(Received for publication, November 13, 1995; and in revised form, January 10, 1996)
From the
We have determined the relative abilities of several members of
the G protein and
subunit families to associate with each
other using the yeast two-hybrid system. We show first that the
mammalian
1 and
3 fusion proteins form a complex in yeast and
that formation of the complex activates the reporter gene for
-galactosidase. Second, the magnitude of reporter activity
stimulated by various combinations of
and
subunit types
varies widely. Third, the reporter activity evoked by a particular
combination of
and
subunit types is not correlated with the
expression levels of these subunit types in the yeast cells. Finally,
the reporter activity shows a direct relationship with the amount of
hybrid
complex formed in the cell as determined by
immunoprecipitation. These results suggest that different
and
subunit types interact with each other with widely varying
abilities, and this in combination with the level of expression of a
subunit type in a mammalian cell determines which G protein will be
active in that cell. The strong preference of all
subunit types
for the
1 subunit type explains the preponderence of this subunit
type in most G proteins.
Most of the neurohormonal signaling pathways in mammals are
mediated by heterotrimeric G proteins(1, 2) . Most
cells contain many different G protein subunit types, and yet
particular agonists evoke a highly specific response in a cell by
activating a defined G protein-mediated signaling pathway(3) .
Various mechanisms can contribute to this specificity. For instance, in
a cell that contains many different ,
, and
subunit
types, only certain types may be capable of forming a heterotrimeric
complex because of the differences in the intrinsic affinity of these
subunit types for one another. It has been shown that interactions
between two different
subunit types (
1 and
2) and three
different
subunit types (
1-
3) are selective,
indicating that this is indeed a mechanism for achieving
specificity(4, 5) . However, these experiments were
performed using in vivo or in vitro systems that
could detect differences in the ability of these subunit types to
interact but were not sufficiently sensitive to detect low level
interaction between some of the subunit types. To obtain a more
sensitive measure of the interaction between various members of the
and
subunit families, we used the yeast two-hybrid system.
In this system, the proteins with the potential to interact are
expressed as hybrids of two different domains of a transcription
factor. If the proteins interact with each other, the transcription
factor domains are in proximity and capable of activating a promoter
that controls reporter activity(6) . We chose this system
because it measures protein-protein interaction in a yeast cell and is
therefore a reasonably accurate reflection of the ability of the
interacting proteins to form a complex in a cell. Furthermore, it is
highly sensitive in comparison with the methods used before to measure
protein-protein interaction. For instance, in an assay of interaction
between p53 and T antigen, it was shown that p53 mutants, whose
interaction with T antigen could not be detected in an
immunoprecipitation assay, were actually capable of interaction with T
antigen since co-expression of the mutant p53 and T antigens as hybrids
resulted in measurable reporter activity in the two-hybrid
system(6) . In addition, the same study showed that in the case
of mutant p53 proteins, whose interaction with T antigen could be
detected by immunoprecipitation, there was a direct relationship
between the amount of protein complex immunoprecipitated and the
reporter activity detected when the same proteins were co-expressed in
the two-hybrid system. This raised the possibility of determining the
efficacy of complex formation between different
and
subunit
types based on the magnitude of reporter activity. Using this system,
we tested interaction between the five
subunit types
(
1-
5) that have been identified so far and six
subunit
types (
1-
5 and
7) with different primary structures and
tissue-specific expression (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .
The interaction between the
and
subunits is of special
interest because these two subunits are tightly bound as a complex and
are thought to act inside the cells in this form(2) . The
transcripts for
1-
4 subunits are expressed
ubiquitously, while
5 is expressed only in the
brain(7, 9, 11, 12, 13) .
The
3 protein has been inferred to be the
subunit associated
with a cone photoreceptor-specific
subunit
type(22, 23) . The
subunits show more variation
than the
subunits in their tissue-specific distribution.
1
is specific to rod photoreceptors. (
)
2 is present in
several tissues(16) .
3 and
4 are
brain-specific(18, 21) .
5 is
ubiquitous(19) .
7 is present in several tissues but
similar to
2 is enriched in brain(20) . By testing the
interaction between a variety of
and
subunit types, we
attempted to determine whether interactions to form the
complex were highly selective (resulting in the formation of complexes
between some subunit types but not others) or variable over a
continuous spectrum of interacting abilities.
Functionally, the
complex has been known for a long time to be required for
interaction of a G protein with various receptors (24, 25, 26) . Recent results indicate that
the
subunit directly and specifically interacts with the
receptor, raising the possibility that different
subunit types
could direct association of G proteins with various
receptors(27, 28) . It is also known that the
complex can directly regulate effector function(2) .
If
complexes made up of different
and
subunit
types had different properties at each of these two points in a G
protein-mediated signaling pathway, the mechanisms that control the
formation of specific forms of the
complex in a cell will
also regulate the signaling properties of a cell.
