(Received for publication, August 24, 1995; and in revised form, October 23, 1995)
From the
Heterotrimeric guanine nucleotide binding proteins (G proteins)
are made up of ,
, and
subunits, the last two forming a
very tight complex. Stimulation of cell surface receptors promotes
dissociation of
from the
dimer, which, in turn, allows
both components to interact with intracellular enzymes or ion channels
and modulate their activity. At present, little is known about the
conformation of the
dimer or about the areas of
that interact with
. Direct information on the orientation of
protein surfaces can be obtained from analysis of chemically
cross-linked products. Previous work in this laboratory showed that
1,6-bismaleimidohexane, which reacts with cysteine residues,
specifically cross-links
to
and
to
(Yi, F.,
Denker, B. M., and Neer, E. J.(1991) J. Biol. Chem. 266,
3900-3906). To identify the residues in
and
involved
in cross-linking to each other or to
, we have mutated the
cysteines in
,
, and
and analyzed the mutated proteins by in vitro translation in a rabbit reticulocyte lysate. All the mutants were
able to form
dimers that could interact with the
subunit. We found that 1,6-bismaleimidohexane can cross-link
to
but not to
.
The cross-link goes from Cys
in
to
Cys
in
. This cysteine is absent from any
of the other known
isoforms and therefore confers a distinctive
property to
. The
subunit in the
dimer can be cross-linked to an
unidentified protein in the rabbit reticulocyte lysate, generating a
product slightly larger than cross-linked
. The
subunit can also be
cross-linked to
, giving rise to two products on
SDS-polyacrylamide gel electrophoresis, both of which were previously
shown to be formed by cross-linking
to Cys
in
(Thomas, T. C., Schmidt, C. J., and Neer, E. J.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10295-10299).
Mutation of Cys
in
abolished one of
these two products, whereas mutation of Cys
abolished the
other. Because both
-
cross-linked products are formed in
approximately equal amounts, Cys
and Cys
in
are equally accessible from Cys
in
. Our findings begin to define intersubunit surfaces,
and they pose structural constraints upon any model of the
dimer.
Heterotrimeric guanine nucleotide binding proteins (G proteins) ()are key components of the transmembrane signaling
machinery. They link cell surface receptors to intracellular enzymes or
ion channels whose modulated activity ultimately leads to the cellular
response. G proteins are made up of three different polypeptides,
,
, and
: the
subunit binds and hydrolyzes GTP;
the
and
subunits form a very tight complex and can
therefore be considered as a functional monomer. In the inactive state,
GDP is bound to the heterotrimer. However, upon receptor stimulation,
GDP is replaced by GTP, and the
subunit becomes activated and
dissociates from the
dimer. The free subunits, both
and
, can then modulate the activity of target effectors
(reviewed by Neer(1995)).
The structural basis for the subunit
function is now better understood because of the recent solution of the
crystal structure of GTP- and GDP-liganded forms of two
subunits:
transducin and
(Noel et al., 1993;
Lambright et al., 1994; Coleman et al., 1994). In
contrast, little is known about the structure of
. The
subunit is predicted to contain an amino-terminal amphipatic
helix that may be involved in coiled-coil interactions with
(Lupas et al., 1992). The remaining sequence is made up of
seven repeating units of approximately 43 amino acids each (Fong et
al., 1986). These repeating units, also found in many other
proteins, consist of a conserved core, usually starting with the
sequence Gly-His (GH) and ending with Trp-Asp (WD) and are therefore
referred to as WD repeats. Each core is predicted to form a
strand-turn-
strand-turn-
strand structure, followed by a
loop of variable length leading to the next core (Neer et al.,
1994). The
subunit is predicted to be largely
helical
(Lupas et al., 1992). In solution it behaves as an
asymmetrical, extended molecule (Mende et al., 1995). A
cysteine residue near its carboxyl terminus is prenylated, and this
lipid modification has been shown to be essential for membrane
attachment of the
dimer (reviewed by Casey(1994) and
Wedegaertner et al.(1995)). There is, however, little
information about the spatial organization of the
repeats and
about the overall configuration of the dimer. The parts of
that interact with
or with effectors have not been identified,
although genetic analysis in yeast has suggested some important
residues (Leberer et al., 1992; Whiteway et al.,
1994). However, because the surface of the
dimer facing the
subunit will presumably be exposed after dissociation of the
subunits, it is likely that this area would be important for
interaction with effectors. Therefore, to understand how signaling by
is regulated, we need to define the regions of the subunits
that face each other and eventually to locate the contact points.
