Journal of Neuruscience Research 26: 16-23 (1990)
Identification of GTP-Binding Proteins in Myelin and Oligodendrocyte Membranes P.E. Braun, E. Horvath, V.W. Yong, and L. Bernier Departments of Biochemistry (P.E.B., E.H.) and Neurology and Neurosurgery (P.E.R., V.W.Y., L.B.), McGill University, Montreal, Quebec, Canada
Myelin membranes purified from mouse and rat brain are associated with alpha subunits of four signal transducing guanosine triphosphate (CTP)binding proteins: Go, Gi, G,, and ras. Four low-molecular-weight (M,) GTP-binding proteins are also present, as demonstrated by the binding of GTP to proteins immobilized in nitrocellulose. This latter group is more prominent at early stages of myelination and remains associated with isolated myelin membranes despite repetitive cycles of purification. At least one nonmyelin subcellular membrane fraction possesses the same proteins. The total membrane fraction of cultured oligodendrocytes is associated with both groups of GTP-binding proteins. None of the well-known myelin proteins bound GTP by the procedure described. Key words: G proteins, GTP-binding proteins, ADP-ribosylation, myelin proteins, oligodendrocyte, myelinogenesis, muscarinic receptor INTRODUCTION A number of observations were recently made concerning thc presence in isolated myelin of cellular and catalytic components that do not accord with the conventional, accepted view of what role the myelin sheath has to play in the nervous system. Among these, we note the presence of cholinergic receptors and adenylate cyclase in highly purified central nervous system (CNS) myelin (Larocca et al., 1987), stimulation by acetylcholine (ACh) of phosphoinositide metabolism in CNS myelin (Kahn and Morell, 1988), evidence for muscarinic receptor-stimulated metabolism of polyphosphoinositides in PNS myelin (Eichberg et al., 1989), regulated phosphorylationidephosphorylation of myelin basic proteins (Ulmer et al., 1987), and myelin-associated glycoprotein (Edwards et al., 1988). Implicit in some or all these events is the possible indirect participation of guanosine triphosphate (GTP)-binding proteins, a potential mechanism that has been generally recognized by the contributors of the above observations. The recent, unexpected discovery that myelin basic protein might serve as a G 1990 Wiley-Liss, Inc.
GTP-binding protein (Chan et al.. 1988) and that 2l.3'cyclic nucleotide 3 '-phosphodiesterase (CNP) posses5es domains that share sequence homology with authentic G proteins (Bernier et al., 1989; Sprinkle and Hancock, 1989) has served to direct our attention to GTP-mediated events in myelination other than cell-surface-mediated signal transduction. We report here a survey of the membrane associated GTP-binding proteins that are present in purified brain myelin and in membranes of cultured oligodendrocytes obtained from brains at an early stage of myelination. We show that alpha-subunits of three major signal transducing G proteins as well as the proto-oncogene product ras are associated with these membranes. In addition, we describe a class of low-molecular-weight proteins that bind GTP when they arc electroblotted onto nitrocellulose. Proteins of this type have been observed in other membranes (Bhullar and Haslam, 1987; 1988; Comerford et al., 1989) and may represent a group of GTP-binding proteins that subserve a role in the mediation of vectorial transport processes (Bourne, 1988) integral to membrane assembly and turnover. A preliminary report of some of these findings has been presented (Braun and Bernier, 1989).
MATERIALS AND METHODS Identification of G, Proteins Proteins that bind GTP when they are immobilized on nitrocellulose were detected by the procedure of Bhullar and Haslam ( 1 987) with minor modifications. Membrane proteins, separated by SDS-PAGE (Laemnili, 1970) in minigels of 10 or 15% acrylamide were electrophoretically transferred to nitrocellulose (Ni troscreen) for 60-70 min at 100 V. This electroblot was then incubated for 15 min in 50 mM Tris buffer, pH 7.5. containing 50 mM MgCl,, 10 FM ATP (as a nonspecific Recelled October 23, 1989, accepted November 9, 1989 Address reprint requests to Dr. Peter E Braun, Departments of Biochemistry and Neurology and Neurosurgery, McGill University. Montreal, Quebcc H3G 146, Canada.
GTP-Binding Proteins in Myelin
blocking agent), and 0.3% Tween 20. It was transferred to the same buffer, with the addition of the isotope (2 pCi of a3’P-GTP; 4 pCi of y3’S-GTP), and incubated for 60 min with gentle shaking at room temp. It was then washed twice with the same buffer for 15 min each. In the case of y3’S-GTP, all buffers also contained 1 niM dithiothreitol. After air drying the blot was exposed for 24-48 hr to Kodak X-OMAT-AR film with a screen for 32P. a3*P= GTP (3,000 Ciimmol) and Y ~ ~ S - G T (1P300 Ci/mmol) were purchased from New England Nuclear.
Identification of Proteins by Jmmunostaining A modification of the immunoelectroblotting procedure of Towbin et al. (1 979) was used to identify specific proteins. We followed this protocol as detailed by Promega Corp. in their 1988 catalog, section 6, pp 1516. Antisera to CNP and MBP have been previously described (Bernier et al.. 1987; Ulmer and Braun. 1983). Polyclonal antibodies to alpha-subunits of G , , Gi, and G, were a generous gift from Dr. Allen M. Spiegel, National Institutes of Health, Bethesda. Anti-ras p 21 (pan) was provided by DuPont Corp.