The ability to assay interactions between two families of proteins with diverse structures also allows us to examine the molecular basis of interactions between protein families that form a multimeric complex.
Figure 1:
1-
3 interaction in the
two-hybrid system.
1,
3, Gpa1, Ste4, Grr1, and Tat were
expressed as fusion proteins (see ``Materials and Methods''). A,
-galactosidase activity from cells of different
genotypes (described under ``Materials and Methods''). Gpa1,
a yeast G protein
subunit, and Ste4, a yeast
subunit,
served as positive controls (34) . As unrelated proteins, Grr1
and Tat served as negative controls. The bars represent the
mean value of
-galactosidase activity determined from three
different transformants. B, formation of the
1
3
complex in yeast. Yeast extracts from the equivalent of 1 A
unit of cells were immunoprecipated with the
3-specific CG antibody. The proteins immunoprecipitated were
detected with same antibody on the immunoblot shown. AD-
1 that
co-immunoprecipitated with the CG antibody was detected with an
antibody specific to the Gal4 activation domain (GAL4 AD mAb). Y190, untransformed control yeast cells. Only portions of
blots containing the hybrid proteins identified based on reactivity and
mobility are shown.
To
examine whether the reporter activation resulted directly from complex
formation between the hybrid and
subunits, transformants
expressing the same combinations of hybrids as those mentioned above
(excepting Gpa1+Ste4) were lysed, and the proteins in the cell
extract were immunoprecipitated with a
3-specific antibody, CG,
which is directed at the N-terminal 14 amino acids of
3(32) . We have shown that the epitope for this antibody
is available in the
1
3 complex so that it is capable of
immunoprecipitating
3 when bound to the
subunit and also
co-immunoprecipitating
1(37) . Immunoprecipitates from
different transformants were electrophoresed and immunoblotted with
specific antibodies. The results of immunoblotting are shown in Fig. 1B. The CG antibody immunoprecipitated the
BD-
3 hybrid protein of expected molecular weight which is
30
kDa (
7-kDa
3+
23-kDa BD-nuclear targeting domain
fusion) (second lane from left), but an unrelated
protein Grr1 was not co-immunoprecipitated with
3, indicating that
these two proteins did not form a complex in the yeast cells. No
protein was specifically immunoprecipitated from untransformed Y190
cells or cells expressing Tat+
1, confirming the specificity
of the CG antibody for
3. Immunoprecipitation of the BD-
3
hybrid from cells co-expressing AD-
1 resulted in the
co-immunoprecipitation of a protein with the expected molecular weight
of the AD-
1 hybrid, which is
60 kDa (
36-kDa
subunit+
24-kDa AD-nuclear targeting domain fusion),
demonstrating complex formation inside the cells between BD-
3 and
AD-
1 (last lane from left). This result showed
that the stimulation of reporter activity in
1+
3 cells (Fig. 1A), is due to complex formation between these
hybrids.
These results also indicated that the mammalian and
subunits formed a complex inside the yeast cells, even though
they were fused to the Gal4 transcription factor domains. Moreover, the
complex was capable of activating the GAL1 promoter, resulting
in significantly enhanced reporter activity.
Figure 2:
Differential interactions among the G
protein and
subunit types. Bars in each panel show
the amounts of
-galactosidase activity detected from cells
containing specific combinations of
and
subunit types. The
genotype of the cell is denoted below each bar. Reporter activity
estimated from cells co-expressing the particular BD-
subunit type
with AD-GRR1 was one of the negative controls and is shown in each
panel. Other negative controls are described in the text. The
-galactosidase activity form all control cells was lower than 0.05
units (data not shown). The bars represent the mean (± S.E.)
derived from six different transformants.
Figure 3:
Comparison of reporter activity with the
expression level of hybrid proteins in cells co-expressing BD-2
and different
subunits. A, mean value of
-galactosidase activity obtained from cells of each genotype.
Activity from cells of each genotype is expressed as a percentile of
activity from
2+
1 (same data as in Fig. 2). B, immunoblot probed with GAL4 AD mAb, showing the relative
amounts of AD-
subunit types in 1 µg of total proteins in the
cell extract from various genotypes. Protein levels based on
densitometry of the bands are expressed for each genotype as a
percentile of the level in AD-
1. C, immunoblot probed
with a
2-specific antibody, BG, showing the relative amounts of
BD-
subunit types in 1 µg of total proteins in the cell
extract from various genotypes. Protein levels based on densitometry of
the bands are expressed for each genotype as a percentile of the level
in BD-
2. Only portions of blots containing the hybrid proteins
identified based on reactivity and mobility are shown. The immunoblots
from B and C were scanned for quantitation using a
laser densitometer and the Image Quant program (Molecular Dynamics).