Analysis of mutated and chimeric molecules can help identify regions
important for protein-protein interactions. However, such an approach
cannot discriminate regions that are near each other from regions that
maintain the conformation necessary for the interaction. More direct
information on the orientation of protein surfaces can be obtained from
analysis of chemically cross-linked products. This laboratory has
previously shown that purified G protein subunits can be cross-linked
using 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linker
reacting with sulfhydryl groups of cysteine residues. BMH specifically
cross-links to
and
to
. Hydrodynamic analysis
demonstrates that the cross-linked product containing
and
is composed of one
and one
subunit and likewise, the
cross-linked
product is composed of only one
and one
subunit (Yi et al., 1991; Thomas et al.,
1993b). However, as is common for cross-linked proteins, these products
migrate anomalously in SDS-PAGE. (
)
We have previously
established that BMH cross-links to
through cysteine 215 in
(Thomas et al.,
1993a). This cysteine residue is found in the
helix,
a region shown to have a different conformation in
-GDP compared
with
-GTP (Lambright et al., 1994). We now report the
identification of the sites in
and
involved in the
formation of the
and
cross-links. These studies
begin to map the regions of
near the
contact surface,
and they set physical limits to the distance between residues in
,
, and
.
Cysteine residues in
position 30 and 50 in (cDNA kindly provided by Dr. M.
I. Simon) were individually mutated by polymerase chain reaction using
primers that included conveniently located restriction sites at the 3`
end of the mutated residue (StyI and BamHI,
respectively). The detailed sequences of the oligonucleotides used as
primers are TGCTGCCTTGGACACCTTTATCCGGGCCAA for mutation of Cys
and GAGGGGATCCTCAGCGGCG for mutation of Cys
. The
inserts were completely sequenced to ensure that no additional
mutations were introduced during the polymerase chain reaction
reaction.
Cross-linking of purified subunits was performed as described
previously (Yi et al., 1991); 5-10 µg of bovine
brain purified or
(Neer et
al., 1984) in HMSE plus 0.4-0.6% Lubrol PX were used for
each reaction.
Figure 1:
Cross-linking of
purified and in vitro translated G protein subunits. A, heterotrimers containing or
dimers purified from bovine brain were cross-linked with BMH
essentially as described under ``Materials and Methods.''
Similarly, in vitro translated and dimerized
subunits were mixed with purified
(
3 µg) and treated with BMH. Cross-linked
samples were then resolved on 9% polyacrylamide gels and stained with
Coomassie Blue. Lanes containing in vitro translated samples
were further processed for autoradiography as detailed under
``Materials and Methods.'' The positions of uncross-linked
and
subunits are indicated by arrowheads.
Cross-linked products are shown by arrows. B, in
vitro translated
and
were
treated separately (sets A and B) or mixed and
allowed to dimerize (set C). The cross-linking reaction was
carried out either in the absence (-
) or
presence (+
) of purified
, and
samples were resolved on 9% SDS-PAGE and subjected to autoradiography.
The arrowhead shows the position of the radioactive band
corresponding to the
subunit; cross-linked products are indicated
by arrows. In the lanes marked - BMH, 20 mM DTT
was added prior to BMH. For cross-linker-specific bands compare -
BMH and + BMH lanes. Set B was exposed three times longer
than sets A and C to make sure that no specific bands
were formed. The
subunit could not be detected in this gel (9%
polyacrylamide), but its presence was confirmed on tricine gels that
resolve low molecular mass proteins (Schägger and
von Jagow, 1987).
Figure 2:
Trypsin digestion of
dimers containing
cysteine mutants. In vitro translated wild type or mutant
subunits were incubated without (first two lanes) or
with
and digested with trypsin to verify
dimerization. The fragments were resolved on 11% polyacrylamide gels
and visualized by autoradiography. The first lane of each pair (-
trypsin) corresponds to control undigested sample. The protected
carboxyl-terminal fragment is indicated by an arrow. The
position of the mutated cysteine in the
sequence is
shown on top of each pair of lanes. The same results were
obtained in three independent experiments. The two radioactively
labeled bands showing up right below the
subunit band in control
lanes correspond to incomplete
proteins generated during the in vitro translation as a result of either a premature
termination or an internal start from methionine residues located
downstream of the initial AUG. These truncated proteins, however,
cannot dimerize with the
subunit and therefore do not interfere
with our experimental procedures.
When dimers
containing different cysteine mutants and wild type
were cross-linked to
and the
resulting products resolved on SDS-PAGE, we observed that most of the
mutants were still able to generate the two
cross-linked
bands (Fig. 3, solid arrows).
