(Received for publication, May 29, 1997)
From the Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, and Regional Primate Research Center, University of Washington, Seattle, Washington 98195-7370
The GTPase Rab3A has been postulated to cycle on and off synaptic membranes during the course of neurotransmission. Moreover, a Rab guanine nucleotide dissociation inhibitor has been shown to cause Rab3A to dissociate from synaptic membranes in vitro. We demonstrate here that Ca2+/calmodulin also can cause Rab3A to dissociate from synaptic membranes in vitro. Like Rab guanine nucleotide dissociation inhibitor, it forms a 1:1 complex with Rab3A that requires both the lipidated C terminus of Rab3A and the presence of bound guanine nucleotide. In addition, a synthetic peptide corresponding to the Lys62-Arg85 sequence of Rab3A can prevent the dissociating effect of each protein and disrupt complexes between each protein and Rab3A. However, Ca2+/calmodulin's effect differs from that of Rab guanine nucleotide dissociation inhibitor not only in being Ca2+-dependent but also in having a less stringent requirement for GDP as opposed to GTP and in involving a less complete dissociation of Rab3A. The functional significance in vivo of Ca2+/calmodulin's effect remains to be determined; it may depend in part on the relative amounts of Ca2+/calmodulin and Rab guanine nucleotide dissociation inhibitor that are available for binding to Rab3A in individual, activated nerve termini.
The opening of voltage-gated Ca2+ channels in active
zones of nerve terminals causes a brief, localized influx of
Ca2+ followed by the secretion of neurotransmitters (1-3).
The molecular basis of this effect is still unclear, but increased
concentrations of intracellular Ca2+ may act at several
levels to trigger fast fusion of pre-docked synaptic vesicles with the
synaptic plasma membrane, promote endocytosis of the vesicle membranes
and subsequent vesicle reformation, and mobilize additional vesicles to
release sites (1, 4). Proteins that bind Ca2+ probably
mediate many of these actions, and a number of candidate proteins have
been identified. They include rabphilin (5, 6); the -,
-II-, and
isoforms of protein kinase C (7); and dynamin (8), all of which
show Ca2+-dependent binding to acidic
phosphoglycerides. They also include calmodulin
(CaM)1 (9), synaptotagmin
(10-13), and calcineurin (14), which bind Ca2+ directly.
CaM that contains bound Ca2+ (Ca2+/CaM) can
activate CaM kinase II and calcineurin (9), and both enzymes may play
important regulatory roles (15-18). Furthermore, Ca2+/CaM
appears to be required for secretion in adrenal chromaffin cells
(19-21). However, how Ca2+- and
Ca2+/CaM-dependent reactions are integrated to
promote and optimize synaptic responses remains to be determined.
In the present investigation we examined the effects of Ca2+ and CaM on the behavior of Rab3A, a low molecular mass, di-geranylgeranylated, guanine nucleotide-binding protein that is attached to neurotransmitter-containing synaptic vesicles (22, 23). Previous investigators had shown that depolarization of rat brain synaptosomes causes a reduction in the contents of both Rab3A and a related guanine nucleotide-binding protein, Rab3C, in crude synaptic vesicles (Refs. 24 and 25 but see Ref. 26 for a conflicting view). Furthermore, action of a Rab guanine nucleotide dissociation inhibitor protein (Rab GDI) had been implicated because of its known ability to form a complex with Rab3A and cause it to dissociate from synaptic membranes in cell-free experiments (27-29). While exploring the possibility that increased concentrations of Ca2+ might affect the Rab3A dissociation process, we discovered that Ca2+/CaM also can cause Rab3A to dissociate from synaptic membranes. Studies of the mechanism of this effect and its relation to that of Rab GDI are described below.2
CaM was obtained from Calbiochem and freshly
dissolved in 50 mM HEPES, pH 7.4, for each experiment.
CaCl2, Suprapur grade, was from EM Science. BS3
was obtained from Pierce. Rab3A peptides
Lys62-Arg85,
Ala2-Asn18, and
Glu177-Asp195 (Table I) and the Rab GDI
peptide, Gly21-Ser45
(GIMSVNGKKVLHMDRNPYYGGESSS), were synthesized by the University of
Washington Biopolymer Facility. The CaM kinase II peptide
Leu290-Ala309 (Table I) was from LC
Laboratories. Stock solutions of peptides were prepared in
Me2SO and then added to incubation mixtures at final
Me2SO concentrations of <5%. GDP, GTPS, and
unprenylated Rab3A were from Calbiochem. Rab GDI was purified from
bovine brain as described (29), except that all buffers used after the
ammonium sulfate precipitation step contained 10% glycerol, 0.25 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml each of
aprotinin and leupeptin, and 1 µg/ml pepstatin A. All other purchased
chemicals were reagent grade from Sigma, and all procedures were
performed at 4 °C unless otherwise indicated.
