From the Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Received for publication, July 10, 2000, and in revised form, January 4, 2001
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ABSTRACT |
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In previous work (Sankaran, B., Osterhout, J.,
Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem.
273, 7148-7154), we showed that overlapping peptides, N20K
(Asn564-Lys583) and E20K
(Glu574-Lys593), from the catalytic domain of
phospholipase C (PLC) Guanine nucleotide-binding proteins (G
proteins)1 are a large group
of structurally similar proteins consisting of three subunits ( Effector-binding sites on the surface of A screen for dominant-negative yeast G In previous work (10) we showed that overlapping peptides, N20K
(Asn564-Lys583) and E20K
(Glu574-Lys593) from the catalytic domain of
phospholipase C (PLC) Materials--
Peptides were purchased from Biosynthesis
(Lewisville, TX) and had a purity of greater than 90% based on high
pressure liquid chromatography analysis, and identities were confirmed
by mass spectrometry. Biotin-labeled peptide (B-N20K) was purchased
from Alpha Diagnostic International (San Antonio, TX). Succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC) and
horseradish peroxidase-conjugated NeutrAvidin were from Pierce. Trypsin
and 2-aminoethanol (monoethanolamine) were from Sigma. PVDF membrane was from PerkinElmer Life Sciences. Nitrocellulose membrane was from
Schleicher & Schuell.
Construction of Recombinant Baculoviruses and Sf9
Culture--
Each Purification of Phospholipase C Purification of Wild Type
For purification of
For purification of endogenous Sf9 Purification of Chemical Cross-linking--
We used two different cross-linking
protocols in this study. Protocol 1, used for the experiments described
in Figs. 1 and 7, has been described previously (10). Here, Cross-linking to Trypsin Digestion of Phospholipase C Assay--
PLC assays were performed as
described previously (12). Briefly, purified PLC PLC
Previous data suggest that cross-linking of N20K to Recombinant Myristoylated Purification of Cysteine-mutated
We obtained the
Each of the mutants was able to bind to His-tagged
Cross-linking of Biotin-labeled Peptide to Wild Type
To confirm that the biotin modification did not affect the activity of
the peptide, we tested whether the B-N20K would block PLC activation by
We went on to determine whether we could detect the cross-linked B-N20K
cross-linked to Panel of Biotin-labeled Peptide Cross-linked to Wild Type and
Cysteine Mutant Cross-linking of Peptide to Trypsin-digested Wild Type and C Mutant
Studies to determine effector-binding sites on This study clearly defines the Cys25 as a site of peptide
cross-linking. Cysteine 25 is located in the 2 block G
-dependent
activation of PLC
2. The peptides could also be directly
cross-linked to
subunits with a heterobifunctional cross-linker
succinimidyl
4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to G
1 was inhibited by PLC
2 but not by
i1(GDP), indicating that the
peptide-binding site on
1 represents a binding site for
PLC
2 that does not overlap with the
i1-binding site.
Here we identify the site of peptide cross-linking and thereby define a
site for PLC
2 interaction with
subunits. Each of the 14 cysteine residues in
1 were altered to alanine. The
ability of the PLC
2-derived peptide to cross-link to each
mutant was then analyzed to identify the reactive sulfhydryl moiety on the
subunit required for the cross-linking reaction. We find that
C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding
to a region near cysteine 25 of
1 which is located in the amino-terminal coiled-coil region of
1 and
identifies a PLC-binding site distinct from the
subunit interaction site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
) that are central molecules coupling seven-transmembrane domain-spanning receptors to downstream effector molecules. Activation of G proteins begins with a ligand-induced conformational change of the
receptor which catalyzes the release of GDP from the
subunit in
exchange for GTP (1, 2). In the GDP-bound heterotrimeric state,
(GDP)·
, neither
(GDP) nor
can regulate effector activity. Upon receptor-catalyzed G protein activation, the
heterotrimer dissociates into free
(GTP) and free
subunits.