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Fig. I . Binding of GTP to brain myelin proteins immobilized on nitrocellulose. Lane 1, Coomassie blue-stained myelin proteins separated by SDS-PAGE (15% acrylamide); lane 2, autoradiograrn of y-3sS-GTP bound to myelin proteins on nitrocellulose (separated as in lane 1); lane 3, autoradiogram of a”P-GTP bound to myelin proteins (as in lane 2). Four isuforms of myelin basic protein are shown in lane 1 . Molecularweight markers are shown in lanes 1 and 2. In lanes 2 and 3, a-cl represent GTP-binding components of approximately 25, 24, 22, and 19 kDa M,,respectively.
Preparation of Membranes Mouse and rat brain myelin wa5 isolated by the procedure of Norton and Poduslo (1973). Primary cul- ’*.P-NAD (30 Ci/mmol; DuPont) in 5 scquential aliquots tures of oligodendrocytes were prepared from myelinat- of 2 FCi each at 10-min intervals. ing rat brain at d19 and purified by the procedure of Cholera toxin (List Biological Labs) was activated Yong et al. (1988). More than 90% of the final cell at 30°C for 30 min in 1 mM potassium phosphate buffer preparations were oligodendrocytes as judged by antiga- (pH 7.5) containing 1 mM dithiothreitol. Pertussis toxin lactocerebroside immunocytocheniistry. A crude mem- (List bologicals) was activated for 30 min at 30°C in 20 brane fraction of thesc cells was obtained as follows. mM Tris buffer pH 7 . 6 containing 2 mM EDTA, 20 mM After removal of culture medium 18 X lo6 cells in dithiothreitol, 1 mM ATP. and 1 % Lubrol-PX (Ribeiro dishes were washed once with HEPES buffer (20 mM, and Rodbell, 1989). pH 7.5) containing KCI ( 3 mM), MgCl, ( 3 mlz?) and protease inhibitors. Cells were then scraped into the same buffer (10 ml total) and homogenized by hand (15 RESULTS strokes of the Dounce pestle). Large cell particulates and Identification of CTP-Binding Proteins nuclei were removed by centrifugation at 3,000g, for 10 Figure 1 demonstrates the binding of GTP to four min, and total membranes were recovered from the su- zones of the electroblot corresponding to polypeptides pernatant by centrifugation for 30 min at 100,000g. The of approximately 25, 24, 22, and 19 kD. a3’P-GTP promembrane-containing pellet was dispersed in sample duces slightly wider, less distinct bands than does buffer in preparation for SDS-PAGE. y”S-GTP, and the two major binding components at -2.5 and 24 kD often appear as one wide band. Since the ADP-Ribosylation of Membrane Proteins G,25 and G , 24 polypeptides co-migrate by SDS-PAGE We used a modification of the procedure of Bokoch with the 21.5K MBP and with PLP M, (25 kD), and et al. (1983). Briefly, membranes (200 pg protein) were since purified human MBP was reported to bind GTP in incubated at 37°C with activated cholera toxin (2.5 pg) solution (Chan et al., 1988), we investigated the possible or pertussis toxin (2 pg) in 200 p1 of a buffer containing GTP binding to the major myelin proteins. Accordingly, 100 mM potassium phosphate (pH 7.5), 10 mM thymi- we isolated the MBPs from rat or mouse brain by caldine, 1 mM ADP, 0.1 mM GTP, 4 niM ADP-ribose, cium extraction (Smith and Braun, 1988) or by acid ex2.5 mM MgCI,, 1 mM dithiothreitol and 0.05% Triton traction (Edwards et al., 1988) and subjected them to thc X-100. The reaction was initiated by the addition of GTP-binding procedure. None of the MBP isoforms
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Fig. 2. Autoradiogram showing specificity of niicleotide binding to brain myelin proteins. Electroblots of myelin proteins were preincubated with unlabeled nucleotjdes (100 pM), then radiolabeled with GTP in the prescnce of the unlabeled nucleotide according to the procedure described in Materials and Methods. Only the region of the gel between 15 and 30 kDaM, is illustrated. Lane 1, contrul (only d2P-GTP);lane 2, GTP;
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lane 3, GDP: lane 4: GMP: lane 5 , ATP.
1 2 bound GTP above background levels (data not shown), and the G, proteins remained associated with the nonextractable myelin membrane components. Normally, little or no PLP is observed in our polypeptide patterns obtained by conventional SDS-PAGE because boiling the samples in SDS with a reducing agent rcsults in irreversible aggregation of both PLP isoforms. Consequently, in comparing samples of myelin that were not boiled with those that were boiled, we found that the GTP-binding pattern and intensity did not change. In addition, several samples of purified PLP failed to bind GTP in this assay (data not shown). Specificity of nucleotide binding was determined by pretreating electroblots of myelin proteins with various unlabeled nucleotides followed by labeled GTP in the presence of the “blocking” nucleotides (Fig. 2). Both ATP and GMP failed to compete out the binding of GTP to the G, polypeptides, but GTP (and yS-GTP, not shown) completely blocked binding of labeled GTP, and GDP partially blocked binding of GTP, as previously observed in many studies of G proteins (Bhullar and Haslam, 1987; Gilinan, 1987). Identical results were obtained with y’*P-GTP as the binding radiolabel, showing that GTPase activity is not a factor in these experiments. Since ADP-ribosylation of polypeptides by cholera or pertussis toxin is often diagnostic of G proteins (Gilman, 1987), we compared the ADP-ribosylated products of the two toxin-catalyzed modifications of myelin. Figure 3 illustrates the major ADP-ribosylated components of adult brain myelin. In agreement with the observation of Chan et al. (1988) that the human 18.5-kD MBP can be ADP-ribosylated by cholera toxin, we show here that all MBP isoforms in mouse (or rat, not shown) br.. din are similarly modified by cholera toxin but not pertussis toxin. Furthermore, we observe two cholera toxin modified polypeptides at -44-46 kD M, that are not ADP-
Fig. 3 . Autoradiogram showing ADP-ribosylation of brain myelin proteins. Mouse brain myelin proteins were ADP-ribosylated by cholera toxin (lane I ) or pertussis toxin (lane 2). After SDS-PAGE, proteins were electroblotted onto nitroccllulose and visualized by autoradiography. Positions o f molecular-weight markers (kDa) are shown on the left.
