(Received for publication, October 30, 1996, and in revised form, November 19, 1996)
From the Department of Anesthesiology and
§ Genetics, Washington University School of Medicine,
St. Louis, Missouri 63110
In the yeast two-hybrid system, a 100-residue
fragment (1A) from the N terminus of the
1 subunit interacts with
domains specific to adenylyl cyclase 2 (AC2), the muscarinic atrial
potassium channel (GIRK1), and phospholipase C-
2 (PLC-
2). Based
on the crystal structure of the G protein,
1A is composed of an
N-terminal
helix, a loop, and five
strands in which the
C-terminal four
strands form a
sheet, the first of seven sheets
that make up the propeller structure of the
subunit. A mutant of
1A (L4P, L7P, and L14P), in which the
helix was potentially
destroyed, interacted poorly with the G protein
subunit but
effectively with domains of AC2, GIRK1, and PLC-
2. In contrast,
another mutant of
1A (S72A, D76A, and W82A), in which a network of
hydrogen bonds was disrupted, interacted poorly with GIRK1 and PLC-
2
domains, but effectively with the
subunit and the AC2 domain. These
results suggest that the proper folding of the first five
strands
in the G protein
subunit is a requirement for appropriately
positioning residues that interact with GIRK1 and PLC-
2.
Furthermore, since mutations that potentially disrupted the folding of
these
strands did not affect interaction with AC2, the structural
determinants on the G protein
subunit for interaction with various
effectors may be different.
The G protein complex plays an important role in modulating
the function of a variety of effectors in cellular signaling. The
effectors regulated by the
complex include adenylyl cyclases, phospholipase C-
2, and potassium channels (1-5). The
complex directly interacts with several effectors, and binding domains for the
complex have been identified in these effectors (6-11). Since
the
and
subunits form a tight complex in mammalian cells, individual roles of the
and
subunits in effector regulation are
unclear. Using the yeast two-hybrid system, we have been investigating interaction of the
and
subunits with effectors. We previously demonstrated that it was the
subunit that interacts with domains specific to adenylyl cyclase type 2 (AC2)1
and the muscarinic atrial potassium channel (GIRK1) and that a
100-residue fragment (
1A) from the N terminus of the
1 subunit interacted with these effector domains as effectively as the whole
subunit (12). These results imply that the
subunit is an important
element in regulating effector activity. The identification of a
subdomain within the N-terminal 100-residue fragment of the
1
subunit will provide further insight into the mechanisms by which the
complex regulates effector function.
To examine interaction between the complex and effectors, we
have specifically used the yeast two-hybrid system because it allows us
to directly measure the effect of mutations in a protein on its ability
to bind another protein in vivo. Biologically relevant
information about the structural basis of interactions in multiprotein
complexes has been obtained through deletion and mutational analysis in
the two-hybrid system (e.g. Refs. 13-15). We have also
successfully used the yeast two-hybrid system to demonstrate that
different members of the
and
subunit families have differential
abilities to form a complex and also to show that an N-terminal domain
on the
subunit interacts with domains specific to two effectors
(12, 16). We showed that the formation of the
complex as fusion
proteins in yeast cells activated a reporter gene, and that the
reporter activity was directly related to the amount of the
complex formed in yeast cells (16). Our finding using the two-hybrid
system that the first 100 residues are important for interaction with
AC2 have been supported by recent results obtained from cross-linking
and modeling experiments with the
complex and the AC2 domain
(17). In this report, we have used the two-hybrid system to analyze
interaction of the
subunits with AC2 and GIRK1 as well as
phospholipase C-
2 (PLC-
2). A domain (residues 580-641) of
PLC-
2 has recently been shown to directly bind to the G protein
complex, and a fragment containing this domain has been shown to
inhibit the
complex-mediated activation of phospholipase C-
2
(9).
The crystal structures for the G protein heterotrimer and the
complex indicate that the N-terminal 100 residues of the
subunit
(
1A) that interacts with AC2 and GIRK1 is made up of distinct
domains in terms of secondary structure (18-20). It consists of an
N-terminal
helix, a loop, and five
strands. The first of these
five strands is the outermost strand of the seventh
sheet in the
subunit. The C-terminal four
strands of
1A form the first
sheet. Based on the crystal structure, hydrogen bonding is
suggested between residues in the loops that connect particular
strands and residues within specific
strands. These hydrogen bonds
are important for stabilizing the folded structure of the multiple
strands of the
subunit. We have introduced mutations into the first
100 residues of the
subunit that potentially disrupt either the
helix or the hydrogen bonds that are important for stabilizing the
folding of these five
strands. The mutants have been examined for
interaction with both the
subunit and the effector domains.
Yeast strain Y190 and the plasmids pAS1 and pACTII have been described before (12, 16).
