(Received for publication, November 23, 1994; and in revised form, January 13, 1995)
From the
The subunit composition of the major bovine brain G
and G
proteins (G
, G
,
G
, G
, and G
) was characterized
using antibodies against specific
isoforms. Each of the purified
G protein heterotrimers contained a heterogeneous population of
subunits, and the profiles of the
subunits found with
G
, G
, and G
were similar. In
contrast, each G
isoform had a distinct pattern of
associated
subunits. These differences were surprising given that
all three
isoforms are thought to share a common
amino-terminal sequence important for the binding of
dimers
and that the
and
proteins may
come from the same
mRNA. The free
and
subunits had unique elution behaviors
during MonoQ chromatography, compatible with differences in their
post-translational processing. These results indicate that both the
and
subunit compositions of heterotrimers define the
structure of an intact G protein. Furthermore, the exact subunit
composition of G protein heterotrimers may depend upon regulated
expression of different subunit isoforms or upon cellular processing of
subunits.
Heterotrimeric G proteins mediate transmission of extracellular
signals from cell-surface receptors to intracellular
effectors(1, 2, 3, 4, 5) .
They are a diverse family of proteins with a common structure composed
of ,
, and
subunits(6, 7, 8) . Historically, they are
named for their
subunit. Receptors catalyze exchange of bound GDP
for GTP on the
subunit and promote dissociation of the G protein
into a separate
subunit and a
dimer(1) . This
suggests simultaneous regulation of multiple cellular responses by G
proteins since both components independently regulate intracellular
effectors(9, 10, 11) .
G protein subunits
exist in a variety of isoforms. To date, 20 , 5
, and 6
subunits have been
described(1, 2, 3, 4, 5, 12, 13, 14) . (
)Random association of these subunits would generate
literally hundreds of different heterotrimeric proteins. Whether
preferred combinations of isoforms combine to form a more limited
number of distinct complexes is not clear(15, 16) .
Regardless, G protein-coupled receptors seem to require heterotrimers
containing specific
,
, and
subunit
isoforms(17, 18, 19) . Thus, receptor
regulation of G protein function must be strongly influenced by how
heterotrimer composition is determined.
We have separated bovine
brain G protein heterotrimers based upon their constituent
subunits and then determined their
subunit composition using
isoform-specific antibodies(12, 20) . All of the
purified G proteins contained a heterogeneous mixture of
subunits, suggesting a great diversity of the number of possible
heterotrimer combinations. However, for the major brain G protein,
G
(21, 22) , the
subunit composition
varied between
splice variants and between
subunits thought to differ because of
post-translational modifications. These results support the idea of
specific
and
subunit associations (23) and indicate
that both subunits are important to define a heterotrimer. Furthermore,
the exact subunit composition of G protein heterotrimers may depend
both upon regulated expression of different subunit isoforms and upon
cellular processing of
subunits.
Isoform-specific subunit antisera
were raised to the sequences NTYEDAAAY(C) (antiserum AO1, residues
295-303 of
), KNNLKDCGLF (antiserum AI12,
COOH-terminal residues of
and
),
and GNLQIDFADPQR (antiserum AI2L, residues 89-100 of
). Peptides AI12 and AI2L were coupled to keyhole
limpet hemocyanin with glutaraldehyde(29) , and peptide AO1 was
coupled to keyhole limpet hemocyanin using sulfo-MBS(30) .
Antisera to the coupled peptides were raised in rabbits according to
Green et al.(30) . The
antibody,
recognizing the sequence NLKEDGISAAKDVK (residues 22-35) common
to all
isoforms(31) , was a generous gift of
Dr. Ravi Iyengar. Anti-
antibodies were affinity-purified using
peptide immobilized on Affi-Gel-15 (AI12 and AI2L) or on Affi-Gel-102
treated with sulfo-MBS (AO1). Bound antibodies were eluted either with
0.1 M glycine, pH 2.5, 0.5 M NaCl neutralized with 2 M ammonium bicarbonate (AO1 and AI12) or with ActiSep elution
medium (AI2L) from Sterogene (Arcadia, CA). Immunoblotting for
subunits followed the methods of Towbin et al.(32) .