ribosylated by pertussis toxin. We have identified these as G,, subunits of the cyclic adenosine monophosphatc (CAMP)-stimulated G-protein complex by immunostaining with anti-G,, antiserum (Fig. 4). Aside from the MBPs, these are the only myelin proteins ADP-ribosylated by cholera toxin. Pertussis toxin-catalyzed ADPribosylation of myelin produced only one prominently labeled band at -40 kD iM,; on prolonged exposure of the autoradiogram! a more weakly labeled component appeared at -46 kD M , (not shown). The prominent ADP-ribosylated band corresponds to G,, and Gi, by immunolabeling of an electroblot (Fig. 4). The other, weakly labeled component aligns exactly with the two CNP Components on a Western blot (data not shown). None of these binds GTP by the ligand blot overlay procedure, in agreement with observations by others.
Subcellular Fractionation and Myelin Purification Since the possibility exists that the observed GTPbinding and ADP-ribosylated proteins in isolated myelin are derived from other cellular components that might co-fractionate with myelin we fractionated brain membranes in several ways and compared successively purified myelin preparations for their complement of G, proteins, as well as for proteins that can be ADP-ribosylated and those that react with anti-ras antibodies on a Western blot. In contrast to the myelin prepared by minor mod-
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Fig. 4. Inimunoblot identification of G proteins in brain myelin. Mouse brain myelin proteins were separated by SDSPAGE ( 1 1-2370 acrylarnide gradient gel) and electrublotted onto nitrocellulose. Primary antisera were used at 1 : 1,000. Lane 1, anti-CNP; lane 2, anti G, (#104): lane 3, anti G, (#41); lane 4, anti-G, (#708); lane 5, anti rus (pan). The antisera lot numbcrs are thosc provided by Dr. A. Spiegel.
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myelin membranes; they show a progressive depletion of polypcptides above -30 kD M , (mainly myelin basic proteins and proteolipid proteins, although little of the latter is evident because samples for electrophoresis are boiled in the presencc of dithiothreitol, producing aggrcgates of proteolipid proteins that accumulate near the top of the gel). Littlc difference is observed between lanes 4, 5 , and 6 showing that Pl,2B" is already a fairly pure myelin fraction. Only minor amounts of niyelin-characteristic polypeptides are evident in P I$, as expectcd. The GTP-binding pattern (Fig. SB) demonstrates that G,, proteins are found in all our membranecontaining brain subfractions. A nonmyelin fraction (PI,&) appears to possess the same ma.jor and minor C, proteins seen in inyelin fractions (P,.,A; P,,,B and successivcly purified myelin P, , $ - I V ) but in varying amounts. When selected fractions were electroblotted onto nitroccllulose and immunoreacted with anti-ras, we observed the prescncc of ras in every membrane-containing fraction (Fig. 6). In each fraction, a major doublet (-21 kI) M,.) was accompanied by a varying abundance of several minor imniunoreactive components both above and bclow the dominant bands. This multiplicity of bands has also been commonly observed by others (Barbacid, 1987). One of these (- 19 kD M , ) corresponded to a weak GTP-binding component (band d in Fig. 1). Even after extensive purification of the myelin fraction (P, ,2B11),ras remained undiminished in its imniunostaining intensity (not shown).
Developmental profiles of GTP-Binding Proteins and ras in Myelin Although we observed a variety of GTP-binding proteins in mature myelin. we wanted to assess possible ifications of the procedure of Norton and Poduslo (1973) developmental fluctuations of their deposition in develthat we use in Figures 1-4, we prepared myelin from oping myelin relative to the presencc of other myelin myelhated axons of adult mouse brain (Pereyra and proteins, during the period of active myelination. AcBraun, 1983) by a series of sequential steps designed to cordingly, we prepared mouse brain myelin at several produced m y e h membranes progressively freer of non- appropriate ages and examined thc appearance of the G, myelin membranes. Figure 5A describes the various sub- class of GTP-binding proteins as well as of ras. Figure fractions and shows profiles of the stained polypeptides 7A shows the expected pattern of myelin proleins below (after electroblotting) at various stages of myelin isola- -40 kD M , (mainly the MBPs, since little of the aggretion, and the corresponding profile of GTP binding to gated PLPs is evident in this gel systcm); Figure 7B electroblotted polypeptides is seen in Figure SB. P, ,?A shows the binding of GTP to low-molecular-weight polyrcpresents mainly large and small myelinated axon frag- peptides immobilized on nitrocellulose at these five ages. ments, with some entrapped cytoplasmic elements. PI,*B Despite the relatively lower abundancc of proteins in this is cornposed largely of loosely ensheathed axons with size class at day 1 I , the greatest amount of GTP binding cytoplasmic inclusions plus large membrane fragments is evident at this age, decreasing slightly as myelination and contains the bulk of compact myelin in adult brain. progresses from day 16 to 23, but persisting in adult P I,,C is composed mainly of mitochondria, synapto- myelin. Likewise, the immunoreactivity of blotted polysoma1 elements, and Golgi and other endomembranes peptides to anti-rus is highest at day 11, but remains (Pereyra and Braun, 1983). The subfractions designated readily apparent at all ages studied (Fig. 7C). We conp ~','I,"l,lv represent a series of progressively purified firmed the identity of the four MBPs by immunoreactiv1.2
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Fig. 5. G , proteins in subcellular membrane fractions of mouse brain. A: Coomassie blue-stained proteins separated by SDS-PAGE (15% acrylamide); 50 tJ.g of protein per lane. R: Autoradiogram showing u3'P-GTP bound to proteins electro-
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blotted onto nitrocellulose from the gel in A . Lane 1, P,,,A; lane 2, P,,,B; lane 3, P,,,B1; lane 4, P,,,B"; lane 5, P,,2B11'; lane 6, P,,,BTV;lane 7. P,,,C. Position of molecular weight markers (kDa) are illustrated.
intensity because of the extensive washes of the blot inherent in the GTP overlay procedure (data not shown).