It is well understood that both
(GTP) and
subunits can
interact with a variety of downstream effector molecules including
enzymes and ion channels. GTP is hydrolyzed to GDP, and reassociation
of
(GDP) with
results in deactivation of
-dependent signaling. Despite detailed knowledge of
- and
subunit functions, the mechanism for how
subunits activate its variety of effectors is not entirely understood.
are beginning to be
mapped. The putative competition between
(GDP) and effectors for
forms the premise for recent studies to map effector-binding sites at the
subunit-binding interface on
. The
three-dimensional structure of the G protein heterotrimer reveals that
the
subunit is a
-propeller with seven "blades" and an
amino-terminal
-helix (3, 4). The
subunit binds to a portion of
the top of the
-propeller and along side one of the blades of the
propeller. Two groups have shown that alanine substitution of
-contacting residues on the top surface of the
-propeller
differentially affected the ability of
to regulate various
target molecules (5, 6). One of these groups tested whether the sides
of the
-propeller may be important for effector interactions.
Residues along the outer strand of each of the 7 blades of the
-propeller were altered, and each
mutant was tested for its
ability to stimulate phospholipase C (PLC)
2 and regulate adenylyl
cyclases (7). Mutations in three of the blades eliminated the ability of
to activate PLC
2, whereas these same mutations did not affect regulation of adenylyl cyclase isoforms 1 and 2. Consistent with
these studies, synthetic peptides from discrete blade regions of
were used in competition experiments to define a region located near the interface between
and
as being important for
activation of PLC
2 (8).
mutants that could interfere
with mating factor signaling identified different regions involved in
subunit-effector interactions (9). In particular, one set of
mutations maps to an amino-terminal region involved in a coiled-coil
interaction with
subunits on the opposite side of the
subunit
from the
subunit-binding site. Taken together, these studies show
that the top surface of
, where many
-contact points are located,
is a region critical for effector binding and regulation. However,
effector-binding sites are being mapped to a variety of regions on the
surface of
. Multiple interaction regions are reported even for
the same effector. It has been suggested that distinct sets of contacts
may define the specificity of the interaction of
with various
effectors (i.e. each effector will have a characteristic
footprint along the sides of the molecule).
2, block PLC activation by
and could be
directly cross-linked to
subunits with a heterobifunctional
cross-linker (SMCC). In the study presented here we have identified the
site of cross-linking of N20K to the
subunit thereby defining a
binding site for PLC
2. This approach identified a site distinct
from those mapped by site-directed mutagenesis but consistent with the
region identified in yeast
subunits as being important for
subunit-effector interactions (9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-Cysteine mutants in pALTER (11) were kindly provided
by Dr. Eva J. Neer.
1-cysteine mutant was subcloned into
the baculovirus transfer vector pFASTBAC (Life Technologies, Inc.).
Recombinant baculoviruses were generated using standard manufacturer's
procedures. Sf9 cells were grown at 27 °C in Sf900
medium (Life Technologies, Inc.).
2--
Baculovirus constructs
directing expression of recombinant His6 PLC
2 were used
to infect 800 ml of Sf9 cells at a density of 2.5 × 106 cells/ml. PLC
2 was purified according to published
procedures (12).
1
2
Cysteine-mutated
1
2 and Sf9
Subunits--
For purification of wild type
1
2, baculovirus constructs encoding
1,
2, and His6-tagged
i1 were obtained from Alfred Gilman's laboratory. 800 ml of Sf9 cells at 2.5 × 106 cells/ml were
simultaneously infected with the three constructs, and the
subunits were purified according to published procedures (13) and
modified as in Romoser et al. (12). The protein was concentrated on a 0.3-ml macro-prep ceramic hydroxyapatite (Bio-Rad) column and eluted into
vehicle (20 mM HEPES, pH 8.0, 100 mM NaCl, 1% octyl glucoside, 200 mM
potassium phosphate, pH 8.0).
1
2-cysteine (C)
mutants, 200 ml of Sf9 cells at 2.5 × 106
cells/ml were simultaneously infected with baculovirus constructs encoding each
1-cysteine mutant,
2, and
His6-tagged
i1. Each
-cysteine mutant
was purified according to the above procedures but modified to batch
style with 0.5 ml of nickel-nitrilotriacetic-agarose. Peak fractions
were pooled and concentrated on a 0.3-ml hydroxyapatite column, and
protein was eluted into
vehicle.
, 1 liter of Sf9
cells at 2.5 × 106 cells/ml was simultaneously
infected with baculovirus constructs encoding His6-tagged
i1 and
2. Sf9
subunits were
purified according to the procedures for the purification of wild type
as described above.