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Two different methods were used to prepare synaptosomes from cerebral cortex of nonhuman primates (Macaca nemestrina), obtained from the tissue distribution program of the Regional Primate Research Center at the University of Washington. In method 1, 50 g of cortex was sliced in ice-cold buffer A (320 mM sucrose; 2 mM EGTA; 0.1 mM phenylmethylsulfonyl fluoride; 1 µg/ml each of leupeptin, aprotinin, and pepstatin A; and 2 µg/ml p-aminobenzamidine). Then a gray matter-enriched portion, diluted with 10 volumes of buffer A, was homogenized successively with an Oster blender and a Potter-Elvehjem homogenizer. Finally, a crude synaptosomal pellet (9000 × gav) (where gav is g force at tube half-length) was prepared and purified on a Ficoll gradient using a modification of the methods of Fisher von Mollard et al. (24) and Barrie et al. (31). In method 2, synaptosomes to be used primarily for depolarization studies were prepared from 15 g of prefrontal and temporal cortex. The tissue was minced with a razor blade in ice-cold buffer A (with 5 mM HEPES, pH 7.4, 0.5 mM EGTA), rinsed twice with buffer, and homogenized in a total volume of 100 ml using a loose-fitting Potter-Elvehjem homogenizer (0.25 mm clearance; Ref. 32). Then a synaptosome-enriched fraction was prepared as described (24).
Preparation of Synaptosomal Lysates and Lysate SubfractionsFor most studies synaptosomal lysates were prepared
and successively centrifuged to yield a synaptosomal plasma
membrane-enriched fraction (28,000 × gav
pellet), a crude synaptic vesicle fraction (176,000 × gav pellet), and a corresponding high speed
supernatant as described (33) except that EGTA (2 mM) was
added to all buffers. The crude synaptic vesicle fraction, which
contained Rab3A-enriched membranes (REM), was resuspended in 2-3 ml of
buffer containing 300 mM glycine, 0.1 mM EGTA,
1 µg/ml aprotinin, 1 µg/ml leupeptin, and 5 mM HEPES,
pH 7.4. Then aliquots were flash-frozen and stored at 70 °C. Both
freshly prepared and freeze-thawed REM gave similar results. The
176,000 × gav supernatant was concentrated
10-fold with a PM10 membrane (Amicon), and aliquots were prepared and frozen as described above.
REM (15 µg of protein) were incubated for 30 min at 30 °C with various additives in 50 µl of buffer B (50 mM HEPES, pH 7.4, containing 0.1 mM CaCl2, 0.5 µM MgCl2, 1 mM DTT, 2 µg/ml aprotinin, and 2 µg/ml leupeptin). For specific incubation conditions, refer to the figure legends. The reaction mixtures were centrifuged for 30 min at 100,000 × gav in a Beckman TLA 45 rotor. The pellets were resuspended in 50 µl of buffer B (but without Ca2+) with brief sonication. For quantitation of Rab3A, aliquots of each pellet and supernatant fraction (5 and 7 µl, respectively) were examined by SDS-PAGE and Western blot analysis with the use of anti-Rab3A antibodies (see below). Corresponding samples of untreated membranes (0.15, 0.3, 0.6, 1.2, 1.8, and 2.4 µg of protein/sample) were used as standards. For quantitation of Rab1A and Rab5A, 10- and 12-µl aliquots of pellet and supernatant fractions were used, respectively; the samples of untreated membranes used as standards were 0.5, 1.0, 1.5, 2.5, 3.0, 3.5, and 4.0 µg of protein/sample. The Western blots were scanned with a Bio-Rad model GS-670 imaging densitometer, and the absorbance values obtained for the Rab protein bands were converted to µg of equivalence of Rab proteins on the basis of the standard curves that were generated. The amount of each Rab protein dissociated from the REM was reported as a percentage of the total Rab protein recovered in the supernatant and membrane fractions. Unless indicated otherwise, data points shown in the figures represent single or duplicate measurements. In general, results of replicate measurements varied by no more than 2%. For Rab3A, the number of experiments represented by each figure is reported in the figure texts. For Rab1A and Rab5A, two dissociation experiments were performed and gave similar results.
AntibodiesRabbit polyclonal antibodies were raised against peptides corresponding to amino acids Ala2-Asn18 of the cDNA-predicted sequence of bovine brain Rab3A (34) as described previously (35) except that a C-terminal cysteine was added when the peptide was synthesized to facilitate conjugation to keyhole limpet hemocyanin (Pierce). A 1/500 dilution of the affinity purified antibody was used for most anti-Rab3A Western analyses. However, for Western analyses in cross-linking experiments with Rab3A peptide Ala2-Asn18 (see below), a 1/500 dilution of a Rab3A-specific antibody that had been raised against Rab3A peptide Lys202-Asp217 (Santa Cruz Biotechnology) was used. The anti-Rab1A and anti-Rab5A antibodies (Santa Cruz Biotechnology) were each used at a 1/125 dilution. Antibodies were also prepared against peptides corresponding to amino acids Gly21-Ser45 of the cDNA-predicted sequence of bovine brain Rab GDI (36). A 1/200 dilution of this affinity purified antibody was used for all anti-Rab GDI Western analyses.