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I 2 3 4 Fig. 6. Immunoblot showing the presence of YUS in several mouse brain subfractions. Lane 1, total homogenate; lane 2, P,,2A; lane 3, P,,,B; lane 4, P,,,C. Protein (SO kg) applied to each lane of the starting gel; prestained molecular-weight markers are shown on the left (kDa).
GTP-Binding Proteins in Cultured Oligodendrocytes Membrane preparations obtained from cultured rat oligodendrocytes were subjected to SDS-PAGE, and the proteins electroblotted onto nitrocellulose. These blots were then probed with antibodies or overlayed with radioactive GTP. In addition, some of the CTP-binding proteins were verified by ADP-ribosylation. Figure XA,B shows that the G, class of GTP-binding proteins found in myelin is also present in oligoendrocyte membranes. although the relative amounts of the individual components is slightly different. The same complement of signal-transducing G proteins seen in CNS myelin (determined as the alpha-subunits of G,, G,, GJ is present in oligodendrocyte membranes (Fig. SC). Nor surprisingly, the ruus polypeptides observed in myelin are also apparent in oligodcndrocytes (Fig. SC).
IIISCUSSION ity to anti-MBP (data not shown). The indicated position of rus in Figure 7B was verified by subjecting this GTPlabeled electroblot to anti-ras antibody, which clearly visualized the major ras bands, although at a diminished
Although some G proteins are probably found in all mammalian cells, not all members of this family are likely to be present in every cell type. Therefore, in order to complement our work on CNS myelin, we sought to define the GTP-binding proteins that are present in cul-
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Fig. 7. Developmental appearance in mouse brain myelin G , proteins and ms. A: Coomassie blue-stained brain myelin proteins separated by SDS-PAGE ( 15% acrylamide). Only the gel region representing - 12-35 kDa-M, is shown. Numbers represent age in days; Ad, adult. B: Autoradiogram showing
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cr3’P-GTP bound to myelin proteins at different ages. The radiolabeled bands correspond to those shown in Figure 1. C: Imniunoelectroblot showing the presence of rus in myelin at different ages.
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Fig. 8. Presence of G, proteins and signal transducing G proteins in membranes of cultured oligodendrocytes. A: Autoradiogram showing a3’P-GTP bound to rat brain myelin proteins (lane I) and oligodendrocyte membranes (lane 2). R: Same as A, except that y%GTP is the bound radiolabel. C: Immunoelectroblot showing the presence of G proteins in membranes
of cultured oligodendrocytes. Proteins (30 k*.gper lane) separated by SDS-PAGE in 15% acrylamide. Antisera (as described in Fig. 4) were used at 1: 250. Lane 1, anti-G,,: lane 2, anti-G,; lane 3, anti-G,: lane 4. anti-CNP; lane 5, anti-ras. Positions of prestained molecular-weight markers are shown on the right (kDa).
tured oligodendrocytes. Not surprisingly, G, and G,, regulatory components of CAMP-dependent signal-transducing networks, are readily apparent in oligodendrocytes and, given the presence of muscarinic receptors and adenylate cyclase in the CNS myelin sheath (Larocca et al., 1987), the demonstration of these two G proteins in association with highly purified myelin membranes strengthens the case for physiologically significant involvement of some myelin compartments (presumably the paranodal loops) in events triggered by ACh. Whether this is related in any way to neuronal conduc-
tion is not clear. Recent studies reported in N a t u r ~how. ever, point to a role for muscarinic receptors in mediating developmental and growth signals leading to stimulation of DNA synthesis in at least one type of glial cell, the astrocytes (AshkcnaLi et al., 1989). It seems reasonable, therefore. for us to suggest that comniunication between neurons and oligodendroglia during development and in maintenance of the axon-myelin unit may also be dcpendent, in part, on molecular machinery encompassing muscarinic receptors and their associated G proteins.
