRL-1-containing carriers, but CD63 and vesicular stomatitis virus G (VSVG) are not. siRNA-mediated depletion of endogenous Rab31 causes collapse of the TGN apparatus and markedly decreases the levels of OCRL-1 in the TGN and endosomes. Our observations indicate that the role of Rab31 in the Golgi/TGN structure and transport of MPRs depends on its capability to recruit OCRL-1 to domains of the TGN where the formation of carriers occurs. The importance of our observations is highlighted by the fact that mutation of OCRL-1 causes demyelination in humans.

Transport of mannose-6-phosphate receptors from the trans-Golgi network to endosomes requires Rab31.

Rab31, a protein that we originally cloned from a rat oligodendrocyte cDNA library, localizes in the trans-Golgi network (TGN) and endosomes. However, its function has not yet been established. Here we show the involvement of Rab31 in the transport of mannose 6-phosphate receptors (MPRs) from TGN to endosomes. We demonstrate the specific sorting of cation-dependent-MPR (CD-MPR), but not CD63 and vesicular stomatitis virus G (VSVG) protein, to Rab31-containing trans-Golgi network carriers. CD-MPR and Rab31 containing carriers originate from extending TGN tubules that also contain clathrin and GGA1 coats. Expression of constitutively active Rab31 reduced the content of CD-MPR in the TGN relative to that of endosomes, while expression of dominant negative Rab31 triggered reciprocal changes in CD-MPR distribution. Expression of dominant negative Rab31 also inhibited the formation of carriers containing CD-MPR in the TGN, without affecting the exit of VSVG from this compartment. Importantly, siRNA-mediated depletion of endogenous Rab31 caused the collapse of the Golgi apparatus. Our observations demonstrate that Rab31 is required for transport of MPRs from TGN to endosomes and for the Golgi/TGN organization.

Vesicle transport in oligodendrocytes: probable role of Rab40c protein.

Intracellular membrane trafficking plays an essential role in the structural and functional organization of oligodendrocytes, which synthesize a large amount of membrane to form myelin. Rab proteins are key components in intracellular vesicular transport. We cloned a novel Rab protein from an oligodendrocyte cDNA library, designating it Rab40c because of its homology with Rab40a and Rab40b. The DNA sequence of Rab40c shows an 843-base pair open reading frame. The deduced amino acid sequence is a protein with 281 amino acids, with a molecular weight of 31,466 Da and an isoelectric point of 9.83. Rab40c presents a number of distinct structural features including a carboxyl terminal extension and amino acid substitutions in the consensus sequence of the GTP-binding motifs. The carboxyl terminal region contains motifs that permit isoprenylation and palmitoylation. Binding studies indicate that Rab40c binds guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S) with a K(d) of 21 microM and has a higher affinity for guanosine triphosphate (GTP) than for guanosine diphosphate (GDP). Rab40c is localized in the perinuclear recycling compartment, suggesting its involvement in endocytic events such as receptor recycling. The importance of this recycling in myelin formation is suggested by the increase in both Rab40c mRNA and Rab40c protein as oligodendrocytes differentiate.

Myelin biogenesis: vesicle transport in oligodendrocytes.

Intracellular trafficking of membranes plays an essential role in the biogenesis and maintenance of myelin. The requisite proteins and lipids are transported from their sites of synthesis to myelin via vesicles. Vesicle transport is tightly coordinated with synthesis of lipids and proteins. To maintain the structural and functional organization of oligodendrocytes it is essential synchronize the various pathways of vesicle transport and to coordinate vesicle transport with reorganization of cytoskeleton. The systems that regulate the targeting of protein to myelin by vesicle transport are now being described. Here we review the current knowledge of these systems including those involved in (a) protein folding, (b) protein sorting and formation of carrier vesicles, (c) vesicle transport along elements of the cytoskeleton, and (d) vesicle targeting/fusion.

Role of rRAB22b, an oligodendrocyte protein, in regulation of transport of vesicles from trans Golgi to endocytic compartments.