Subunits--
Recombinant myristoylated
i1 and
o were purified from
Escherichia coli coexpressing
i1 or
o with N-myristoyltransferase according to
published procedures (14) with some modification. Briefly,
i1 or
o were expressed in 1 liter of
E. coli containing the N-myristoyltransferase
plasmid for 16 h. After preparation of the lysate, the protein was
applied to a 50-ml column of Q Sepharose fast flow (Amersham Pharmacia
Biotech) that had been equilibrated with 50 mM Tris, pH
8.0, 2 mM dithiothreitol, 2 µM GDP. The
column was washed with 150 ml of equilibration buffer followed by
elution of the protein with a 400-ml linear gradient to 250 mM NaCl in equilibration buffer. Fractions containing
subunits were further purified by fast protein liquid chromatography phenyl-Superose chromatography resulting in two peaks of
subunits eluting from the column. The second peak corresponding to myristoylated
subunit was pooled and concentrated; 50 µM GDP was
added, frozen in aliquots in liquid N2, and stored at
80 °C. Functionality of the
subunits was confirmed by the
ability to be ADP-ribosylated by pertussis toxin and the ability to
inhibit
-mediated activation of PLC
2.
subunits at the indicated concentrations were mixed with N20K
(NRSYVISSFTELKAYDLLSK) peptide followed by addition of SMCC to 200 µM from a 2 mM stock solution. The reaction
was allowed to proceed for 5 min. For protocol 2, N20K was reacted with
SMCC for 10 min followed by addition of 500 mM
ethanolamine, pH 7.3, to inactivate the unreacted succinimide moiety.
subunits were then added, and the reaction was allowed to
proceed at room temperature for 1.5 min. For both protocols reactions
were quenched with SDS sample buffer (32 mM Tris, pH 6.8, 5% glycerol, 1% SDS, 350 mM 2-mercaptoethanol, bromphenol blue (final concentrations)), resolved on SDS-PAGE, and transferred overnight onto a PVDF membrane (PerkinElmer Life Sciences). For some
experiments (as indicated) cross-linked species were detected with
horseradish peroxidase (HRP)-conjugated NeutrAvidin (Pierce). In other
experiments cross-linked
subunits were detected with the
carboxyl-terminal anti-
subunit antibody B600 and/or amino-terminal specific
subunit antibody followed by secondary antibody linked to
HRP and development with Amersham Pharmacia Biotech chemiluminescence reagents.
Subunits--
Wild
type or cysteine mutant
1
2 was digested
with trypsin at a
:trypsin ratio of 75:1, for 30 min at 30 °C.
The digestion was stopped by addition of 62.5 µM
phenylmethylsulfonyl fluoride. Digested
(80 nM), 100 µM N20K, and cross-linker were mixed as in cross-linking
protocol 1 and analyzed by immunoblotting with an amino-terminal
specific
subunit antibody.
2 was mixed with
sonicated phospholipid vesicles containing 50 µM
phosphatidylinositol 4,5-bisphosphate, 200 µM phosphatidylethanolamine, and
[3H]phosphatidylinositol 4,5-bisphosphate
(6000-8000 cpm/assay), with or without 100 nM
purified
1
2 or mutants and/or peptides. Reactions were allowed to proceed from 3 to 5 min at 30 °C. Intact lipids and proteins were precipitated with bovine serum albumin and
10% trichloroacetic acid and removed by centrifugation. Supernatant containing soluble [3H]inositol 1,4,5-trisphosphate was
analyzed by liquid scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 Inhibits Peptide Cross-linking to
Subunits but
Subunits Do Not--
We have previously shown that two peptides
derived from PLC
2 (N20K and E20K) are specifically and chemically
cross-linked to both G protein
and
subunits using a
heterobifunctional cross-linking reagent (SMCC) (10). SMCC contains
maleimide- and succinimide-reactive groups, which react with SH- and
NH- moieties, respectively. Since the peptides have no cysteine
residues, cross-linking must be through a primary amine on the peptide
and a sulfhydryl group in the
subunit. The cross-linking reaction is equally effective if the amino terminus of the peptide is acetylated indicating that one of the two lysine residues within the N20K peptide
sequence is involved in the reaction.
subunits
could be blocked by PLC
2 but not
(GDP). To analyze this in
greater detail, the concentrations of
subunits and PLC
2 required to inhibit N20K cross-linking to the
subunit were examined (Fig. 1). Cross-linking of
subunit to
the peptide is indicated by the increase in apparent molecular weight
of the immunoreactive
subunit after incubation with peptide and
SMCC. The uppermost band is
cross-linked to
(this band reacts
with
subunit antibodies (not shown)); the bottom band is
uncross-linked
, and the two bands in between are
cross-linked
to the peptide. There is one prominent peptide cross-linked species and
a second minor peptide cross-linked species. PLC
2 (90 nM) significantly inhibited cross-linking of 10 µM peptide (N20K) to 30 nM
and
completely inhibited the cross-linking at 150 nM PLC
2.