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Although the function of G,, protein may overlap that of other G proteins (Spiegel, 1987), there are indications that it may regulate ion channels (Freissmuth et al., 1989), in addition to other functions it might have. Since not all cell types possess this G protein (Gilman, 1987), we think it is significant that oligodendrocytes do and that the association of this protein with myelin membranes persists despite numerous procedures designed to rid myelin of adventitiously associated cellular elements. Participation of G, in thc regulation of ion channels and its putative linkage to the action of inositol phospholipids in signal transduction accords with current developments in studies of polyphosphoinositide metabolism in paranodal loops (Kahn and Morell, 1988; Eichberg et al., 1989) and of ion channels in oligodcndroglia (Banes et al., 1988). The proto-oncogene product, r m , is also thought to participate in the inositol phospholipid-linked mode of signal transduction (Barbacid, 1987; Kamata and Kung, 1988), although its prccisc role is not clear. The association of ras with isolated and purified myelin helps support the argument for signal responsive receptors or ion channcls in paranodal compartments of the myelin sheath. Numerous small (
Identification of GTP-Binding Proteins in Myelin and Oligodendrocyte Membranes P.E. Braun, E. Horvath, V.W. Yong, and L. Bernier Departments of Biochemistry (P.E.B., E.H.) and Neurology and Neurosurgery (P.E.R., V.W.Y., L.B.), McGill University, Montreal, Quebec, Canada
Myelin membranes purified from mouse and rat brain are associated with alpha subunits of four signal transducing guanosine triphosphate (CTP)binding proteins: Go, Gi, G,, and ras. Four low-molecular-weight (M,) GTP-binding proteins are also present, as demonstrated by the binding of GTP to proteins immobilized in nitrocellulose. This latter group is more prominent at early stages of myelination and remains associated with isolated myelin membranes despite repetitive cycles of purification. At least one nonmyelin subcellular membrane fraction possesses the same proteins. The total membrane fraction of cultured oligodendrocytes is associated with both groups of GTP-binding proteins. None of the well-known myelin proteins bound GTP by the procedure described. Key words: G proteins, GTP-binding proteins, ADP-ribosylation, myelin proteins, oligodendrocyte, myelinogenesis, muscarinic receptor INTRODUCTION A number of observations were recently made concerning thc presence in isolated myelin of cellular and catalytic components that do not accord with the conventional, accepted view of what role the myelin sheath has to play in the nervous system. Among these, we note the presence of cholinergic receptors and adenylate cyclase in highly purified central nervous system (CNS) myelin (Larocca et al., 1987), stimulation by acetylcholine (ACh) of phosphoinositide metabolism in CNS myelin (Kahn and Morell, 1988), evidence for muscarinic receptor-stimulated metabolism of polyphosphoinositides in PNS myelin (Eichberg et al., 1989), regulated phosphorylationidephosphorylation of myelin basic proteins (Ulmer et al., 1987), and myelin-associated glycoprotein (Edwards et al., 1988). Implicit in some or all these events is the possible indirect participation of guanosine triphosphate (GTP)-binding proteins, a potential mechanism that has been generally recognized by the contributors of the above observations. The recent, unexpected discovery that myelin basic protein might serve as a G 1990 Wiley-Liss, Inc.
GTP-binding protein (Chan et al.. 1988) and that 2l.3'cyclic nucleotide 3 '-phosphodiesterase (CNP) posses5es domains that share sequence homology with authentic G proteins (Bernier et al., 1989; Sprinkle and Hancock, 1989) has served to direct our attention to GTP-mediated events in myelination other than cell-surface-mediated signal transduction. We report here a survey of the membrane associated GTP-binding proteins that are present in purified brain myelin and in membranes of cultured oligodendrocytes obtained from brains at an early stage of myelination. We show that alpha-subunits of three major signal transducing G proteins as well as the proto-oncogene product ras are associated with these membranes. In addition, we describe a class of low-molecular-weight proteins that bind GTP when they arc electroblotted onto nitrocellulose. Proteins of this type have been observed in other membranes (Bhullar and Haslam, 1987; 1988; Comerford et al., 1989) and may represent a group of GTP-binding proteins that subserve a role in the mediation of vectorial transport processes (Bourne, 1988) integral to membrane assembly and turnover. A preliminary report of some of these findings has been presented (Braun and Bernier, 1989).
MATERIALS AND METHODS Identification of G, Proteins Proteins that bind GTP when they are immobilized on nitrocellulose were detected by the procedure of Bhullar and Haslam ( 1 987) with minor modifications. Membrane proteins, separated by SDS-PAGE (Laemnili, 1970) in minigels of 10 or 15% acrylamide were electrophoretically transferred to nitrocellulose (Ni troscreen) for 60-70 min at 100 V. This electroblot was then incubated for 15 min in 50 mM Tris buffer, pH 7.5. containing 50 mM MgCl,, 10 FM ATP (as a nonspecific Recelled October 23, 1989, accepted November 9, 1989 Address reprint requests to Dr. Peter E Braun, Departments of Biochemistry and Neurology and Neurosurgery, McGill University. Montreal, Quebcc H3G 146, Canada.
GTP-Binding Proteins in Myelin
blocking agent), and 0.3% Tween 20. It was transferred to the same buffer, with the addition of the isotope (2 pCi of a3’P-GTP; 4 pCi of y3’S-GTP), and incubated for 60 min with gentle shaking at room temp. It was then washed twice with the same buffer for 15 min each. In the case of y3’S-GTP, all buffers also contained 1 niM dithiothreitol. After air drying the blot was exposed for 24-48 hr to Kodak X-OMAT-AR film with a screen for 32P. a3*P= GTP (3,000 Ciimmol) and Y ~ ~ S - G T (1P300 Ci/mmol) were purchased from New England Nuclear.
Identification of Proteins by Jmmunostaining A modification of the immunoelectroblotting procedure of Towbin et al. (1 979) was used to identify specific proteins. We followed this protocol as detailed by Promega Corp. in their 1988 catalog, section 6, pp 1516. Antisera to CNP and MBP have been previously described (Bernier et al.. 1987; Ulmer and Braun. 1983). Polyclonal antibodies to alpha-subunits of G , , Gi, and G, were a generous gift from Dr. Allen M. Spiegel, National Institutes of Health, Bethesda. Anti-ras p 21 (pan) was provided by DuPont Corp.
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Fig. I . Binding of GTP to brain myelin proteins immobilized on nitrocellulose. Lane 1, Coomassie blue-stained myelin proteins separated by SDS-PAGE (15% acrylamide); lane 2, autoradiograrn of y-3sS-GTP bound to myelin proteins on nitrocellulose (separated as in lane 1); lane 3, autoradiogram of a”P-GTP bound to myelin proteins (as in lane 2). Four isuforms of myelin basic protein are shown in lane 1 . Molecularweight markers are shown in lanes 1 and 2. In lanes 2 and 3, a-cl represent GTP-binding components of approximately 25, 24, 22, and 19 kDa M,,respectively.