However, two of the mutations affected the cross-linking to
; mutation of Cys
blocked the formation
of the upper band, whereas mutation of Cys
abolished the
lower band. The fact that each of these mutant
dimers can
still form one of the two cross-linked
products shows that
each is still able to interact with
. It was previously
established in this laboratory that both cross-linked products are
generated through a single cysteine residue in the
subunit (Cys215), because mutation of that amino acid prevented
the formation of both bands (Thomas et al., 1993a). Therefore,
each one of the two bands is due to cross-linking Cys
in
to a different cysteine in
:
Cys
for the upper band and Cys
for the
lower one. The possibility existed that
might be present in one
of the two products, presumably the one with the larger apparent
molecular mass. However, because each cross-linked product is affected
by mutation of only one cysteine residue in
(Cys
) and one cysteine residue in
(Cys
or Cys
), we conclude that
neither product contains an additional cross-linked component, which
would have required the involvement of yet another cysteine residue.
The distinct mobilities on SDS-PAGE of the two cross-linked products of
the same composition are probably due to differences in SDS binding or
in the shape of the denatured molecules resulting from the two
alternate cross-linking sites in
.
Figure 3:
Cross-linking of
dimers containing
cysteine
mutants to
. In vitro translated wild type or
mutated
subunits were dimerized with in vitro translated
and treated with BMH in the presence
of purified
as described. Reactions were stopped by
the addition of DTT and/or Laemmli sample buffer, and samples were
subjected to SDS-PAGE on 9% polyacrylamide gels followed by
autoradiography. Control, uncross-linked samples (- BMH) were
incubated with BMH in the presence of excess DTT. The two cross-linked
products are shown by arrows, and the numbers on top of each pair of lanes indicate which
cysteine mutant was used. The arrowheads point to the
mutants that did not behave like the wild type. Shown here is a
representative example of three independent
experiments.
Figure 4:
Cross-linking of
dimers containing
cysteine mutants. Dimers of
containing wild type or mutated
subunits were
incubated with BMH, and both treated (+ BMH) and untreated
(- BMH, 20 mM DTT added before BMH) samples were
subjected to SDS-PAGE on 9% polyacrylamide gels followed by
autoradiography as described previously. The number of the cysteine
residue mutated to alanine in
is indicated on top of each lane. The closed arrowhead indicates the position
of
. The
50-kDa cross-linked product is shown by
an arrow. For simplicity only some of the mutants are shown;
the rest were indistinguishable from the wild type. The open
arrowhead points to the mutant that failed to generate that
cross-linked product. This experiment was repeated three times with
similar results.
Figure 5:
Characterization of cysteine mutants. A, in vitro translated wild
type
subunits were incubated with either wild type
or a double mutant
with no
cysteines in its sequence. Dimerization was subsequently checked by
trypsin digestion as described previously. The fragments were resolved
on 11% polyacrylamide gels and visualized by autoradiography. The arrow points to the protected carboxyl-terminal
fragment indicative of dimer formation. Shown here is an example
of four independent experiments. B, the same dimers containing
wild type or mutated
were cross-linked with BMH.
Treated (+ BMH) and untreated (- BMH, 20 mM DTT
added before BMH) samples were resolved on 9% polyacrylamide gels and
processed for autoradiography. The cross-linked product is indicated by
an arrow. This experiment was repeated four times with similar
results.
Figure 6:
Comparison of the position of cysteine
residues in ,
, and
sequences. The complete aligned amino acid sequences of bovine
,
, and
(accession
numbers: K03255, M37183, and M58349) are shown. The position of the
cysteine residues in each subtype is indicated by arrowheads.
Figure 7:
Comparison of cross-linked products
obtained with dimers purified from brain and
or
dimers translated in vitro.
Purified
subunits or in vitro translated dimers of
defined composition (
or
) were treated in parallel with or
without cross-linker, and the resulting products were resolved on 9%
polyacrylamide gels and stained with Coomassie Blue. Lanes containing in vitro translated subunits were further processed for
autoradiography as described. The arrowhead points to the
radioactive band corresponding to
. The position of
the cross-linked products is indicated by closed and open
arrows. The same experiment was repeated three times with similar
results.
Figure 8:
Characterization of cross-linked products
obtained with dimers.
and
subunits were separately translated in
vitro in the presence (*) or the absence of
[
S]methionine, and dimers were formed in which
either both subunits (B) or just one of them (C and D) were radioactively labeled. The dimers were then mixed with
purified
and incubated with cross-linker (+ BMH)
or with 20 mM DTT added before the cross-linker (- BMH).