This concentration dependence is consistent with the EC50
of ~50-100 nM for PLC activation by
subunits
(15). On the other hand
i1(GDP) did not inhibit cross-linking of the peptide up to 250 nM. This same
preparation of
subunit was able to block
-mediated activation
of PLC
2 by 95% at a 2:1 ratio of
to
(not shown). These
results indicate that the peptide and
subunit-binding sites on
are distinct and that the peptide-binding site is within the
binding site for PLC
2 on
.
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Fig. 1.
Effects of PLC 2
and
i1(GDP) on peptide
cross-linking to G
1.
1
2 (30 nM) was incubated with
the indicated concentrations of PLC
2 or
i1 in the
presence of 10 µM GDP and 0.1%
C12E10 for 5 min at 23 °C. The N20K peptide
(10 µM) was added followed by 200 µM SMCC
according to cross-linking protocol 1 under "Experimental
Procedures."
subunit and cross-linked species of
subunit were
detected with a carboxyl-terminal specific antibody (B600).
Arrows indicate the positions of molecular mass markers in
kDa. This experiment was performed multiple times with similar
results.
Subunits Do Not Prevent PLC from
Inhibiting Cross-linking of the Peptide--
As discussed,
subunits have been suggested to inhibit
subunit-mediated PLC
2 activation by competing for binding of the PLC
2 to the
subunits. To test whether
(GDP) inhibits the binding of PLC
2 to
subunits, we tested whether excess
o(GDP) (250 nM) altered the ability of PLC
2 to block peptide cross-linking to
(Fig. 2). For
this experiment the cross-linking protocol was modified to eliminate
intermolecular
cross-linking to
and reduce the number of
cross-linked species (see "Experimental Procedures" cross-linking
protocol 2 and the figure legend). Although PLC
2 did not completely
inhibit peptide cross-linking to
subunits in the absence of
o in this experiment (compared with Fig. 1), cross-linking was still significantly inhibited when PLC
2 was added. In the presence of the
subunit, PLC
2 also inhibited cross-linking, but a higher concentration of PLC was required to
observe the inhibition. Similar results were observed with
i(GDP). This suggests that PLC
2 can bind to
subunits at the peptide interaction site even in the presence of
subunits, but
subunits may alter the affinity of PLC for
.
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Fig. 2.
o(GDP) does not
alter the ability of PLC
2 to block peptide
cross-linking. Peptide N20K at 10 µM was
cross-linked to
1
2 (30 nM) at
the indicated concentrations of PLC
2 in the presence or absence of
250 nM
o. Incubations, reactions, and
electrophoresis are as described in cross-linking protocol 2 under
"Experimental Procedures." Arrows indicate the positions
of molecular mass markers in kDa. This experiment was performed five
times with similar results.
1 Subunits--
To
identify the location of peptide cross-linking and thus map a region
for PLC
2 binding that does not coincide with
subunit binding,
we used a sulfhydryl mutagenesis strategy similar to that designed by
Garcia-Higuera et al. (11). Since cross-linking can only
occur between a cysteine residue from
and lysine from the peptide,
we characterized cross-linking of the peptide to a series of
subunit mutants where each cysteine residue had been individually
mutated to alanine.
1-cysteine mutant cDNAs from Dr. Eva
J. Neer and created baculovirus constructs for expression and
purification of each cysteine mutant from Sf9 insect cells. Fig.
3A displays a
Coomassie-stained panel of 11 of the 14 purified
1
2-cysteine mutants. The numbers indicate
the positions of the cysteine residue in the
1 primary
sequence. The Cys166, Cys149, and
Cys317 proteins are not shown but are of similar purity.