Preparation of Membranes Mouse and rat brain myelin wa5 isolated by the procedure of Norton and Poduslo (1973). Primary cul- ’*.P-NAD (30 Ci/mmol; DuPont) in 5 scquential aliquots tures of oligodendrocytes were prepared from myelinat- of 2 FCi each at 10-min intervals. ing rat brain at d19 and purified by the procedure of Cholera toxin (List Biological Labs) was activated Yong et al. (1988). More than 90% of the final cell at 30°C for 30 min in 1 mM potassium phosphate buffer preparations were oligodendrocytes as judged by antiga- (pH 7.5) containing 1 mM dithiothreitol. Pertussis toxin lactocerebroside immunocytocheniistry. A crude mem- (List bologicals) was activated for 30 min at 30°C in 20 brane fraction of thesc cells was obtained as follows. mM Tris buffer pH 7 . 6 containing 2 mM EDTA, 20 mM After removal of culture medium 18 X lo6 cells in dithiothreitol, 1 mM ATP. and 1 % Lubrol-PX (Ribeiro dishes were washed once with HEPES buffer (20 mM, and Rodbell, 1989). pH 7.5) containing KCI ( 3 mM), MgCl, ( 3 mlz?) and protease inhibitors. Cells were then scraped into the same buffer (10 ml total) and homogenized by hand (15 RESULTS strokes of the Dounce pestle). Large cell particulates and Identification of CTP-Binding Proteins nuclei were removed by centrifugation at 3,000g, for 10 Figure 1 demonstrates the binding of GTP to four min, and total membranes were recovered from the su- zones of the electroblot corresponding to polypeptides pernatant by centrifugation for 30 min at 100,000g. The of approximately 25, 24, 22, and 19 kD. a3’P-GTP promembrane-containing pellet was dispersed in sample duces slightly wider, less distinct bands than does buffer in preparation for SDS-PAGE. y”S-GTP, and the two major binding components at -2.5 and 24 kD often appear as one wide band. Since the ADP-Ribosylation of Membrane Proteins G,25 and G , 24 polypeptides co-migrate by SDS-PAGE We used a modification of the procedure of Bokoch with the 21.5K MBP and with PLP M, (25 kD), and et al. (1983). Briefly, membranes (200 pg protein) were since purified human MBP was reported to bind GTP in incubated at 37°C with activated cholera toxin (2.5 pg) solution (Chan et al., 1988), we investigated the possible or pertussis toxin (2 pg) in 200 p1 of a buffer containing GTP binding to the major myelin proteins. Accordingly, 100 mM potassium phosphate (pH 7.5), 10 mM thymi- we isolated the MBPs from rat or mouse brain by caldine, 1 mM ADP, 0.1 mM GTP, 4 niM ADP-ribose, cium extraction (Smith and Braun, 1988) or by acid ex2.5 mM MgCI,, 1 mM dithiothreitol and 0.05% Triton traction (Edwards et al., 1988) and subjected them to thc X-100. The reaction was initiated by the addition of GTP-binding procedure. None of the MBP isoforms
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Fig. 2. Autoradiogram showing specificity of niicleotide binding to brain myelin proteins. Electroblots of myelin proteins were preincubated with unlabeled nucleotjdes (100 pM), then radiolabeled with GTP in the prescnce of the unlabeled nucleotide according to the procedure described in Materials and Methods. Only the region of the gel between 15 and 30 kDaM, is illustrated. Lane 1, contrul (only d2P-GTP);lane 2, GTP;
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lane 3, GDP: lane 4: GMP: lane 5 , ATP.
1 2 bound GTP above background levels (data not shown), and the G, proteins remained associated with the nonextractable myelin membrane components. Normally, little or no PLP is observed in our polypeptide patterns obtained by conventional SDS-PAGE because boiling the samples in SDS with a reducing agent rcsults in irreversible aggregation of both PLP isoforms. Consequently, in comparing samples of myelin that were not boiled with those that were boiled, we found that the GTP-binding pattern and intensity did not change. In addition, several samples of purified PLP failed to bind GTP in this assay (data not shown). Specificity of nucleotide binding was determined by pretreating electroblots of myelin proteins with various unlabeled nucleotides followed by labeled GTP in the presence of the “blocking” nucleotides (Fig. 2). Both ATP and GMP failed to compete out the binding of GTP to the G, polypeptides, but GTP (and yS-GTP, not shown) completely blocked binding of labeled GTP, and GDP partially blocked binding of GTP, as previously observed in many studies of G proteins (Bhullar and Haslam, 1987; Gilinan, 1987). Identical results were obtained with y’*P-GTP as the binding radiolabel, showing that GTPase activity is not a factor in these experiments. Since ADP-ribosylation of polypeptides by cholera or pertussis toxin is often diagnostic of G proteins (Gilman, 1987), we compared the ADP-ribosylated products of the two toxin-catalyzed modifications of myelin. Figure 3 illustrates the major ADP-ribosylated components of adult brain myelin. In agreement with the observation of Chan et al. (1988) that the human 18.5-kD MBP can be ADP-ribosylated by cholera toxin, we show here that all MBP isoforms in mouse (or rat, not shown) br.. din are similarly modified by cholera toxin but not pertussis toxin. Furthermore, we observe two cholera toxin modified polypeptides at -44-46 kD M, that are not ADP-
Fig. 3 . Autoradiogram showing ADP-ribosylation of brain myelin proteins. Mouse brain myelin proteins were ADP-ribosylated by cholera toxin (lane I ) or pertussis toxin (lane 2). After SDS-PAGE, proteins were electroblotted onto nitroccllulose and visualized by autoradiography. Positions o f molecular-weight markers (kDa) are shown on the left.