Intracellular membrane trafficking plays an essential role in the biogenesis and maintenance of myelin. Members of the Rab protein family are important components of the systems that regulate intracellular vesicle transport. We examine the function of rRab22b, a novel rat Rab protein cloned from an oligodendrocyte cDNA library, by visualizing and identifying in living Hela cells the organelles that contain rRab22b. Our results show that rRab22b is present in the trans Golgi/TGN and endocytic compartments. Trafficking of membranes from trans Golgi to endocytic compartments takes place via small tubulo vesicular organelles containing rRab22b. The formation of vesicles in the trans Golgi also appears to be regulated by rRab22b. Additionally, our results suggest that rRab22b controls the transport of vesicles from the trans Golgi to endocytic compartments that localize in oligodendrocyte processes. That rRab22b is involved in the transport of certain proteins from trans Golgi to myelin is suggested by the evidence that certain proteins being targeted to the plasma membrane are first transported from trans Golgi to endocytic compartments.

Pharmacological and functional characterization of muscarinic receptor subtypes in developing oligodendrocytes.

This study focused on the molecular and pharmacological characterization of muscarinic acetylcholine receptors expressed by progenitors and differentiated oligodendrocytes. We also analyzed the role of muscarinic receptors in regulating downstream signal transduction pathways and the functional significance of receptor expression in oligodendrocytes. RT-PCR analysis revealed the expression of transcripts for M3, and to a lesser extent M4, followed by M1, M2 and M5 receptor subtypes in both progenitors and differentiated oligodendrocytes. Competition binding experiments using [(3)H]N-methylscopolamine and several antagonists, as well as inhibition of carbachol-mediated phosphoinositide hydrolysis, showed that M3 is the main subtype expressed in these cells. In progenitors the activation of p42/44-mitogen-activated protein kinase (MAPK) and cAMP-response element binding protein (CREB) as well as c-fos mRNA expression were blocked by the M3 relatively selective antagonist, 4-DAMP, and its irreversible analogue, 4-DAMP-mustard. Carbachol increased proliferation of progenitors, an effect prevented by atropine and 4-DAMP, as well as by the MAPK kinase inhibitor PD98059. These results indicate that carbachol modulates oligodendrocyte progenitor proliferation through M3 receptors, involving activation of a MAPK signaling pathway. Receptor density and phosphoinositide hydrolysis are down-regulated during oligodendrocyte differentiation. Functional consequences of these events are a reduction in carbachol-stimulated p42/44(MAPK) and CREB phosphorylation, as well as induction of c-fos.

Characterization of the signal transduction pathways mediating noradrenaline-stimulated MAPK activation and c-fos expression in oligodendrocyte progenitors.

In examining the signaling transduction pathway of adrenoceptors in oligodendrocyte progenitors, we have found that stimulation of alpha(1)-adrenoceptors with norepinephrine (NE), in the presence of 3 microM propranolol, increased the activity of mitogen-activated protein kinases (MAPKs). This stimulation was concentration- and time-dependent, with maximal response after 10 min of exposure to 10 microM NE. Pertussis toxin (PTX) blocked NE-mediated MAPK activation, suggesting that alpha(1)-adrenoceptor activates MAPK through a PTX-sensitive G-protein. In the presence of U73122, an inhibitor of phospholipase C (PLC), MAPK activation was blocked. In oligodendrocyte progenitor cultures, chronic treatment with phorbol-12-myristate-13-acetate (PMA) down-regulated protein kinase C (PKC) and blocked NE-mediated MAPK activation. The response to NE was also significantly decreased by the PKC inhibitors H7 and bisindolylmaleimide GF109203X. Similarly, the effect of NE on MAPK activation was not observed in a calcium-free medium. Furthermore, attenuation of MAPK activity was observed when cultures were pretreated with LY294002 and wortmannin, inhibitors of phosphatidylinositol-3 kinase (PI3K). These results suggest that alpha(1)-adrenoceptor-mediated activation of MAPK involves a PTX-sensitive G-protein, PLC, PI3K, and 1,2-diacyl glycerol (DAG)-dependent PKC isozyme. Stimulation of oligodendrocyte progenitors with NE also resulted in an increase in c-fos expression, which was mediated by both alpha(1)- and beta-adrenoceptor and was calcium-, PKC-, and protein kinase A (PKA)-dependent. Interestingly, in the presence of PD 098059, a specific inhibitor of MAPK kinase (MEK), both MAPK activity and c-fos expression were blocked. This suggests that MAPK is implicated in the transmission of the signal from alpha(1)-adrenoceptor to c-fos gene expression.