1 Cys103, Cys114,
Cys121, Cys148, and Cys271 are
doublets in which the top band of each doublet is Sf9
that copurifies. It will be shown in later experiments that Sf9
does not cross-link to N20K, and therefore, any cross-linking that is
observed must be to the expressed mutant
subunit. The concentration of each
1-cysteine mutant was estimated by immunoblot
analysis because the preparations contained Sf9
, and we
wanted to base the analysis on equal amounts of the expressed
mutants. The concentration of each
1-cysteine mutant
(lower band in the case of doublets) was estimated by comparing the
band intensities to a range of intensities produced by known amounts of
wild type
1 protein (an example of such an estimation
for Cys114 is shown in Fig. 3B).
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Fig. 3.
1
2
cysteine mutants purified on nickel-nitrilotriacetic column.
A, each
1
2 cysteine mutant was
run on SDS-PAGE (12%) followed by staining with Coomassie Brilliant
Blue. wt, wild type; numbers, positions of the mutated
cysteines in the
1 sequence. Arrows indicate
the positions of molecular mass markers in kDa. B,
immunoblot quantitation of Cys114. The position of the
expressed
1 subunit is indicated by the arrow
on the right. The 38-kDa molecular mass marker is indicated
by the arrow on the left.
i and elute with addition of a G protein activator
(AlF
2 in an in vitro
phospholipase C assay (Fig. 4). All of
the mutants that were tested were able to activate PLC
2 between 3- and 5-fold compared with 7-fold for wild type
1
2. Thus, while their efficacy is
slightly diminished (by ~1/2) relative to wild type
1
2, in general these mutants are properly
folded and capable of interacting with
subunits and PLC
2. It is
not clear that why these mutants have a reduced ability to activate PLC
2, but we suspect that it is not a result of the mutations
themselves but rather is a consequence of the difficulties in
quantitating the mutants as well as repeated freezing and thawing of
the samples. Regardless, this cannot be responsible for any differences
observed in the cross-linking experiments since there is no correlation between the activities and the degree of cross-linking.
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Fig. 4.
Ability of the purified cysteine mutants to
activate PLC 2. Each
cysteine mutant (100 nM) was assayed in the presence of 1 ng of PLC
2. CaCl2 was 2.8 mM in the
presence of 3 mM EGTA at pH 7.2, and reactions were for 3 min at 30 °C. Each point represents duplicate determinations, and
the experiment was repeated twice.
1
2 Dimer--
For the survey of the
ability of the peptide to cross-link to the mutants, we modified
cross-linking protocol 2 (see "Experimental Procedures") to use an
amino-terminal biotin-modified form of the N20K peptide (B-N20K) which
we could detect with horseradish peroxidase-conjugated avidin
(HRP-NeutrAvidin from Pierce). With this modified peptide our procedure
for monitoring cross-linking to
subunits allows only the peptide
cross-linked species of
to be detected.
. Consistent with our previous report (10), B-N20K inhibited
-stimulated PLC activity with an IC50 of ~50 µM with 88% inhibition of activity occurring at 100 µM (Fig. 5A). B-N20K was cross-linked to wild type
1
2
and immunoblotted with
subunit antibodies to confirm that this
peptide behaved like unmodified N20K in the cross-linking reaction
(Fig. 5B). A shift to higher molecular weight was observed
as has been seen for unmodified N20K. One major cross-linked species
was observed, and one very minor species appeared with longer reaction
times, confirming that the procedure could be used to monitor specific
reaction of the peptide with the
subunit at a single site.
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Fig. 5.
Effects of biotin modification on
peptide-binding properties. A, PLC 2 (2 ng) activity
was measured in the presence (
) or absence (
) of
1
2 (100 nM).
subunits
and biotin-labeled peptide (concentrations as indicated) were
preincubated together at 1.5 times the final concentrations in the
presence of 0.15% octyl glucoside. The
/peptide preincubation
mixture was then diluted 1.5-fold into a PLC assay such that the final
concentration of
subunits was 100 nM, the peptide
concentrations as indicated, and octyl glucoside was 0.1%.