ribosylated by pertussis toxin. We have identified these as G,, subunits of the cyclic adenosine monophosphatc (CAMP)-stimulated G-protein complex by immunostaining with anti-G,, antiserum (Fig. 4). Aside from the MBPs, these are the only myelin proteins ADP-ribosylated by cholera toxin. Pertussis toxin-catalyzed ADPribosylation of myelin produced only one prominently labeled band at -40 kD iM,; on prolonged exposure of the autoradiogram! a more weakly labeled component appeared at -46 kD M , (not shown). The prominent ADP-ribosylated band corresponds to G,, and Gi, by immunolabeling of an electroblot (Fig. 4). The other, weakly labeled component aligns exactly with the two CNP Components on a Western blot (data not shown). None of these binds GTP by the ligand blot overlay procedure, in agreement with observations by others.
Subcellular Fractionation and Myelin Purification Since the possibility exists that the observed GTPbinding and ADP-ribosylated proteins in isolated myelin are derived from other cellular components that might co-fractionate with myelin we fractionated brain membranes in several ways and compared successively purified myelin preparations for their complement of G, proteins, as well as for proteins that can be ADP-ribosylated and those that react with anti-ras antibodies on a Western blot. In contrast to the myelin prepared by minor mod-
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Fig. 4. Inimunoblot identification of G proteins in brain myelin. Mouse brain myelin proteins were separated by SDSPAGE ( 1 1-2370 acrylarnide gradient gel) and electrublotted onto nitrocellulose. Primary antisera were used at 1 : 1,000. Lane 1, anti-CNP; lane 2, anti G, (#104): lane 3, anti G, (#41); lane 4, anti-G, (#708); lane 5, anti rus (pan). The antisera lot numbcrs are thosc provided by Dr. A. Spiegel.
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myelin membranes; they show a progressive depletion of polypcptides above -30 kD M , (mainly myelin basic proteins and proteolipid proteins, although little of the latter is evident because samples for electrophoresis are boiled in the presencc of dithiothreitol, producing aggrcgates of proteolipid proteins that accumulate near the top of the gel). Littlc difference is observed between lanes 4, 5 , and 6 showing that Pl,2B" is already a fairly pure myelin fraction. Only minor amounts of niyelin-characteristic polypeptides are evident in P I$, as expectcd. The GTP-binding pattern (Fig. SB) demonstrates that G,, proteins are found in all our membranecontaining brain subfractions. A nonmyelin fraction (PI,&) appears to possess the same ma.jor and minor C, proteins seen in inyelin fractions (P,.,A; P,,,B and successivcly purified myelin P, , $ - I V ) but in varying amounts. When selected fractions were electroblotted onto nitroccllulose and immunoreacted with anti-ras, we observed the prescncc of ras in every membrane-containing fraction (Fig. 6). In each fraction, a major doublet (-21 kI) M,.) was accompanied by a varying abundance of several minor imniunoreactive components both above and bclow the dominant bands. This multiplicity of bands has also been commonly observed by others (Barbacid, 1987). One of these (- 19 kD M , ) corresponded to a weak GTP-binding component (band d in Fig. 1). Even after extensive purification of the myelin fraction (P, ,2B11),ras remained undiminished in its imniunostaining intensity (not shown).
Developmental profiles of GTP-Binding Proteins and ras in Myelin Although we observed a variety of GTP-binding proteins in mature myelin. we wanted to assess possible ifications of the procedure of Norton and Poduslo (1973) developmental fluctuations of their deposition in develthat we use in Figures 1-4, we prepared myelin from oping myelin relative to the presencc of other myelin myelhated axons of adult mouse brain (Pereyra and proteins, during the period of active myelination. AcBraun, 1983) by a series of sequential steps designed to cordingly, we prepared mouse brain myelin at several produced m y e h membranes progressively freer of non- appropriate ages and examined thc appearance of the G, myelin membranes. Figure 5A describes the various sub- class of GTP-binding proteins as well as of ras. Figure fractions and shows profiles of the stained polypeptides 7A shows the expected pattern of myelin proleins below (after electroblotting) at various stages of myelin isola- -40 kD M , (mainly the MBPs, since little of the aggretion, and the corresponding profile of GTP binding to gated PLPs is evident in this gel systcm); Figure 7B electroblotted polypeptides is seen in Figure SB. P, ,?A shows the binding of GTP to low-molecular-weight polyrcpresents mainly large and small myelinated axon frag- peptides immobilized on nitrocellulose at these five ages. ments, with some entrapped cytoplasmic elements. PI,*B Despite the relatively lower abundancc of proteins in this is cornposed largely of loosely ensheathed axons with size class at day 1 I , the greatest amount of GTP binding cytoplasmic inclusions plus large membrane fragments is evident at this age, decreasing slightly as myelination and contains the bulk of compact myelin in adult brain. progresses from day 16 to 23, but persisting in adult P I,,C is composed mainly of mitochondria, synapto- myelin. Likewise, the immunoreactivity of blotted polysoma1 elements, and Golgi and other endomembranes peptides to anti-rus is highest at day 11, but remains (Pereyra and Braun, 1983). The subfractions designated readily apparent at all ages studied (Fig. 7C). We conp ~','I,"l,lv represent a series of progressively purified firmed the identity of the four MBPs by immunoreactiv1.2
Braun et al.