Bound antibody was visualized using the ECL reagents from Amersham
Corp.
Figure 1:
Separation of bovine brain G protein
heterotrimers. A, 10-µl aliquots of 1-ml fractions were
analyzed on a 13% acrylamide, 0.4% bisacrylamide SDS-polyacrylamide gel
and stained with Coomassie Blue. B, the elution of the G
protein isoforms was continuously monitored by changes in the
absorbance at 280 nm. The NaCl gradient is indicated (- -
-). C, the intensity of each subunit band in the
gel in A was analyzed by scanning densitometry to determine
the elution profiles of specific
subunit isoforms. The elution
positions of five G protein isoform peaks (based upon
subunits)
are indicated:
, G
isoforms (39 kDa
) including
G
, G
, and G
;
, G
(41 kDa
);
, G
(40 kDa
).
One or more additional FPLC steps were required to
isolate each of five distinct G protein heterotrimers corresponding to
G, G
, G
, G
, and
G
(Fig. 2A). These assignments were
confirmed by immunoblotting with
subunit-specific antibodies (Fig. 3). G
and G
were both
recognized by antibodies specific for an
sequence (39) . This agrees with the conclusion of Shibasaki et al.(38) that G
and G
are
both translated from the same
splice variant. Silver-stained gels (Fig. 2A) and immunoblots (Fig. 2B)
revealed minor cross-contamination of the G protein heterotrimers,
particularly G
and G
. Nevertheless, each G
protein was at least 80% pure. G
, G
, and
G
were nearly homogeneous. The stoichiometry of GTP
S
binding to the purified heterotrimers was 0.9:1 (assuming an M
of 80,000) for all of the isoforms except
G
, which was 0.8:1. These data suggest that these proteins
are purified as intact viable heterotrimers.
Figure 2:
A,
analysis of the and
subunit composition of the purified G
protein isoforms by gel electrophoresis. Purified G protein isoforms (1
µg) were loaded in each lane of a 10-20% gradient
SDS-polyacrylamide gel. The resolved
and
subunits were
visualized by silver staining. B, analysis of the
subunit composition of the purified G protein isoforms by gel
electrophoresis. The gel represents a typical analysis of three sets of
G protein heterotrimers. Purified G protein isoforms (1 µg) were
resolved on a 10-20% gradient acrylamide, 0.8% bisacrylamide gel
using a Tricine buffer system. The protein bands were visualized by
silver staining after first staining with Coomassie
Blue.
Figure 3:
Analysis of G isoform preparations using
antibodies against specific isoforms. Purified G protein isoforms
(1 µg) were separated on 13% acrylamide, 0.4% bisacrylamide gels,
transferred to nitrocellulose, and immunoblotted with antibodies with
the indicated specificity for
isoforms (see ``Experimental
Procedures'').
Figure 4:
Immunoblot analysis of subunit
distribution among G isoforms. Purified G protein isoforms (1 µg)
were resolved on 15% polyacrylamide gels and immunoblotted with
antisera of the indicated
subunit specificity as described under
``Experimental Procedures.'' The immunoblots shown are
typical of the results of the analysis of three sets of heterotrimers,
with the exception of
3, as described in the
text.
Both
techniques verified the observation that G protein heterotrimers,
defined by their subunits, contain multiple
subunit
isoforms and hence multiple combinations of
dimers(15, 16) . Furthermore, G
and
G
had very similar
subunit compositions that were
not substantially different from that of G
. Although
interesting and potentially important, this conclusion may have to be
refined as
subunit heterogeneity is further characterized. In
addition, such nonspecific association of subunits could have resulted
from random recombination of the subunits during the isolation of the
heterotrimers. Surprisingly, the three G
isoforms had
different
subunit profiles (Fig. 4). For example, G
had considerably more
than did G
or G
. Even more striking was the minimal amount of
in G
and the virtual absence of
in G
. In contrast, the contribution of
and
to each of the different
heterotrimers was the same (data not shown). These results cannot be
explained by random association of
subunits with
dimers, either in vivo or during G protein purification.