CaCl2 was 2.8 mM in the presence of 3 mM EGTA at pH 7.2, and reactions were for 5 min at
30 °C. Each point represents duplicate determinations. B,
cross-linking of biotin-labeled peptide to wild type
1
2 dimer. 50 µM biotin-N20K
was reacted with 200 µM cross-linker and
as in
Fig. 2, and the
subunit was detected by Western blotting
(cross-linking protocol 2). The 38-kDa molecular mass marker and the
bands representing
1 subunit cross-linked to peptide are
indicated with arrows. C, biotin-labeled peptide
and SMCC were reacted and quenched as in B. Wild type
1
2 subunits (30 nM) were
preincubated with the indicated concentrations of PLC
2. The
cross-linking reactions were initiated by addition of the
1
2/PLC
2 mixtures as in cross-linking
protocol 2 for 1.5 min, quenched by addition of SDS sample buffer, and
resolved on SDS-PAGE (12% gel). Proteins were then transferred
overnight onto PVDF membrane and detected with HRP-NeutrAvidin. The
38-kDa molecular mass marker and the bands representing
1 subunit cross-linked to biotin-labeled peptide are
indicated with arrows.
using HRP-NeutrAvidin. This method detected only
one cross-linked species at a molecular weight corresponding to the
cross-linked species detected with
subunit-specific antibody (Fig.
5C, indicated by the arrow on the
right). Consistent with our previous report (10), PLC
2
inhibits cross-linking of B-N20K peptide to
1, further
demonstrating that the biotin modification does not affect the binding
properties of the peptide and that the site of peptide cross-linking
represents a binding site for PLC
2 on
subunits.
1
2 Dimers--
Fig.
6A displays the results of a
comprehensive cross-linking screen of B-N20K to wild type
1
2 and all 14 cysteine mutants. Only the
cysteine 25 alteration eliminated peptide cross-linking, whereas the
remaining 13 cysteine mutations did not significantly affect peptide
cross-linking to
1, indicating that the major peptide
cross-linking site is cysteine 25 of
1. Whereas the
other mutations may have had a minor effect on cross-linking, it is difficult to make conclusions as to their significance with this method
since it is not strictly quantitative. Clearly, however, in multiply
repeated experiments the only mutation that consistently resulted in a
clearly significant reduction in cross-linking of the peptide was
Cys25. As discussed, Cys103,
Cys114, Cys121, Cys148, and
Cys271 show double bands with the upper band corresponding
to Sf9
that copurifies with the mutant
subunit (Fig. 3).
We purified Sf9
(see under "Experimental Procedures") to
determine whether the peptide cross-links to this
subunit. We were
unable to detect cross-linking of B-N20K to Sf9
subunit
(Fig. 6B). This indicates that it is not Sf9
contamination of the preparation that is responsible for the
cross-linking observed with any of the cysteine mutants.
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Fig. 6.
Panel of biotin-labeled peptide cross-linked
to wild type and cysteine mutant
1
2
dimers. A, cross-linking to cysteine mutants.
B, cross-linking to Sf9
. Cross-linking
reactions and membrane blotting are as in Fig. 5C. The
38-kDa molecular mass marker is indicated with an arrow. The
numbers indicate the positions of the mutated cysteines in
the
1 sequence.
1
2 Dimers--
To confirm further that
peptide cross-linking to the Cys25 mutant is eliminated, we
performed trypsin cleavage to isolate the amino-terminal fragment of
1 where cysteine 25 is located and assayed for a shift
in the molecular weight of this fragment in the presence or absence of
N20K (not biotinylated) and cross-linker. In the native state only one
of the 32 potential trypsin cleavage sites in
1
(arginine at position 129) is accessible to trypsin. Thus trypsin
cleavage of the native
complex produces two fragments of 14- and
24-kDa (amino and carboxyl termini, respectively) (16). Fig.
7 shows immunoblots from a cross-linking
experiment of trypsin-digested wild type and cysteine mutant
1
2 complexes in the presence and absence
of N20K, detected with an amino-terminal specific anti-
1 antibody. The cross-linking reaction resulted in appearance of an
immunoreactive species at ~17 kDa (top arrow), only in the presence of peptide, for wild type
1
2,
Cys103, and Cys114. The apparent molecular
weight of the cross-linked band corresponds to what would be expected
if one peptide of ~2.5 kDa is covalently attached to the
amino-terminal fragment of 14 kDa. No cross-linking to the
amino-terminal fragment was observed for Cys25 confirming
that Cys25 is a site of N20K cross-linking.
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Fig. 7.