The cross-linked products were resolved on 9% polyacrylamide gels and
subjected to autoradiography. For comparison a
sample treated similarly is included (A). Cross-linked products are indicated by arrows.
With
(but not
), we observed an
additional cross-linked product at about 60 kDa. It contains both
and
and is not dependent on the presence of
. It may
represent
cross-linked to another protein in the
reticulocyte lysate or multimers of
. It was not
characterized further. This is a representative example of three
independent experiments. Shown in the inset are the bands
obtained after cross-linking with BMH purified
to
purified
subunits labeled with
I-Bolton-Hunter
(Thomas et al., 1993b). The samples were run on a 9%
polyacrylamide gel, which was subsequently stained with Coomassie Blue,
dried, and exposed to autoradiographic film for 2 days. Only the
fragment of the gel including those cross-linked products is
shown.
In order to determine which cysteine
residue in is really cross-linked to
, we tested the
cysteine mutants
again but this time using
instead of
(Fig. 9, lower part; the effect of mutations on
the
cross-link will be discussed below). The ability of all
mutants to dimerize with
was
confirmed with a trypsin digestion assay (data not shown). Fig. 9shows the cross-linking results obtained with the most
significant
mutants, the rest being indistinguishable
from the wild type. The C271A mutant, which failed to generate the
50-kDa cross-linked product in experiments with
(see Fig. 4), could however be cross-linked to
. Conversely, mutation of Cys
in
, which showed no phenotype when tested with
(data not shown), completely abolished the
cross-linking. Therefore,
is cross-linked to
through
Cys
, which lies within the 14-kDa amino-terminal
tryptic fragment as shown in our previous studies with purified protein
(Thomas et al., 1993b).
Figure 9:
Cross-linking of
dimers containing
cysteine mutants. In vitro translated wild type or mutant
subunits were dimerized with
and
cross-linked in the presence of
. Both treated (+
BMH) and untreated (- BMH, 20 mM DTT added before BMH)
samples were subjected to SDS-PAGE on 9% polyacrylamide gels followed
by autoradiography. Uncross-linked
is indicated by an arrowhead. Cross-linked products are shown by arrows.
The position of the mutated cysteine in the
sequence
is specified on top of each pair of lanes. Shown here is a
representative example of three independent
experiments.
We next sought to establish which
one of the two additional cysteine residues in (Cys
or Cys
; see diagram in Fig. 6) conferred the ability to be cross-linked to
by
BMH. Following the same strategy, we individually mutated both
cysteines to alanines and used tryptic protection assays to confirm
that the mutants could still dimerize with
(Fig. 10A). Subsequently, we cross-linked
dimers containing either wild type
or mutant
subunits and showed that mutation of
Cys
but not Cys
prevents the formation of the
cross-linked
band (Fig. 10B, lower
part). This cysteine residue in
(Cys
) is not present in any of the other
subtypes and therefore confers a distinctive property to
.
Figure 10:
Characterization of
cysteine mutants. A, in vitro translated wild type
subunits were incubated with either wild type or
mutated
subunits. Dimerization was subsequently
checked on a trypsin digestion assay as described previously. The
fragments were resolved on 11% polyacrylamide gels and visualized by
autoradiography. The arrow points to the protected
carboxyl-terminal
fragment indicative of dimer
formation. B, the same dimers containing wild type or mutated
were mixed with
and used in a
cross-linking assay. Treated (+ BMH) and untreated (- BMH,
20 mM DTT added before BMH) samples were resolved on 9%
polyacrylamide gels and processed for autoradiography. Cross-linked
products are indicated by arrows. The stronger intensity of
the radioactive bands corresponding to the two
cross-linked products in the lanes containing the Cys
mutant was only observed in this particular experiment and does
not reflect an increased affinity of the
mutant for
the
subunit. The position of the mutated cysteine in the
sequence is shown on top of each pair of
lanes. The arrowhead signals the mutant that failed to
generate a
cross-link. The same result was obtained in three
separate experiments.
Figure 11:
Schematic representation of the major
BMH-specific bands obtained with purified or in vitro translated subunits after SDS-PAGE. Shown on the left side is the composition of each one of the cross-linked
products. The cysteine residues in
involved in each
cross-link are indicated on the right
side.