ConstructspAS1 contains the Gal4 binding domain, and
pACTII contains the Gal4 activation domain. cDNAs encoding a
protein or a protein domain were subcloned downstream of either the
Gal4 binding domain in pAS1 or the activation domain in pACTII.
cDNAs for the subunits were subcloned in pAS1 and cDNAs for
the
subunits, and
1A-
1D were subcloned in pACTII, as
described previously (12, 16). A synthetic oligonucliotide cassette
encoding AC2Q (residues 956-982 of adenylyl cyclase 2) was subcloned
into NcoI/BamHI cut pAS1 (12). DNA encoding GKN
(residues 1-83 of GIRK1) was amplified by polymerase chain reaction
using specific primers and subcloned into
NcoI/BamHI cut pAS1 (12). DNA encoding the PLC62
domain (residues 580-641 of PLC-
2) was amplified by polymerase
chain reaction using specific primers and subcloned into both pAS1 and pACTII which were cut with NcoI/BamHI. DNAs
encoding two mutants of
1A fragment,
1Am1 (L4P, L7P, and L14P)
and
1Am2 (S72A, D76A, and W82A), were amplified by polymerase chain
reaction using specific primers and subcloned into
NcoI/BamHI cut pACTII. Correct reading frames and
nucleotide sequence after polymerase chain reaction were confirmed by
nucleotide sequencing. Control plasmids expressing BD-Tat and AD-Grrl
have been described (16).
The liquid culture
assay of -galactosidase activity has been described (16).
Since the G protein subunits were targets for domains specific
to two effectors (12), we wanted to know whether the
subunits were
also able to bind to a domain specific to another effector,
phospholipase C-
2. PLC62 is a domain with 62 residues specific to
phospholipase C-
2. This domain was able to bind to the
complex directly as a fusion protein (9). We first examined whether the
PLC62 domain was able to interact with the G protein
subunits. In
our study, PLC62 was a hybrid with the DNA binding domain of the Gal4
transcription factor, and the
subunits were hybrids with the
activation domain of Gal4. These hybrids were co-expressed in a yeast
strain containing a reporter gene that encodes
-galactosidase. If
PLC62 interacts with the
subunits, the Gal4 transcription factor
domains will be brought together to activate the expression of the
reporter gene. When the PLC62 domain was co-expressed with the
subunits in yeast cells, the reporter gene was significantly activated,
with higher reporter activities in cells expressing
1,
2, and
5 subunits (Fig. 1A). This is consistent
with reports that a
complex containing either
1 or
5 was
able to activate phospholipase C-
2 (4, 21). The different reporter
activities elicited by PLC62 with different
subunit types may be
due to differential interaction between PLC62 and the
subunit
types. Co-expression of PLC62 with different
subunit types did not
significantly activate the reporter gene (Fig. 1B). We then
tried to identify a region of the
subunit that interacts with PLC62
by co-expression of this effector domain with various regions of the
1 subunit. The
1 subunit was split into four different fragments
using an appropriate restriction site as described before (12).
Fragments included the following regions of the protein.
1A,
residues 1-100;
1B, residues 101-187;
1C, residues 188-261;
and
1D, residues 262-340. The N-terminal fragment (
1A, residues
1-100) of the
1 subunit interacted with PLC62 as effectively as the
whole
subunit (Fig. 1C).
1A also interacted more
effectively with
subunits than the entire
1 subunit (Ref. 12 and
data not shown). Although PLC62 did not interact with other regions of
the
1 subunit, this does not rule out the possibility that these
fragments may be folded improperly in yeast. Together with our previous
finding (12), these results strongly indicate that the N-terminal 100 residues from the
1 subunit contain subdomains that interact with
three different effectors. Are these putative subdomains in
1A
distinct structural determinants or overlapping regions?
Based on the crystal structure of the G protein, the subunit is a
propeller composed of seven
sheets with an N-terminal
helix.
1A, a 100-residue fragment from the N terminus of the
1 subunit,
encompasses an N-terminal
helix, five
strands, and a loop
connecting the
helix and the
strands (18, 20). The N-terminal
helix of the
1 subunit forms the beginnings of a parallel
coiled-coil with the N-terminal
helix of the
subunit. The first
of the
strands is the outer strand of the seventh
sheet in the
propeller structure of the
1 subunit. The next four
strands form
the first
sheet. Both the
helix and/or the
strands could be
involved in interaction with these effector domains.
To test if the N-terminal helix is involved in interaction with
effectors, we introduced mutations into
1A that have the potential
to destroy the
helix. The
helix is stabilized by hydrogen bonds
between the NH and CO groups of the main chain. The proline side chain
is different from others in that its side chain is bonded to both the
nitrogen and
-carbon atoms to form a ring structure. This prevents
the N atom of proline from participating in hydrogen bonding and should
form steric obstacles to the formation of an
-helix. The
helix
situated at the N terminus of the
subunit is amphipathic and forms
part of a coiled coil with a similar amphipathic helix from the N
terminus of the
subunit. In the first mutant of
1A,
1Am1, we
therefore chose to substitute three leucines in the
helix along the
hydrophobic surface of the helix (Fig. 2A).