Cross-linking of peptide to trypsin-digested
wild type and cysteine mutant
1
2
dimers. Wild type or C mutant
1
2
were digested with trypsin prior to incubation with N20K peptide
(pep) and cross-linker as described under "Experimental
Procedures." After transfer to nitrocellulose the proteins were
immunoblotted with G
1 amino-terminal (N-term)
specific antibodies. Molecular mass markers are in kDa. The
G
1 amino-terminal fragment (14 kDa) and peptide
cross-linked fragment bands are indicated by arrows. wt,
wild type
1
2; numbers,
positions of the mutated cysteines in the
1 amino acid
sequence.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
suggest that
effectors bind to various regions along the
surface and that
each effector will have its own characteristic set of contact points or
footprints along the sides of the
complex. These contact points
may serve to define the specificity of interactions between
and
its effectors. All of the mapping studies to define these footprints
are based on measurement of
-mediated stimulation of effector
activity to monitor the effects of site-directed mutation within the
subunit at sites predicted to be important for effector binding
(5-7). Here, we take an alternative approach that does not require
prediction of the binding site but rather directly assesses the
location of a specific cross-linking site. Although we used
site-directed mutagenesis, the mutations in the
1
subunit were not intended and did not disrupt the function of the
complex. Our mutations were used only as a tool to monitor
direct protein-protein interactions while retaining the function of the
protein. That the Cys25
mutant stimulated PLC
2
activity and supported PTX-catalyzed
i subunit
ADP-ribosylation similarly to wild type
indicates that the
complex is likely to be properly folded and that the PLC interaction
site is intact.
amino-terminal
coiled-coil region (Fig. 8). The
cross-linker SMCC can link the sulfhydryl groups on cysteine residues
to
-amino groups on lysine that are separated by a maximum of 11.6 Å (Pierce (17)) (for perspective, the distance between
Cys25 to Leu30 of 15 Å is shown in Fig. 8).
This defines a surface area on the
complex with cysteine 25 situated in the center of 450 A2. The
subunit has
approximate dimensions of 40 Å in diameter by 30 Å high. Given these
dimensions, if the
subunit is modeled as a cylinder with a smooth
surface, it would have a surface area of 6248 Å2. Thus we
can restrict the area that can be reached by this cross-linker to less
than 10% of the
subunit surface that includes part of the
amino-terminal coiled coil as well as the sides of propeller blade 5 and possibly blade 4. Our previously published work (10) has
shown that the region of the peptide involved in binding to the
subunits is within the carboxyl-terminal 10 amino acids (ELKAYDLLSK) of
the peptide where the lysines that are involved in cross-linking are
located. Thus the peptide-binding site must be within or very near to
the area that can be reached by the cross-linker.
View larger version (47K):
[in a new window]
Fig. 8.
Location of cysteine 25. This
representation is a "top" view of the structure of
1
2 generated from coordinates deposited
at the Brookhaven protein data bank (4) using Protein Explorer (Eric
Martz, University of Massachusetts). The
subunit is displayed as a
ribbon, and
2 is in yellow. The
blades in the
-propeller are numbered according the
nomenclature of Sprang and co-workers (4).
Cys25 and
Leu40 are shown in a space-filling representation, and the
distance between the Cys25 sulfur atom and an
Leu30 methyl carbon is indicated. The amino-terminal helix
of
involved in a coiled-coil interaction with
is in
red.
Cross-linking of the peptide to cysteine 25 is not consistent with its
binding to three previously identified PLC-binding sites, blades 2, 6, and 7. The closest of these three blades to Cys25 is blade
6. The most proximate amino acid to Cys25 on propeller
blade 6 is 25 Å away. Given a lysine side chain length of 5 Å and the
11.6-Å cross-linker length, the peptide could not be binding to this
region and cross-linking to cysteine 25. The closest amino acid on the
surface of another putative PLC-binding region, blade 2, is 40 Å away
if measured in a direct line from cysteine 25 through the protein. The
distance along the surface of the protein is of course even greater.
Thus the peptide cannot be binding to blade 2 and cross-link to
cysteine 25. A similar argument can be made for blade 7, which is 50 Å away from Cys25. Given the length of the cross-linker the
most reasonable sites for binding are the amino-terminal coiled-coil
region and the sides of blade 5 or 4. We do not conclude that the
results from our study and the results from site-directed mutagenesis
are mutually exclusive. Given the size of PLC 2, it is possible that
the enzyme can interact with multiple regions of the
subunit. Our
study simply identifies an interaction site that was not implicated in
other studies of mammalian
subunits.