Which are the cysteine residues involved in the formation
of those two new cross-linked products? Our hypothesis was that the two
new products were the result of attaching to the two previously
described forms of cross-linked
. If this were the case, the
same
cysteine mutations that prevented the formation of the two
lower bands (Cys
and Cys
) should also
affect the two additional upper bands. That is exactly what we observed
when we tested the
mutants dimerized with
in a
cross-linking assay in the presence of
(see Fig. 9, upper part). The C204A mutant failed to produce
the upper of the two
bands (as we had previously
established) as well as the upper of the two
bands (for
schematic representation of the four products, see Fig. 11).
Conversely, C271A mutant failed to produce the lower of the two
bands and the lower of the two
bands. As
expected, mutation of Cys
blocked the formation of the
three bands containing
leaving the other three (the two
cross-linked
bands and
X) unaffected. Finally, the
results with the
cysteine mutants (see Fig. 10B, upper part) confirmed that the same
cysteine involved in the
cross-linking (Cys
) is
responsible for the formation of the two cross-linked
bands.
Fig. 11summarizes the results obtained during the
characterization of the six major cross-linked products obtained with in vitro translated G protein subunits; starting from below
the first band corresponds to cross-linked to
through Cys
,
being in turn
covalently bound to
through Cys
. Right above is
cross-linked to a rabbit reticulocyte lysate protein (X) through
Cys
. The higher molecular mass bands, starting from the
bottom, correspond to
cross-linked to
through Cys
(lower band) and through Cys
(upper band) followed
by
cross-linked to both
and
again through Cys
or Cys
(to
) and through Cys
(to
).
The residues in and
that are
cross-linked to each other (Cys
in
and
Cys
in
) lie within regions believed to
be important for dimerization. The minimum sequence of
necessary
to form a native
dimer is not known. However, removal of 15
amino acids from the amino terminus diminishes but does not entirely
block dimerization, whereas removal of 13 amino acids from the carboxyl
terminus has little effect (Mende et al., 1995). Therefore,
residues important to contact
probably lie between amino acids 15
and 59. The sequence that defines the selectivity of
interactions is also located in this central region (Spring and Neer,
1994; Lee et al., 1995). Mutations in the putative
amino-terminal
-helix in
, where Cys
is located, can inhibit
dimerization (Garritsen et
al., 1993). However, selectivity for different
subunits is
determined by multiple sites within the WD repeat region of the
subunit (Pronin and Gautam, 1992; Garritsen and Simonds, 1994; Katz and
Simon, 1995).
and
cannot be
cross-linked to
by BMH. Neither they nor any of the
other known
subtypes including the newly cloned isoforms (Ong et al., 1995; Ryba and Tirindelli, 1995; Ray et al.,
1995) contain a cysteine at a position equivalent to Cys
of
. Therefore, in any preparation, formation of
a cross-linked
product after treatment with BMH suggests the
presence of
dimers. This distinctive property
could in turn be used to probe for interaction of
dimers
containing
with other proteins.
The two cross-linked
cysteines are located in the carboxyl-terminal portion of the
subunit, in agreement with our previous observation with purified
subunits that after tryptic digestion of the cross-linked products the
subunit remains associated with the 24-kDa carboxyl-terminal
fragment (Yi et al., 1991). Cys
is found
in the fourth WD repeat, whereas Cys
lies within repeat
six. Both are likely to be found in turns. The spatial organization of
the seven WD repeats in
is not known. It is clear however that
forms a very tightly associated globular structure (Thomas et al. 1993b). Any model of the
dimer must be
compatible with Cys
, located in the
helix of the
subunit, reaching Cys
in the fourth
repeat and Cys
in the sixth repeat through an arm of
approximately 16 Å (see Fig. 12). The two
cysteines are probably not providing bonds to hold
and
together because they can be mutated without disrupting the
association. Our findings, however, begin to define an area in
that faces the
subunit, and they pose structural constraints to
any model of the configuration of the
subunit.
Figure 12:
Schematic representation of the
structure: location of the cysteine residues involved in cross-linking
to
and
subunits. The predicted structure of the
subunit is schematically depicted. Each WD repeat is indicated by a circle. The positions of Cys
and Cys
are indicated. The helical and GTPase domains of the
subunit are indicated by two ovals. Cys
is the
residue in the GTPase domain of
which is cross-linked to
(Thomas et al., 1993a). The putative
-helical
amino-terminal segment in
is shown as a bar. The
subunit is shown as a long bar. The site of the
Cys
to
Cys
cross-link
is indicated by a line. The carboxyl-terminal prenyl group is
indicated by the zig-zag line.