Antibody specificity was determined by two-dimensional gel electrophoresis (37, 38) followed by Western analysis (35). In the case of Rab3A, 20 µg of of purified synaptic vesicle protein (33) were analyzed, and only one immunoreactive band, representing >95% of the antibody-reactive material, was found. In the case of Rab GDI, 100 µg of nonhuman primate brain cytosol (9200 × gav × 20 min supernatant as described (33)) were similarly analyzed and found to contain two adjacent proteins (representing >90% of the antibody-reactive material) that most likely represented two charged isoforms of Rab GDI (39).
Sucrose Density GradientsThe apparent molecular mass of the Rab3A·CaM complex was determined by sucrose gradient centrifugation (27) modified as follows. Supernatants from Rab3A dissociation experiments with 90 µg of REM, 100 µM CaCl2, and 50 µM CaM in 250 µl of buffer B were overlaid onto a 5-ml, 5-25% continuous sucrose gradient containing 5 mM MgCl2, 1 mM DTT, 0.1 mM CaCl2, 50 mM HEPES, pH 7.4, and protease inhibitors as described for buffer B. After centrifugation for 7 h at 173,000 × gav in a Beckman SW50.1 rotor, fractions were analyzed for Rab3A with an immuno-dot blot assay. The high speed supernatant fraction from synaptosomal lysates (1 mg/250 µl) was similarly analyzed.
Cross-linking StudiesFor cross-linking experiments, REM (15-25 µg of protein) were washed twice by centrifugation in 50 mM HEPES, pH 7.4, containing 100 mM NaCl, 1 mM MgCl2, 1 mM DTT, and protease inhibitors as described for buffer B. The washed membranes were mixed with Ca2+/CaM in 25 µl of buffer B and incubated for 30 min at 30 °C as described in the figure legends. The incubation mixtures were centrifuged for 30 min at 100,000 × gav; the supernatants were treated for 30 min at 30 °C with freshly prepared 1 mM BS3 (40), and the reactions were quenched with Tris buffer and analyzed as described above (41). A similar BS3 treatment procedure was used in all other cross-linking experiments.
Radiolabeling of CaM and Ca2+/125I-CaM-binding ExperimentsCaM (225 µg) in 50 µl of 50 mM HEPES buffer, pH 7.6, was modified by reaction with 0.5 mCi of 125I-labeled Bolton-Hunter reagent (2200 Ci/mmol; NEN Life Science Products) according to the manufacturer's directions. The 125I-labeled CaM was then separated from unreacted reagent with the use of a Bio-Gel P-6 DG (Bio-Rad) spin column as described (42). After centrifugation, a mixture of 50 µl of 125I-labeled CaM (10 nmol) and 50 µl of REM (450 µg) was incubated for 45 min at 37 °C in the presence of 100 µM CaCl2 and 0.1 mM GDP. After incubation the mixture was centrifuged for 30 min at 100,000 × gav, half of the supernatant was treated with BS3, and half was reserved as control. An aliquot of each half (50 µl) was then immunoprecipitated with 50 µl of anti-Rab3A IgG (1 µg) for 24 h at 0 °C. After immunoprecipitation, 40 µl of immobilized protein A on Trisacryl beads (Pierce; 50% slurry), which had been pretreated with bovine serum albumin, were added, and the sample was mixed for 20 h at 4 °C in a tube rotator. The protein A beads were then washed four times by a procedure that involved suspension in 50 mM HEPES, pH 7.4, containing 100 mM NaCl, 0.1 mM CaCl2, 0.1% Tween 20, 0.1 mM DTT, and 1 µg/ml each of leupeptin and aprotinin, followed by centrifugation for 4 min at 350 × gav. The washed beads were boiled for 4 min in 30 µl of 2% SDS and prepared for SDS-PAGE analysis (described below).
Preparation of Rab3A-depleted Synaptic Membranes and Transfer of Rab3A to These MembranesTo generate Rab3A-depleted membranes, REM (30 µg) were incubated for 30 min at 30 °C with 1.6 µM Rab GDI in 50 µl of buffer B (but without Ca2+), pelleted by centrifugation for 30 min at 100,000 × gav, and suspended in 5 µl of buffer B (but without Ca2+). To study the transfer of Rab3A to these membranes, they were mixed with medium containing Rab3A·Ca2+/CaM complex (prepared by incubating REM with buffer containing 75 µM CaM and 100 µM CaCl2) and incubated for 30 min at 30 °C in the presence or absence of one of the peptides listed in Table I. Then the incubation mixtures were subfractionated by centrifugation and analyzed as described above.