G and G
were separated by us (Fig. 1) and by others (15, 34) based upon
their differing behavior on a MonoQ column. Antibody and proteolytic
peptide mapping data suggest that G
and
G
come from the same
mRNA(15, 38) , and our results agree with this
conclusion (Fig. 3). However, since G
and G
differ in their
subunit composition (Fig. 4), it is
possible that their
subunits are in fact identical and that their
different elution during MonoQ chromatography is due solely to their
different
dimer compositions. To test this possibility,
and
were separated from their
dimers and individually examined on a MonoQ column (Fig. 5). The position of the protein in each case was
determined by GTP
S binding activity and immunoreactivity with an
-specific antibody (AO1).
eluted
as a single major peak with a shoulder probably indicative of some
contamination with
. Interestingly,
eluted as two fairly sharp peaks in two of three preparations,
suggesting additional
heterogeneity. Regardless, the
two isolated
subunits,
and
,
still eluted differently on the MonoQ column. Thus, not only are the
dimers of G
and G
different, but
their
subunits differ as well. Although these results do not
exclude the possibility that
and
are internal splice variants of the
gene,
existing evidence suggests that they have the same coding sequence.
Four mRNAs have been shown to be generated from the bovine
gene(45) . One corresponds to
and
would generate
. The other three differ in their
3`-untranslated regions, but code for identical
proteins. Presumably,
and
are derived from these three mRNAs. This suggests that they are
proteins with the same sequence, but different post-translational
modifications.
Figure 5:
Comparison of the elution profiles of
and
on a MonoQ FPLC column. The
(
) and
(
) isoforms
(100 µg each) were loaded in separate experiments onto a MonoQ
column and eluted at 1 ml/min using a linear NaCl concentration
gradient as indicated(- - - ). The elution profiles
of the
isoforms were determined by GTP
S binding activity in
the fractions indicated and by immunoblotting of sample aliquots using
the
-specific antibody characterized in Fig. 3.
As discussed above, the different patterns of
subunit expression observed with G
and G
could result from different preferences of
subunits for
specific
dimers. The variable interaction of
isoforms
with
subunits (46) might support such a conclusion.
Alternatively, our results could be explained by the regulated
coexpression of
and
subunit isoforms in different cells, or
perhaps even the same cell. This could be related to the multiple
transcripts coding for the
protein(45) .
Future studies will be required to define the relative contributions of
these two possibilities. Immunocytochemistry with site-specific
antibodies and in situ hybridization determining the
expression of variable
and
subunit proteins and transcripts
will be important to resolve the possible colocalization of these
proteins. Complementary biochemical studies of the G
isoforms will help determine the preferential interaction of
subunits with
dimers and the functional significance
of these unique heterotrimer combinations.
Muscarinic and
somatostatin receptors are thought to couple specifically to the
and
heterotrimers,
respectively, in their regulation of Ca
channels in
GH
cells(17, 18, 19) . These
results suggest exquisite specificity in the recognition of G protein
heterotrimers by receptors. It remains unclear whether specific G
protein subunit combinations occur randomly or if there are mechanisms
that dictate the composition of heterotrimers. Such mechanisms would
have great significance for the regulation of cellular function by G
protein-coupled receptors. More important, in addition to the
previously reported heterogeneity in
/
subunit
associations(15, 16) , our results indicate that these
subunits do exist in preferred combinations. This is not only true for
the association of
isoforms with splice variants of a single
subunit, such as
and
, but
also for
subunits that may differ only by their
post-translational modifications, which may be the case for
and
. The nature of the putative
modifications that distinguish these proteins is not yet known;
however, many potential modifications, such as palmitoylation,
carboxymethylation, and phosphorylation, are reversible and
regulatable. Thus, it may be possible that cellular regulation of the
modification of G protein subunits could ultimately affect the
composition of G protein heterotrimers and direct their subsequent
receptor and effector interactions.