Molecular pathways mediating activation by kainate of mitogen-activated protein kinase in oligodendrocyte progenitors.

Oligodendroglial cells express ionotropic glutamate receptors of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid hydrobromide (AMPA) and kainate (KA) subtypes. Recently, we reported that AMPA receptor agonists increased 45Ca2+ uptake and phospholipase C (PLC) activity. To further elucidate the intracellular signaling mechanisms, we examined the effects of AMPA and KA on mitogen-activated protein kinase (MAPK). KA caused a time- and concentration-dependent increase in MAPK activity (predominantly the p42mapk or ERK2) and the effect was blocked by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), a competitive AMPA/KA receptor antagonist. Furthermore, the noncompetitive antagonists of AMPA receptor GYKI 52466 and LY 303070 prevented the actions of the agonists, indicating that the effect of KA on MAPK activation is mediated through AMPA receptors in oligodendrocyte progenitors. Chelation of extracellular Ca2+ by EDTA or inhibition of PLC with U73122 abolished MAPK activation by KA. In addition, KA-stimulated MAPK activation was reduced by the protein kinase C (PKC) inhibitors, H7 and bisindolylmaleimide, as well as downregulation of PKC by prolonged exposure to phorbol esters. The involvement of PKC in the signal transduction pathways was further supported by the ability of KA to induce translocation of PKC measured by [3H]PDBu binding. Interestingly, a wortmannin-sensitive phosphatidylinositol 3-kinase and a pertussis toxin (PTX)-sensitive G protein form part of the molecular pathways mediating MAPK activation by AMPA receptor. A specific inhibitor of MAPK kinase, PD 098059, blocked MAPK activation and reduced KA-induced c-fos gene expression. All together, these results indicate that MAPK is implicated in the transmission of AMPA signaling to the nucleus and requires extracellular Ca2+, and PLC/PKC activation.

Molecular analysis of the monomeric GTP-binding proteins of oligodendrocytes.

Vesicle transport plays an important role in the formation of myelin. Transport of proteins, including proteolipid protein and myelin associated glycoprotein, from their site of synthesis in the endoplasmic reticulum in the perikaryon of the oligodendrocytes, to myelin, takes place via carrier vesicles. The mechanisms that regulate vesicle transport in oligodendrocytes are largely unknown. The presence of monomeric GTP-binding proteins in myelin and oligodendrocytes suggested the hypothesis that these proteins participate in the regulation of vesicle transport. In an attempt to identify the Rab and Rho GTP-binding proteins present in oligodendrocytes, a cDNA library specific for these proteins was generated using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach. Twelve different clones containing sequences that coded for members of the Rab and Rho families of GTP-binding proteins were isolated. This group includes Rab1, -1b, -2, -5b, -5c, -7, -8, -12, -14, -23 and Rho A. One additional clone revealed a novel cDNA sequence. Analysis of the effector loop motif indicated that this sequence encodes for a member of the Rab family. We refer to this new sequence as Rab0. Comparison of Rab0 with the most similar rat Rab sequences, Rab 14 and Rab 22, and with a recently cloned human Rab22b, showed a 71%, 72% and 94% identity, respectively. By RT-PCR analysis the Rab0 mRNA was found to be mainly expressed in oligodendrocytes and to a lesser extent in oligodendrocyte precursors, astrocytes and microglia. Moreover, the highest levels of Rab0 mRNA were observed in areas of the brain that are heavily myelinated. Rab0 mRNA was also detected in other tissues such as kidney, liver, skeletal muscle. These data provide initial evidence regarding signal transduction pathways that regulate intracellular transport in oligodendrocytes.

Induction of oligodendrocyte apoptosis by C2-ceramide.