Depolarization AssaysThe ability of synaptosomes to
secrete glutamate was measured after KCl-induced depolarization (24) or
treatment with 4-aminopyridine + 4-phorbol dibutyrate (31). A
modification of the method previously described (24) was used. Briefly,
control and depolarized samples were incubated for 10 min at 37 °C
and then placed in ice water for 2 min and centrifuged for 2 min at
13,000 × gav. The amount of NADPH that had
been produced was determined by measuring the absorbance of the
supernatant at 360 nm on a Beckman DU 640 spectrophotometer using 390 nm as the reference wavelength.
SDS-PAGE was performed as described by Laemmli (43) but with 14% gels. In some cases, EGTA was added to the samples just before they were boiled. Proteins were transferred from SDS-PAGE gels to Immobilon P membranes for 30 min at 80 V for Western analysis of Rab3A or transferred for 60 min to identify cross-linked products of higher molecular mass. Immunoblots were performed as described (35). Protein concentrations were determined with the Bradford method (44) (Bio-Rad) or, for SDS-containing samples, the micro-BCA method (Pierce). Free calcium ion concentrations were varied in the presence of 1 mM EGTA, on the basis of established binding constants (45).
The initial aim of this investigation was to determine
whether Ca2+ and CaM influence the dissociation of Rab3A
from synaptic membranes. To examine this possibility, we isolated
synaptosomes from samples of macaque cerebral cortex, lysed the
synaptosomes in hypotonic medium, and prepared Rab3A-enriched membranes
(REM) from the lysates by ultracentrifugation. Then we suspended the
REM in medium containing Ca2+ and/or various other
additives, incubated the mixtures for 30 min at 30 °C, separated the
membranes from the medium by centrifugation, and separately measured
the amounts of Rab3A recovered in the membrane and supernatant
fractions. The results of these experiments demonstrated that medium
containing both Ca2+ and CaM, i.e. a
Ca2+/CaM complex, caused Rab3A to dissociate from the
membranes but that medium containing either 100 µM
Ca2+ or 60 µM CaM alone did not (Fig.
1). The dissociation of Rab3A occurred in
the absence of added ATP, and half-maximal effects were observed when
the concentrations of Ca2+ and CaM were about 0.5 and 20 µM, respectively. Maximal dissociation of Rab3A
(approximately 65%) was obtained when the concentrations of
Ca2+ and CaM were about 10 and 65 µM,
respectively (data not shown).
Mechanism of the Effects of Ca2+/CaM
To examine
the mechanism of the Rab3A-dissociating effect of Ca2+/CaM,
we first incubated REM in the presence of medium that contained Ca2+/CaM and then recovered the medium and subfractionated
it by sucrose gradient ultracentrifugation. Upon measuring the content
of Rab3A in the subfractions, we detected a peak of material that had
an apparent molecular mass of about 40 kDa (Fig.
2A). This peak could be
distinguished easily from the peak of Rab3A-containing material detected in sucrose gradient ultracentrifugation experiments with a
high speed supernatant fraction from a synaptosomal lysate (Fig. 2A). The peak from the lysate supernatant had a considerably
larger apparent molecular mass and probably corresponded to a complex of Rab3A and Rab GDI (28).
In subsequent studies we incubated REM with Ca2+/CaM, recovered the medium, and added the cross-linking agent, BS3, to it. Then we analyzed the cross-linked material by SDS-PAGE and Western blotting with an antibody to Rab3A. In agreement with the sucrose gradient experiments, the results revealed the presence of Rab3A-containing material that had an apparent molecular mass of 43 kDa (Fig. 2B, lane 2). The combined results of these experiments suggested that Ca2+/CaM could form a 1:1 molar complex with Rab3A.
To obtain additional evidence concerning this possibility, we incubated REM with Ca2+/CaM that contained 125I-labeled CaM, recovered the medium, and added BS3 to it. Then we immunoprecipitated Rab3A-containing material, analyzed it by SDS-PAGE, and identified 125I-CaM-containing bands by autoradiography (Fig. 2C). The results revealed 125I-CaM-containing material that had an apparent molecular mass of 43 kDa and therefore provided direct evidence for the presence of a 1:1 molar complex of Rab3A and CaM.