Results shown are representative of two independent
experiments.
Figure 4:
Relationship between reporter activity and
complex formation. A, mean value of -galactosidase
activity from cells containing
3+
1 and
3+
3 (same data as in Fig. 2) expressed as
relative percentiles. B, immunoblot with bands corresponding
to the
subunit hybrids and the relative amounts of proteins in
these bands as determined by densitometry. The immunoblot contains the
immunoprecipitate obtained with a
3 antibody, CG, from 25 µg
of total proteins extracted from each of the transformants,
3+
1 and
3+
3. Conditions used for
immunoprecipitation from cell extracts of both genotypes were identical
to allow quantitative comparison of the amount of
complex
present. The proteins co-immunoprecipitated with BD-
3 were blotted
and probed with GAL4 AD mAb. The BD-
3 immunoprecipitated with
3 antibody is not shown since a proportion of it may not be bound
to
. Bars above the bands show the relative amount of
AD-
1 and AD-
3 co-immunoprcipitated with BD-
3 as
determined by densitometry. The amount of AD-
3 is expressed as a
percentage of AD-
1. C, immunoblot probed with GAL4 AD
mAb, showing the relative amounts of AD-
1 and AD-
3 in 1
µg of total proteins obtained from cells containing
3+
1 and
3+
3. The amount of AD-
3 is
expressed as a percentage of AD-
1. D, immunoblot probed
with
3 antibody, CG, showing the relative amount of BD-
3 in 1
µg of total proteins obtained from cells containing
3+
1 and
3+
3. The amount of BD-
3 from
cells containing
3+
3 is expressed as a percentage of
BD-
3 from cells containing
3+
1. Only portions of
blots containing the hybrid proteins identified based on reactivity and
mobility are shown. Quantitation of protein bands on the immunoblots in B, C, and D were performed as described in
the legend to Fig. 3. Results shown are representative of two
independent experiments.
The reporter activity resulting from interaction between two
hybrids may be affected by several different factors: efficiency of
complex formation, expression levels of the hybrids, and accessibility
of the activation domain to the transcription machinery. It is also
possible that the and
subunit types influence the
localization of individual hybrids to the nucleus, although both
transcription factors possess nuclear targeting signals upstream.
Results shown in Fig. 3indicate that the differential reporter
activity from cells containing various combinations of
and
subunit types is not related to the expression levels of the hybrid
proteins, implying that it results from the different levels of
complex formation. The results presented in Fig. 4support this implication. These results show that there is
a direct correlation between the reporter activity and the amount of
complex formed in the cells. The differential reporter
activity noted among the different genotypes is therefore mainly due to
different levels of hybrid
complexes formed in these cells
and not any of the other reasons mentioned above. This confirms a
previous report of a positive correlation between the amount of
immunoprecipitated complex of two hybrids and reporter activity
stimulated by those hybrids(6) . Many combinations of
and
subunit types have significant reporter activity in comparison
with the negative control,
subunit type+Grr1 (Fig. 2). This indicates that complex formation does occur
between many of the
and
subunit types examined here.
However, the extent of complex formation differs considerably among the
different combinations of
and
subunit types (Fig. 2).
The G protein subunits are
post-translationally modified by the attachment of a prenyl group to
the C terminus(38) . There is evidence that yeast cells possess
the same enzymes as mammalian cells that modify various proteins with
the two types of prenyl groups, farnesyl and
geranygeranyl(39) . It is therefore most likely that all of the
mammalian
subunits expressed in yeast are prenylated
appropriately. However, since it is known that prenylation of the
subunits is not required for interaction with the
subunit(4, 40) , even if a mammalian
subunit
type was inefficiently prenylated it would not affect
complex formation in yeast cells. The results from the
immunoprecipitation of the
complex in Fig. 1and Fig. 4support this directly.
In comparison with the other
subunit types, the
3 subunit type is consistently poorer at
interacting with all of the
subunit types tested. This would
indicate that differentially high levels of expression of this subunit
type would be required to ensure its appropriate interaction with a
subunit type. This may be the reason for its restricted
expression in cone photoreceptors compared with rods (22, 23) . There is also the possibility that
3
selectively interacts much better with a
subunit specific to
cones,
c, compared with other
subunit types. The predominant
subunit types in the G protein
complexes purified from
a variety of mammalian tissues are
1 and
2, although G
contains only
1(41) . This preference for the
1
and
2 subunits is explained by the data in Fig. 2; all of
the
subunit types, other than
1, have a strong preference
for these subunit types.
Recent evidence indicates that the
subunit interacts directly with the receptor(27, 28) .
If different
subunit types specifically interacted with various
receptors, this intrinsic ability of
and
subunit types to
favor complex formation with certain types and not others would be a
primary level of control over which G protein would be active in a
cell.