These leucines (Leu-4, Leu-7, and Leu-14) in
1A were substituted
with prolines. We then co-expressed
1Am1 with the
subunit and
each of the effector domains in yeast cells. The reporter activities
induced by
1Am1 in combination with the
subunit or the effector
domains are shown in Fig. 2, B-E. As expected based on the
three-dimensional structure for the
complex, the ability of
1Am1 to interact with the
subunit was dramatically decreased
(Fig. 2B). The reporter activity stimulated by co-expression of
1Am1 and
5 is about one-tenth of that seen in cells
co-expressing
1A and
5. It is highly unlikely that this result is
due to altered expression of the mutant protein since we have
repeatedly examined the expression of a variety of hybrids and found
them to be expressed at similar levels (12, 16). The reduced
interaction could result from both the loss of leucines at the binding
surface and the disruption of the helix due to proline substitutions.
We do not have direct biophysical results that show disruption of the N-terminal
helix of the
subunit, but, based on the effect of
proline substitutions on the formation of an
helix, it is most
likely that the helix is disrupted. Although the N-terminal
helix
that is required for interaction with the
subunit was potentially
destroyed by the proline substitutions, the ability of
1Am1 to
interact with AC2Q, GKN, and PLC62 was decreased less than 20% (Fig.
2, C-E). This indicates that the regions of
1A that
interact with the effector domains were not significantly affected by
potential disruption of the amphipathic
helix. The ability of the
mutations in
1Am1 to affect interaction with the
subunit is
consistent with previous results from mutant analysis of
-
interaction (22).
Since the results above indicated that residues in the N-terminal helix of
1A were unlikely to be involved in the interaction with
AC2Q, GKN, and PLC62, we investigated whether the appropriate folding
of the
strands in
1A was a requirement for interaction with the
effector domains. To address this question, we made the second mutant
of
1A-
1Am2. The
subunit is made up of seven WD repeats of
about 40 amino acids each (23). Based on the crystal structure of the
subunit, four residues that are largely conserved within each of
the seven WD repeats have been implicated in forming hydrogen bonds
that are important for stabilizing the characteristic folded structure
of
strands (18, 20). In the mutant
1Am2, three of these
residues, Ser-72, Asp-76, and Trp-82, which form hydrogen bonds were
replaced with alanines. In the crystal structure of the
complex,
the
strands 2-5 of the
subunit form the first
sheet in the
G protein
subunit. Ser-72 is within the third
strand, Trp-82 is
within the fourth
strand, and Asp-76 is within the loop connecting
the third and the fourth
strand (Fig.
3A). Hydrogen bonding occurs between Asp-76
and His-54 which is located in the loop connecting the first and second
strands (Fig. 3A). Thus, a turn in the first
sheet
is coupled to the first
strand. Ser-72 is hydrogen-bonded with
Trp-82 as well as His-54. This bonding couples the third with the
fourth
strand as well as the loop downstream of the first strand.
Trp-82 is also in the hydrophobic core of the first
sheet and in
contact with several residues. The folded structure formed by the five
strands in
1A is therefore likely to be disturbed significantly by altering Ser-72, Asp-76, and Trp-82 to alanines in the mutant
1Am2.
We first tested whether the 1Am2 mutant was able to interact with
the
subunit by co-expressing
1Am2 with
5 in yeast cells.
1Am2 interacted with
5 as effectively as
1A (Fig.
3B), indicating that the mutations in the region of the
strands do not affect complex formation with the
subunit. Combined
with the results from the
1Am1 mutant, this implies that residues in
the
helical region of
1A are major determinants for interaction
with the
subunit. Wild type
1A elicited significant reporter
activity with the three effector domains, AC2Q, GKN, and PLC62 (Fig. 3, C, D, and E). The magnitude of
activity induced by different combinations was very different,
consistent with our previous results (12). The higher activity induced
by
1A with AC2Q compared to the other two domains is most likely due
to differential binding. This is based on our previous results that
show direct relationship between reporter activity elicited by a
particular combination of hybrids and efficiency of complex formation
between the same hybrids as determined by immunoprecipitation (16).