Ca2+/CaM is known to form complexes with many different
proteins, and generally similar mechanisms appear to be involved. When Ca2+ binds to CaM, it induces a conformational change in
CaM that exposes binding sites for both hydrophobic and basic amino
acids (46-48). To obtain further evidence that the 1:1 complex of
Rab3A and CaM involves Ca2+/CaM, we incubated REM with
Ca2+ and CaM, recovered the medium, and incubated aliquots
of it in the presence of different concentrations of EGTA. Then we
treated the incubation mixtures with BS3 and analyzed the
cross-linked products by SDS-PAGE. The results revealed that incubation
of the medium in the presence of 1 mM EGTA decreased the
electrophoretic mobility of the Rab3A·CaM complex (apparent molecular
mass 43 50 kDa) (Fig. 3, compare
lanes 2 and 3), whereas incubation with 10 mM EGTA greatly reduced the amount of the complex that
could be detected (Fig. 3, compare lanes 2 and
4). The binding of Ca2+ to CaM is known to
increase its electrophoretic mobility (49; see also Fig.
4, lanes 8 and 9 and Fig. 7B, lanes 1 and 2). Therefore, both
results provided evidence for the formation of a
Rab3A·Ca2+/CaM complex.
To investigate the role of hydrophobic interactions in forming the Rab3A·Ca2+/CaM complex, we used two different approaches. First, we incubated REM with Ca2+/CaM, recovered the medium, and incubated aliquots of it in the presence or absence of the neutral detergent, Triton X-100. Then we added BS3 to the incubation mixtures and analyzed the cross-linked products by SDS-PAGE. Treatment with 0.1% Triton X-100 completely disrupted the complex (data not shown). In the second approach we incubated unmodified, recombinant Rab3A with Ca2+/CaM, then added BS3, and analyzed the cross-linked material. The fact that no complex of recombinant Rab3A with Ca2+/CaM could be detected (Fig. 4, compare lanes 5 and 8) provided evidence that the lipidated C terminus of Rab3A is required for binding to Ca2+/CaM.
Among the many proteins that form complexes with Ca2+/CaM is CaM kinase II (50). Furthermore, a basic- and hydrophobic amino acid-containing binding site for Ca2+/CaM on this kinase, Leu290-Ala309 (Table I), has been identified (50, 51). Rab3A also contains a sequence that is enriched in basic and hydrophobic amino acids, Lys62-Arg85. To investigate the possibility that the Rab3A Lys62-Arg85 sequence might include a binding site for Ca2+/CaM, we synthesized a peptide that corresponded to it (Table I) and then compared the effects of this peptide with those of other synthetic peptides in the following experiments.
First, we incubated REM with Ca2+/CaM in the presence of
each peptide and then measured the amount of Rab3A that dissociated to
the medium. The results revealed that a 100 µM
concentration of the Rab3A Lys62-Arg85 peptide
or of a peptide corresponding to the CaM kinase II
Leu290-Ala309 sequence blocked the
Rab3A-dissociating effect of Ca2+/CaM (Fig.
5A); half-maximal values were
observed at concentrations of 42 and 18 µM, respectively.
In contrast, 100 µM concentrations of peptides that
respectively corresponded to regions near the Rab3A N terminus or
unmodified C terminus (Rab3A Ala2-Asn18 or
Glu177-Asp195; Table I) had no effect.
Second, after incubating REM with Ca2+/CaM, we recovered
the medium and incubated aliquots of it with the Rab3A
Lys62-Arg85 peptide, the CaM kinase II
Leu290-Ala309 peptide, or the Rab3A
Ala2-Asn18 peptide, then added BS3
to each incubation mixture, and analyzed the content of cross-linked Rab3A·Ca2+/CaM complex. Both the Rab3A
Lys62-Arg85 peptide and the CaM kinase II
Leu290-Ala309 peptide reduced the amount of
complex that could be detected, but the Rab3A
Ala2-Asn18 peptide had no effect (Fig.
6A).
Third, we incubated the Rab3A Lys62-Arg85, Ala2-Asn18-, or Glu177-Asp195 peptides with CaM in the presence of Ca2+ or EGTA, then added BS3, and analyzed the products by SDS-PAGE. The results showed that the Lys62-Arg85 peptide could form a Ca2+-dependent complex with CaM but the other Rab3A peptides could not (Fig. 7). Taken together, the results of these experiments provided strong evidence that Ca2+/CaM promotes the dissociation of Rab3A from synaptic membranes by binding to amino acids within the Rab3A Lys62-Arg85 sequence.
Transfer of Rab3A from Rab3A·Ca2+/CaM to MembranesHaving shown that the Rab3A
Lys62-Arg85 and CaM kinase II
Leu290-Ala309 peptides could separately disrupt
Rab3A·Ca2+/CaM complexes, we examined the possibility
that disruption of the complexes might promote the transfer of Rab3A to
membranes. We did this by incubating REM with Ca2+/CaM to
generate a Rab3A·Ca2+/CaM complex, recovering the medium,
and incubating aliquots of it with one or the other of the peptides in
the presence of Rab3A-depleted synaptic membranes. After the
incubations we separated the membranes from the medium by
centrifugation and measured the contents of Rab3A in the pellet and
supernatant fractions. The results demonstrated that each peptide could
cause Rab3A to translocate from the medium to the membranes, whereas a
control peptide had no effect (Fig. 8).