Tumor necrosis factor-alpha induces oligodendrocytes apoptosis, and is known to stimulate the hydrolysis of sphingomyelin to form the lipid mediator, ceramide. These data encouraged us to determine whether ceramide itself is able to induce apoptosis in oligodendrocytes. For this purpose the cell-permeable ceramide analog, C2-ceramide was used. Treatment of bovine oligodendrocyte cell cultures with this compound induced cell death in a time- and concentration-dependent manner. The induction of cell death was specifically associated with the action of C2-ceramide and could not be elicited by dioctanoylglycerol (DC8) or phorbol 12-myristate 13-acetate (PMA). Treatment of the cultures with neutral sphingomyelinase, which increased the hydrolyses of endogenous sphingomyelin, resulted in oligodendrocyte death, whereas exposure of the cells to phospholipase C and A2 did not. C2-ceramide treatment caused DNA fragmentation. Morphologic analysis of the cells showed that C2-ceramide treatment resulted in a loss of their processes, reduction of cell volume, chromatin condensation, and formation of apoptotic bodies. These results indicate that ceramide can induce oligodendrocyte apoptosis, and suggest that sphingolipid metabolism plays a key role in the regulation of this process.

Expression and signal transduction of the glucagon receptor in betaTC3 cells.

The expression and signal transduction of the glucagon receptor (GR) have been studied in betaTC3 cells. Northern blot and RT-PCR analysis indicated the expression of the GR gene in betaTC3 cells. One-5 nM glucagon stimulated a 2.5-fold increase in the IP(S) production. At glucagon concentrations higher than 5 nM, the production of IP(S) was blunted but not abolished. The accumulation of intracellular cAMP was observed following the stimulation with 5 nM of glucagon. A maximal 4.5-fold increase in cAMP was observed using 250 nM glucagon and higher. Comparative studies using a glucagon anatogonist, des-His1[Glu]9glucagon, showed no effect on intracellular cAMP and IPs in betaTC3 cells. Our data shows that the GR gene is expressed in betaTC3 cells. The GR in betaTC3 cells transmits its intracellular signal by causing the accumulation of both IP(S) and cAMP.

Study of the interaction of the myelin monomeric GTP-binding proteins with other brain proteins.

Although several monomeric GTP-binding proteins have been found in myelin, the signaling pathways in which they operate are not known. To define these signaling pathways we searched for specific target proteins that interact with the myelin monomeric GTP-binding proteins. A blot overlay approach was used. Bovine white matter homogenate, myelin, and oligodendrocyte proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The presence of proteins that interact with the myelin GTP-binding proteins was explored by incubating those blots with an enriched fraction of 22- and 25-kDa myelin GTP-binding proteins labeled with radioactive guanine nucleotides. When the GTP-binding proteins were in the inactive state (GDP-bound) they interacted with 28-, 47-, and 58-kDa oligodendrocyte polypeptides. Only the 28-kDa protein was present in myelin. In the active state (GTP-bound), they interacted only with a 47-kDa protein in myelin but with 31-, 38-, 47-, 58-, 60-, 68-, and 71-kDa proteins in oligodendrocytes and total homogenate. Under these experimental conditions the 28-kDa protein did not interact with the GTP-binding proteins. The fact that the myelin GTP-binding proteins in the active state formed complexes with a different set of proteins than when in the inactive state is a strong indication that these proteins are effector proteins. With the exception of the 31- and 38-kDa proteins that were detected only in the cytoplasmic fraction, these polypeptides were detected in the cytosolic fraction and total membrane fraction. The 25-kDa GTP-binding protein was present in all the complexes. Immunoblot analysis indicated that the 28-kDa polypeptide is RhoGDI, an effector protein that is known to regulate the activation and movement of several GTP-binding proteins between different cellular compartments. Thus, this study opens the way to identify the macromolecules participating in the myelin signaling pathway involving monomeric GTP-binding proteins.

Acetylcholine agonists stimulate mitogen-activated protein kinase in oligodendrocyte progenitors by muscarinic receptors.

Oligodendrocytes, the myelin-producing cells of the central nervous system, express muscarinic acetylcholine receptors (mAChR). Activation of this neurotransmitter receptor by the stable acetylcholine analog carbachol (CCh) triggers transducing events, modulating c-fos expression and cellular proliferation. To elucidate the signal transduction pathways involved in the transmission of these cellular events, we examined the ability of CCh to activate mitogen-activated protein kinase (MAPK) in primary cultures of oligodendrocyte progenitors prepared from newborn rat brain. CCh produced a concentration- and time-dependent increase in MAPK activity (predominantly the p42mapk or ERK2) as determined by in-gel MBP kinase assays. Using the non-selective muscarinic antagonist atropine we determined that MAPK-activation by CCH is mediated by muscarinic receptors. In the presence of PD098059, a specific inhibitor of MAPK kinase (MEK), MAPK activity was blocked. Similarly, the presence of extracellular calcium was required for CCh-mediated MAPK activation. To further elucidate the mechanisms involved in MAPK activation by CCh, the role of PKC was studied. In cells in which protein kinase had been downregulated by chronic treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA), the effect of carbachol on MAPK activation was maintained. In contrast, the response to CCh was blocked by the PKC inhibitors H7 and bisindolylmaleimide GF109203X. Our results suggest that MAPK is implicated in the transmission of the signal for mACh receptors and involves a TPA-insensitive PKC pathway. Further work is required to define the upstream and downstream events which result in CCh-mediated MAPK activation and proliferation of oligodendrocyte progenitors.