Supporting these ideas is the observation that none of the other
cysteine mutations significantly affect cross-linking. In the
cross-linking study by Garcia-Higuera et al. (11) that
utilized this same approach to identify subunit interaction sites,
Cys204 and Cys271 were both found to be
responsible for cross-linking of
o to
1
by bismaleimidohexane (16-Å cross-linker). The mutation at cysteine 25 had no affect on
subunit cross-linking but did prevent cross-linking to
2 in their study. Thus two highly
accessible cysteine residues are present at the
subunit interaction
interface, but apparently they are not the major site of cross-linking
of the N20K peptide. In particular Cys271 is in the loop
region connecting blades 5 and 6. If the peptide were binding to blade
6 one might expect this cysteine to be a cross-linking site. This also
suggests that if the peptide were binding in the
subunit
interaction region it would be detected. Within or near blade 2 of the
-propeller are cysteines 114, 121, 148, 149, and 166, none of which
appear to be critical for cross-linking of the peptide. However, these
amino acids may not be accessible to the cross-linker since they are
mostly buried and not clearly accessible to solvent. The critical data
supporting a binding site other than blades 2 and 6 is that a
cross-linking site at Cys25 has been identified that is not
within range of these regions. The lack of a major effect of mutation
of cysteine residues that are within range of these regions supports
this result and speaks to the specificity of the cross-linking of the peptide.
This general location is in contrast with what has been found in some
mutagenic studies but correlates well with an effector interaction
domain identified in a screen for dominant negative yeast G mutants
(9). Recent studies show that mutation of some of the residues in the
amino terminus of yeast
subunits responsible for the dominant
negative phenotype eliminates binding to Ste20p, the yeast homologue of
mammalian PAK (18). Other mutants in the coiled-coil region of yeast
subunits (Leu65 and Leu49) are unable bind
to the scaffolding protein Ste5p (19). These data strongly suggest that
the amino-terminal coiled-coil region is important for effector binding
in yeast. Evidence for involvement of this region in interactions of
mammalian
subunits with effectors is lacking. One study (20) of
this region with a series of single point mutations found that some of
the mutations had dramatic effects on assembly with
subunits,
whereas those mutants that did assemble had little effect on the
ability of
subunits to activate JNK in a COS cell transfection
assay. A possible explanation for this is that mammalian effectors may
have contacts with other regions of the
subunit, and as such single
point mutations in this region are not sufficient to disrupt effector
interactions in overexpression assays. Our data support the idea that
this region is important for binding of effectors to mammalian
subunits and that this interaction is important for regulation since
binding of a peptide to this region blocks the ability of
to
activate effectors.
The amino-terminal coiled-coil region of the subunit is far from
the
-interacting region located at the top of the
subunit complex making contact with blades 2 and 7 of the propeller. This general location for peptide cross-linking is consistent with our
previous finding that
subunit does not block cross-linking of
peptide to
(10), and our data show that
(GDP) subunits do not
completely prevent PLC from binding to
and thereby preventing cross-linking of peptide to
. This latter finding suggests that perhaps PLC could bind near the amino-terminal region of
even in
the presence of the
subunit. This idea is supported by the results
of another group (21) who propose
(GDP) can bind to
·PLC
2 complex to form
(GDP)·
·PLC
2. Thus, the site we have mapped may represent a region where PLC can remain bound to
even in the presence of
. We can envision a model in which effector
and G protein heterotrimer are precomplexed and poised for immediate
activation. The top surface of the
complex may be critical for
effector activation and may become available to the effector upon
activated receptor-induced activation of
subunits without a
requirement for a potentially rate-limiting dissociation of
subunits or association of PLC
2. Deactivation could involve occlusion of the effector-binding site on
by
(GDP) subunits with
the
·PLC
2 complex without dissociation of PLC
2 from
. Identification of a binding site for PLC that does not overlap with
subunits supports this hypothesis and provides a starting point for understanding molecular interactions that could allow PLC to
remain associated with
subunits in the presence of bound
subunits.
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FOOTNOTES |
---|
* This work was supported by Grant GM 53536 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology
and Physiology, University of Rochester School of Medicine and
Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-0892; Fax: 716-273-2652; E-mail: Alan_Smrcka@URMC.rochester.edu.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M006073200
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ABBREVIATIONS |
---|
The abbreviations used are: G protein, GTP-binding protein; PLC, phospholipase C; SMCC, succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; HRP, horseradish peroxidase.
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