Relation between the Effects of Ca2+/CaM and Those of Rab GDI
Rab GDI can form a 1:1 complex with digeranylgeranylated
Rab3A and cause it to dissociate from synaptic membranes (22, 24). To
examine the relation between this effect and that of
Ca2+/CaM, we first sought to determine whether the two
proteins bind to similar sites on Rab3A. In one set of experiments we
incubated REM with Ca2+/CaM to generate a soluble
Rab3A·Ca2+/CaM complex or used the high speed supernatant
from a synaptosomal lysate as a source of Rab3A-Rab GDI. Then we
incubated the Rab3A·Ca2+/CaM complex for 30 min at
30 °C with Rab GDI or incubated the synaptosomal lysate supernatant
under similar conditions with Ca2+/CaM. Following the
incubations, we treated the incubation mixtures with BS3
and analyzed the Rab3A-containing material (Fig.
9). The results of these experiments
showed that Ca2+/CaM and Rab GDI could compete with each
other for binding to Rab3A. In a second set of experiments we incubated
REM with Rab GDI in the presence of the Rab3A
Lys62-Arg85 peptide, the CaM kinase II
Leu290-Ala309 peptide, the Rab3A
Ala2-Asn18 peptide, or the Rab3A
Glu177-Asp195 peptide. Then we recovered the
medium and measured the content of Rab3A in the supernatant by
immunoblotting. The Rab3A Lys62-Arg85 and CaM
kinase II Leu290-Ala309 peptides separately
blocked the Rab GDI-dependent dissociation of Rab3A from
REM (Fig. 5B); half-maximal values were observed at
concentrations of 46 and 41 µM, respectively (not shown).
In contrast, the Rab3A Ala2-Asn18 and
Glu177-Asp195 peptides were inactive (Fig.
5B). In a third set of experiments we incubated REM with Rab
GDI, isolated the medium, and incubated it for 30 min at 30 °C in
the presence or absence of the Rab3A Lys62-Arg85 peptide or the Rab3A
Ala2-Asn18 peptide. Then we added
BS3 to the incubation mixtures and analyzed the
Rab3A-containing cross-linked material (Fig. 6B). The Rab3A
Lys62-Arg85 peptide reduced the amount of
cross-linked Rab3A·GDI complex recovered but the Rab3A
Ala2-Asn18 peptide did not. The similarity
between these results and those obtained in the corresponding
experiments with Ca2+/CaM (compare Fig. 5, A
with B, and Fig. 6, A with B) provided strong evidence that Rab GDI and Ca2+/CaM interact with
similar binding sites within the Rab3A
Lys62-Arg85 sequence.
The Rab GDI-induced dissociation of Rab3A from membranes is known to be
under the control of guanine nucleotides (27). To determine whether
guanine nucleotides also control the Ca2+/CaM-induced
dissociation of Rab3A, we preincubated REM for 1 h at 37 °C in
the absence of added guanine nucleotides, treated the REM with
Ca2+/CaM or Rab GDI, then measured the amount of Rab3A that
dissociated from the REM to the medium (Fig.
10). Only a small response to Ca2+/CaM or Rab GDI could be detected (compare preincubated
samples with nonpreincubated controls). In contrast, Rab3A dissociated from REM that had been preincubated in the presence of GDP or GTPS
before being treated with Ca2+/CaM or Rab GDI, and GDP was
more effective than GTP
S. Notice that Rab GDI had a more stringent
requirement for GDP than Ca2+/CaM did.
The mechanism of these guanine nucleotide-dependent effects
remains to be determined. However, the conformation of Ras-GDP is known
to differ from that of Ras-GTP (52), and guanine nucleotides are
presumed to have similar effects on Rab proteins and other Ras-related
proteins. Furthermore, a series of experiments with BS3-treated REM provided direct evidence that guanine
nucleotides alter the conformation of Rab3A (Fig.
11). First, analyses of untreated control REM by SDS-PAGE and Western blotting revealed a single major
band of Rab3A that had an apparent molecular mass of about 28 kDa, but
analyses of BS3-treated control REM revealed two major,
Rab3A-containing bands that had respective apparent molecular masses of
about 28 and 23 kDa (Fig. 11, compare lanes 1 and
3). Second, REM that had been preincubated for 1 h at
37 °C in the absence of guanine nucleotides before being treated
with BS3 contained an increased amount of the 28-kDa band
but a reduced amount of the 23-kDa band (Fig. 11, compare lanes
3 and 4). Third, REM that had been preincubated in the
presence of GDP or GTPS before being treated with BS3
showed a distribution of 23- and 28-kDa bands which resembled that in
BS3-treated control membranes (Fig. 11, compare lanes
3, 5, and 6). These results suggest that treatment with
BS3 can stabilize a guanine
nucleotide-dependent conformation of Rab3A that has an
increased electrophoretic mobility. Thus, it seems reasonable to
postulate that the Rab3A of control REM or REM that have been incubated
in the presence of added GDP or GTP
S contains bound guanine
nucleotides but that these nucleotides dissociate from Rab3A when REM
are incubated in the absence of added GDP or GTP
S or when Rab3A is
analyzed by SDS-PAGE (see also Ref. 53). Furthermore, it seems likely
that Rab3A that contains bound GDP or GTP has a more compact
conformation than guanine nucleotide-free Rab3A does and that this
compact conformation can be stabilized by
BS3-dependent, intramolecular cross-linking
reactions.