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Fig. 5. G , proteins in subcellular membrane fractions of mouse brain. A: Coomassie blue-stained proteins separated by SDS-PAGE (15% acrylamide); 50 tJ.g of protein per lane. R: Autoradiogram showing u3'P-GTP bound to proteins electro-
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blotted onto nitrocellulose from the gel in A . Lane 1, P,,,A; lane 2, P,,,B; lane 3, P,,,B1; lane 4, P,,,B"; lane 5, P,,2B11'; lane 6, P,,,BTV;lane 7. P,,,C. Position of molecular weight markers (kDa) are illustrated.
intensity because of the extensive washes of the blot inherent in the GTP overlay procedure (data not shown).
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I 2 3 4 Fig. 6. Immunoblot showing the presence of YUS in several mouse brain subfractions. Lane 1, total homogenate; lane 2, P,,2A; lane 3, P,,,B; lane 4, P,,,C. Protein (SO kg) applied to each lane of the starting gel; prestained molecular-weight markers are shown on the left (kDa).
GTP-Binding Proteins in Cultured Oligodendrocytes Membrane preparations obtained from cultured rat oligodendrocytes were subjected to SDS-PAGE, and the proteins electroblotted onto nitrocellulose. These blots were then probed with antibodies or overlayed with radioactive GTP. In addition, some of the CTP-binding proteins were verified by ADP-ribosylation. Figure XA,B shows that the G, class of GTP-binding proteins found in myelin is also present in oligoendrocyte membranes. although the relative amounts of the individual components is slightly different. The same complement of signal-transducing G proteins seen in CNS myelin (determined as the alpha-subunits of G,, G,, GJ is present in oligodendrocyte membranes (Fig. SC). Nor surprisingly, the ruus polypeptides observed in myelin are also apparent in oligodcndrocytes (Fig. SC).
IIISCUSSION ity to anti-MBP (data not shown). The indicated position of rus in Figure 7B was verified by subjecting this GTPlabeled electroblot to anti-ras antibody, which clearly visualized the major ras bands, although at a diminished
Although some G proteins are probably found in all mammalian cells, not all members of this family are likely to be present in every cell type. Therefore, in order to complement our work on CNS myelin, we sought to define the GTP-binding proteins that are present in cul-
GTP-Binding Proteins in Myelin
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Fig. 7. Developmental appearance in mouse brain myelin G , proteins and ms. A: Coomassie blue-stained brain myelin proteins separated by SDS-PAGE ( 15% acrylamide). Only the gel region representing - 12-35 kDa-M, is shown. Numbers represent age in days; Ad, adult. B: Autoradiogram showing
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cr3’P-GTP bound to myelin proteins at different ages. The radiolabeled bands correspond to those shown in Figure 1. C: Imniunoelectroblot showing the presence of rus in myelin at different ages.
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Fig. 8. Presence of G, proteins and signal transducing G proteins in membranes of cultured oligodendrocytes. A: Autoradiogram showing a3’P-GTP bound to rat brain myelin proteins (lane I) and oligodendrocyte membranes (lane 2). R: Same as A, except that y%GTP is the bound radiolabel. C: Immunoelectroblot showing the presence of G proteins in membranes
of cultured oligodendrocytes. Proteins (30 k*.gper lane) separated by SDS-PAGE in 15% acrylamide. Antisera (as described in Fig. 4) were used at 1: 250. Lane 1, anti-G,,: lane 2, anti-G,; lane 3, anti-G,: lane 4. anti-CNP; lane 5, anti-ras. Positions of prestained molecular-weight markers are shown on the right (kDa).
tured oligodendrocytes. Not surprisingly, G, and G,, regulatory components of CAMP-dependent signal-transducing networks, are readily apparent in oligodendrocytes and, given the presence of muscarinic receptors and adenylate cyclase in the CNS myelin sheath (Larocca et al., 1987), the demonstration of these two G proteins in association with highly purified myelin membranes strengthens the case for physiologically significant involvement of some myelin compartments (presumably the paranodal loops) in events triggered by ACh. Whether this is related in any way to neuronal conduc-
tion is not clear. Recent studies reported in N a t u r ~how. ever, point to a role for muscarinic receptors in mediating developmental and growth signals leading to stimulation of DNA synthesis in at least one type of glial cell, the astrocytes (AshkcnaLi et al., 1989). It seems reasonable, therefore. for us to suggest that comniunication between neurons and oligodendroglia during development and in maintenance of the axon-myelin unit may also be dcpendent, in part, on molecular machinery encompassing muscarinic receptors and their associated G proteins.
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Braun et al.
Although the function of G,, protein may overlap that of other G proteins (Spiegel, 1987), there are indications that it may regulate ion channels (Freissmuth et al., 1989), in addition to other functions it might have. Since not all cell types possess this G protein (Gilman, 1987), we think it is significant that oligodendrocytes do and that the association of this protein with myelin membranes persists despite numerous procedures designed to rid myelin of adventitiously associated cellular elements. Participation of G, in thc regulation of ion channels and its putative linkage to the action of inositol phospholipids in signal transduction accords with current developments in studies of polyphosphoinositide metabolism in paranodal loops (Kahn and Morell, 1988; Eichberg et al., 1989) and of ion channels in oligodcndroglia (Banes et al., 1988). The proto-oncogene product, r m , is also thought to participate in the inositol phospholipid-linked mode of signal transduction (Barbacid, 1987; Kamata and Kung, 1988), although its prccisc role is not clear. The association of ras with isolated and purified myelin helps support the argument for signal responsive receptors or ion channcls in paranodal compartments of the myelin sheath. Numerous small (