Muscarinic receptor-dependent activation of phospholipase C in human fetal central nervous system organotypic tissue culture.

The coupling of muscarinic-cholinergic receptors (mAchR) with the phospholipase C (PLC) second messenger system has been demonstrated in central nervous system (CNS) tissue of many animal species. However, little information exists regarding this association in the developing human CNS. Due to the suggested role of acetylcholine in the regulation of development and differentiation of neural cells, the knowledge of these relationships during human fetal development acquires singular importance. Because of this, we examined the cholinergic stimulation of PLC in human fetal CNS organotypic tissue cultures. Agonist treatment of cultures, in the presence of lithium, resulted in a 4-6-fold increase in inositol phosphates formation. This increase was caused principally by the formation of inositol phosphate (IP). However, kinetic studies demonstrated that the levels of IP2, IP3 and IP4 also increased rapidly after stimulation reaching maximum levels before IP. These results support the hypothesis that muscarinic receptor activation results in an increase in the hydrolysis of PIP2. The inositol phosphate formation was dependent on agonist concentration. The obtained EC50 values were approximately 57 +/- 15 microM for carbachol, 8 +/- 2 microM for acetylcholine and 49 +/- 15 microM for oxotremorine. The agonist-dependent formation of inositol phosphates was inhibited by the muscarinic antagonists atropine and pirenzepine. Pirenzepine inhibited carbachol stimulation with high affinity (Ki = 2.90 +/- 1.15 nM), indicating that PLC activation is the result of activation of the m1 subtype of muscarinic receptors. Treatment of cultures with pertussis toxin did not result in inhibition of agonist-dependent activation of PLC. This result suggests that the m1 muscarinic receptor is coupled to PLC through Gq.

Muscarinic receptor-dependent activation of phospholipase C in the developing human fetal central nervous system.

The coupling of muscarinic-cholinergic receptors (mAChR) to adenylate cyclase and phospholipase C (PLC) second messenger systems has been demonstrated in many animal species. However, little is known about this association in the developing human central nervous system. Because of the proposed role of acetylcholine in the regulation of development and differentiation of neural cells, an understanding of these relationships during human fetal development gains importance. We report, in this communication, the coupling of mAChR with PLC in the human fetal brain. This coupling was determined using two independent approaches that relied upon estimating the accumulation of inositol phosphates (IPs) and cytidine diphosphate diacylglycerol (CDP-DAG). Carbachol treatment of brain slices, in the presence of lithium, resulted in the accumulation of IPs. Analysis of the kinetics of this accumulation showed that IP3 and IP2 increased rapidly, reaching a peak or plateau before IP. The results also showed that agonist-stimulated PLC produced two second messengers, IP3 and DAG. The production of DAG was strongly supported by the carbachol-dependent increase of CDP-DAG. The accumulation of IP and CDP-DAG was dependent on agonist concentration. The obtained EC50 values were approximately: carbachol 47 microM; acetylcholine 6 microM; and oxotremorine 25 microM. Unexpectedly, all three agonists demonstrated a similar efficacy. The cholinergic stimulation of inositide hydrolysis appears to be the result of activation of the m1 muscarinic receptor.

Isoprenylated proteins in myelin.