The interaction of Ca2+/CaM with Rab3A resembles that of Rab GDI with Rab3A in several respects. Both proteins form soluble 1:1 complexes with Rab3A and cause it to dissociate from synaptic membranes. Formation of each of the complexes requires both the lipidated C terminus of Rab3A and the presence of guanine nucleotides. Both Ca2+/CaM and Rab GDI evidently bind to sites within the Rab3A Lys62-Arg85 sequence.
The interactions of Ca2+/CaM and Rab GDI with Rab3A also differ in several respects. Importantly, the interaction of Ca2+/CaM with Rab3A depends on Ca2+. In addition, half-maximal effects of Ca2+/CaM on the dissociation of Rab3A from REM or the dissociation of Rab3A from Rab3A-Rab GDI occur at concentrations of Ca2+/CaM (~20 µM) that are much higher than the concentrations of Rab GDI required to dissociate Rab3A from REM or Rab3A-Ca2+/CaM (<0.5 µM; data not shown). The Ca2+/CaM-dependent dissociation of Rab3A from REM is less extensive than the Rab GDI-dependent dissociation of Rab3A. And the Rab3A-dissociating effect of Ca2+/CaM has a less stringent requirement for GDP than does that of Rab GDI.
The precise mechanism of the Rab3A-dissociating effect of Ca2+/CaM remains to be determined, but it is of interest that the Rab3A Lys62-Arg85 peptide contains a cluster of basic amino acids toward its N terminus, while its hydrophobic amino acids are more evenly distributed (Table I). Furthermore, a helical wheel projection of the peptide's sequence suggested that the clustered, basic amino acids may be located on one side of an amphipathic helix (not shown). The CaM kinase II Leu290-Ala309 peptide has similar characteristics (Table I and Ref. 47); and a recent crystallographic study of its interaction with Ca2+/CaM has shown that the latter can "wrap around" the peptide to make close contact with its basic and hydrophobic amino acids (48). Ca2+/CaM may conceivably interact with the Rab3A peptide in the same way. However, Ca2+/CaM may interact differently with native Rab3A because its binding to the protein appears to require the presence of the modified C terminus. Molecular modeling studies of Rab3A might provide some insight into this issue.
Modeling studies of the GDP-bound form of human Rab5A have suggested that the Rab5A Gln60-His83 sequence, QTVCLDDTTVKFEIWDTAGQEGYH, which is homologous to the Rab3A Lys62-Arg85 sequence, may be partially exposed on the protein's surface (54). The cluster of hydrophilic amino acids toward the N terminus of the Rab5A Gln60-His83 sequence occupies an exposed position adjacent to loop 3 of the Rab5A molecule, but the hydrophobic amino acids of the sequence are generally much less exposed and interact with other amino acids in the protein. If modeling studies of Rab3A suggest that the amino acids of the Lys62-Arg85 sequence (Table I) occupy similar positions related to the protein's surface, the possibility that the clustered basic amino acids in the sequence may be available for binding to Ca2+/CaM would be worth examining.
Experimental tests of the role of individual basic amino acids in the
sequence might be done by site-directed mutagenesis. A similar approach
has been used to examine the regulatory role of amino acids in the
Rab3A Asp77-Glu82 sequence, which corresponds
to the G2 guanine nucleotide-binding region. A Gln81 Leu mutation altered the koff (GDP) and
koff (GTP) of Rab3A and greatly reduced the
ability of Rab3A to respond to Rab3A guanine nucleotide releasing
factor (55). In addition, a recent study of a Rab6-v-Ha-Ras chimera
showed that the Rab6 Arg60-Trp67 sequence
(-RTVRLQLW-), which is homologous to the Rab3A
Lys69-Trp76 sequence (-KRIKLQIW-), includes
binding sites for Rab GDI and Rab geranylgeranyl transferase (56).
It might also be of interest to examine the effects of site-directed mutagenesis within the corresponding regions of several Rab proteins. Thus, the first portion of the Rab3A Lys62-Arg85 sequence contains five clustered basic amino acids, but the first portions of the corresponding sequences of Rab1A and Rab5A contain one and three basic amino acids, respectively. Furthermore, these differences may correlate with differences in the dissociating effects of Ca2+/CaM on the three Rab proteins. In a representative experiment, we incubated REM (from synaptosomes prepared using method 2; see "Experimental Procedures") for 30 min at 30 °C in the presence of 1 mM GDP and 80 µM Ca2+/CaM. Measurements by quantitative densitometry revealed that this caused the dissociation of 55% of the membrane-bound Rab3A but only 10% of the Rab1A and 20% of the Rab5A (data not shown). Therefore, mutation experiments designed to alter the number and/or distribution of basic amino acids within the Lys62-Arg85-like regions of these proteins might be informative.
Interestingly, the GTPase Rad, which was recently shown to bind Ca2+/CaM by a GDP-dependent mechanism, contains an unprenylated C-terminal sequence, Lys279-Lys308, that is enriched in basic amino acids (57). Moreover, both selective truncation experiments involving this sequence and experiments with synthetic peptides have provided evidence that this sequence contains the binding site for Ca2+/CaM. The functional significance of the binding of Ca2+/CaM to Rad has yet to be determined.
The myristoylated, alanine-rich, protein kinase C substrate, MARCKS, also can form a complex with Ca2+/CaM (58, 59). MARCKS contains a region, enriched in basic amino acids, that promotes its binding to vesicles containing acidic phosphoglycerides. In vitro experiments have shown that MARCKS dissociates from such vesicles when serine residues in this region are phosphorylated by protein kinase C (60, 61). Alternatively, Ca2+/CaM may cause MARCKS to dissociate from membranes (60). Ca2+/CaM apparently binds to the unmodified region because its affinity for MARCKS is greatly reduced by the same protein kinase C-dependent phosphorylation reactions (58).
How important is the Rab3A-dissociating effect of Ca2+/CaM likely to be during neurotransmission in vivo? The answer to this question may depend on the amounts of Ca2+/CaM and Rab GDI that are available for binding Rab3A in activated nerve terminals. The amount of Ca2+/CaM available for binding is likely to be a complex function not only of the total, local concentration of Ca2+/CaM, but also of the rates and affinities of Ca2+/CaM binding to other nerve-terminal proteins including CaM kinase II (see particularly Ref. 62). The amount of Rab GDI available for binding to Rab3A presumably depends in part on the concentration of unbound Rab GDI in the nerve terminal, i.e. that fraction of the total, soluble Rab GDI that contains no bound Rab proteins. The amounts of available Ca2+/CaM and Rab GDI remain to be determined. However, the concentration of Ca2+ surrounding activated Ca2+ channels in nerve terminals may be as high as 100 µM (63), the content of total CaM in brain may be ~500 mg/kg (~30 µM; Ref. 64), and the concentration of Rab GDI in brain may be ~270 mg/kg (~5 µM; Ref. 65).
It is noteworthy that some regions of the brain contain a membrane-associated protein called neuromodulin, GAP43, or B50 (66-69), that binds CaM in vitro in the absence of Ca2+. Importantly, neuromodulin releases CaM in the presence of high concentrations of Ca2+ or when one of its serine residues, Ser41, is phosphorylated by protein kinase C (70, 71). Furthermore, neuromodulin has been shown to be attached to the inner surface of the plasma membrane in rat frontal cortex nerve terminals (72), has been implicated in the control of neurotransmission (73), and may be phosphorylated by protein kinase C in activated synaptosomes (74). Therefore, it is possible that activation of nerve terminals may be followed by release of bound CaM from neuromodulin and that the released CaM may increase the local amount of CaM available for Ca2+-dependent binding to nerve terminal proteins.
Unpublished experiments in our laboratory have shown that neuromodulin is present in macaque frontal cortex synaptosomes. In addition, these synaptosomes (prepared by method 2, see "Experimental Procedures") show glutamate responses to K+-induced depolarization or treatment with 4-aminopyridine + phorbol ester, comparable to those observed by others for rat and guinea pig synaptosomes (24, 75). Therefore, the macaque synaptosomes might provide a useful model for examining the functional significance of the Rab3A-dissociating effect of Ca2+/CaM. Experiments designed to examine the effects of depolarization on the distribution of Rab3A are in progress.
In summary, we have shown that Ca2+/CaM can cause Rab3A to dissociate from synaptic membranes and studied the mechanism of this effect. In addition, we have identified similarities and differences between the effects of Ca2+/CaM and those of Rab GDI. These results raise a number of questions. For example, how important is the Rab3A-dissociating effect of Ca2+/CaM in vivo? If important in vivo, what is the significance of the differences between this effect and that of Rab GDI? Does the effect of Ca2+/CaM complement that of Rab GDI in some unknown way? Do complementary Rab3A-dissociating effects of Ca2+/CaM and Rab GDI increase the efficiency of synaptic vesicle recycling? These questions may suggest directions for future research.
We thank Ken Applegate for help in analysis of the Rab5A model.