Incubation of rat brainstem slices with [3H]-mevalonate ([3H]MVA) in the presence of lovastatin resulted in the incorporation of label into three groups of myelin-associated proteins with molecular masses of 47, 21-27, and 8 kDa, as revealed on sodium dodecyl sulfate-polyacrylamide rod gel electrophoresis. Although the gel patterns of [3H]MVA-derived prenylated proteins were similar, the relative level of 3H incorporated into each protein species differed between myelin and the brainstem homogenate. Immunoprecipitation studies identified the 47-kDa prenylated protein as a 2'-3'-cyclic nucleotide phosphodiesterase, whereas the 8-kDa protein proved to be the gamma subunit of membrane-associated guanine nucleotide regulatory protein. The 3H-labeled 21-27-kDa group in myelin corresponds to the molecular mass of the extensive Ras-like family of monomeric GTP-binding proteins known to be prenylated in other tissues. Increase in lovastatin concentration resulted in reduced levels of [3H]MVA-labeled species in myelin and concomitantly increased levels in the cytosol. A cold MVA chase restored to normality the appearance of [3H]MVA-labeled proteins in myelin. Furthermore, a high lovastatin concentration in the brainstem slice incubation mixture altered the appearance of newly synthesized nonprenylated myelin proteins, including proteolipid protein and the 17-kDa subspecies of myelin basic protein. Because other myelin proteins were unaffected by the high lovastatin concentration, restricting the availability of MVA in myelin-forming cells may selectively alter processes required for myelinogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrolysis of inositol trisphosphate by purified rat brain myelin.

Highly purified rat brain myelin was found to hydrolyze inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, but subsequent hydrolysis of the latter, characteristic of whole brainstem, did not occur. Inositol 1,4,5-trisphosphate 5-phosphatase in myelin was approximately 33% of the level in microsomes and 127% that of the cytosolic fraction from brainstem. The myelin and microsomal enzymes had similar properties, as follows: activation by saponin, requirement for Mg2+ and similar Kact (0.16 and 0.13 mM), Km (8.7 +/- 2.5 and 7.0 +/- 1.0 microM), and pH optima (6.6-6.8). Vmax values were 11.2 +/- 1.0 and 26.3 +/- 2.0 nmol/mg/min for myelin and microsomes, respectively. A possible role for this enzyme in phosphoinositide-mediated signal transduction within myelin and its subcompartments is discussed.

Detection of G proteins in purified bovine brain myelin.

Following a previous report on detection of muscarinic receptors in myelin with the implied presence of G proteins, we now demonstrate by more direct means the presence of such proteins and their quantification. Using [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) as the binding ligand, purified myelin from bovine brain was found to contain approximately half the binding activity of whole white matter (138 +/- 9 vs. 271 +/- 18 pmol/mg of protein). Scatchard analysis of saturation binding data revealed two slopes, a result suggesting at least two binding populations. This binding was inhibited by GTP and its analog but not by 5'-adenylylimidodiphosphate [App(NH)p], GMP, or UTP. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of myelin proteins and blotting on nitrocellulose, [alpha-32P]GTP bound to three bands in the 21-27-kDa range in a manner inhibited by GTP and GTP gamma S but not App(NH)p. ADP-ribosylation of myelin with [32P]NAD+ and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholate extract of myelin subjected to chromatography on a column of phenyl-Sepharose gave at least three major peaks of [35S]GTP gamma S binding activity. SDS-PAGE and immunoblot analyses of peak I indicated the presence of Go alpha, Gi alpha, and Gs alpha. Further fractionation of peak II by diethyl-aminoethyl-Sephacel chromatography gave one [35S]GTP gamma S binding peak with the low-molecular-mass (21-27 kDa) proteins and a second showing two major protein bands of 36 and 40 kDa on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphoinositide breakdown in isolated myelin is stimulated by GTP analogues and calcium.

Purified myelin from rat brainstem, prelabeled in vivo by intracerebral injection of [3H]myoinositol, showed enhanced breakdown of phosphoinositides on treatment with 5'-guanylylimidodiphosphate [Gpp-(NH)p] and Ca2+. Concentration variation of the former in the presence of Ca2+ showed a dose-dependent release of inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3), while inositol 1-phosphate (IP) release was erratic. Concentration-dependent release of IP2 and IP3 was also observed with Ca2+ as the variable in the presence of Gpp(NH)p. Carbachol, when present, did not enhance the stimulatory effect of Gpp(NH)p alone. Addition of diphosphoglycerate during incubation enhanced IP3 at the expense of IP2, suggesting the presence of IP3 phosphatase in myelin.