Furthermore, the lower reporter activity elicited by
1A with GKN and
PLC62 should result from significant protein-protein interaction
because these effector domains do not show significant reporter
activity when co-expressed with a variety of control proteins,
unrelated proteins,
subunit types, or the
1B-D fragments. The
differences in reporter activities induced by the GKN and PLC62 domains
with
1A in comparison with a variety of controls are clearly
apparent in filter assays where yeast cells have been examined for
-galactosidase activity (Ref. 12 and data not shown). When the
mutant,
1Am2, was co-expressed with GKN or PLC62 in yeast cells,
1Am2 was significantly affected in its interaction with these two
effector domains, and the efficiency of interaction was decreased
3-4-fold (Fig. 3, D and E). As in the case of
1Am1, it is highly unlikely that this result is due to altered
expression of the mutant protein since we have examined the expression
of a variety of hybrids and found them to be expressed approximately at
similar levels (12, 16). It is possible that the residues (Ser-72,
Asp-76, and Trp-82) themselves are directly involved in binding to both
GKN and PLC62. Altering these residues in
1A would therefore result
in defective interaction with the effector domains. However, we think
it is more likely that interaction of
1Am2 with GKN and PLC62 is
affected because the folding of the
strands in
1A is disrupted.
This inference is based on the following. (i) Ser-72 and Trp-82 are
located within the
sheet formed by
strands 2-5 where they are
unlikely to be accessible to an effector domain. (ii) It seems unlikely
that we could have at random mutated precisely those residues that are
critical for binding with GKN and PLC62. Moreover, the insignificant
effect of the L4P, L7P, and L14P mutations on
1Am1 binding with GKN and PLC62 show that mutational alterations of
1A do not in general lead to gross misfolding that renders the protein incapable of interaction with other proteins. The effective interaction of
1Am2
with AC2Q (Fig. 3C) further emphasizes the specificity of the effect of mutations in
1A on effector domain interactions.
Thus, the results from the analysis of the 1Am2 mutant imply that
residues in
1A that interact with GKN and PLC62 require appropriate
folding of the first five
strands of the
subunit to be able to
bind these effector domains. Based on the ability of the
subunit to
disrupt binding of an effector with the
complex, candidates for
such residues are those that are positioned in the bends between the
strands 1-2, 3-4, and 5-6 as well as the exposed portions of the
strands located at the same surface of the
complex which
interacts with the
subunit.
We have identified an N-terminal fragment of 100 residues from the G
protein subunit that is able to interact with domains specific to
three different effectors, AC2, GIRK1, and PLC-
2. Within this
fragment, the structural determinant for interaction with AC2 seems to
be different from that for interaction with GIRK1 and PLC-
2 because
of the following reason. Potential disruption of the folded structure
of the
strands that should occur in the
1A fragment of the
subunit has no significant effect on the interaction of the mutant
1A with AC2Q. But potential disruption of the folded structure of
the
strands in the mutant
1Am2 significantly affects interaction
of this mutant
1A with GKN and PLC62. It is still likely that the
structural determinant(s) for interaction with AC2Q are in the exposed
surfaces formed by the folding of the five
strands. A domain
(residues 52-100) of the
1 subunit that contains
strands 2-5
(which form the first
sheet) was able to interact with AC2Q in the
two-hybrid system, although not as effectively as
1A (data not
shown). Cross-linking and modeling implicate residues 75-165 of the
subunit in binding with the AC2Q peptide (17). A peptide specific
to residues 86-105 of the
1 subunit has also been shown to inhibit
interaction of the
complex with adenylyl cyclase 2 in a
sequence-specific manner.2 These results
further confirm a role for residues in the structure formed by the
first five
strands in interaction with AC2. The simplest
interpretation of our results is that these residues do not require the
formation of the folded structure of interacting
strands to be able
to bind AC2Q.
The presence of different structural determinants for AC2Q
versus GKN/PLC62 in 1A suggests that this may be one
mechanism that the
complex uses to discriminate between
structurally distinct effectors. Similarly, mutations at the C terminus
of the
subunit affected PLC
2 activation but not activation of the mitogen-activated protein kinase pathway (24). The specificity of
interaction of the
complex with GIRK1 and PLC-
2 could be affected by domains other than those identified here. It was reported that two domains of GIRK1 were able to bind to the
complex, an
N-terminal domain and a C-terminal domain (7, 8). GKN is the N-terminal
domain of GIRK1 that is able to bind to the N-terminal fragment (
1A)
of the
1 subunit. The C-terminal domain of GIRK1 could bind to
another site on the
complex that has not been identified yet.
This second site on the
complex may be important for specific
interaction between the
complex and GIRK1.
We have begun to understand the individual roles of the G protein and
subunits in cellular signaling. It is likely that the
subunit plays an important role in receptor interaction (25, 26). The
results here together with our previous findings (12) indicate an
important role for the
subunits in effector interaction. Results
from other laboratories also support this role. The AC2Q peptide was
chemically cross-linked to the
subunit (17), and the extreme
C-terminal domain of the
subunit was important for activation of
phospholipase C-
2 in COS cells (24). Since prenylation of the
subunit is essential for appropriate interaction with effectors
(e.g. Refs. 4 and 27), the lipid group at the C terminus of
the
subunits could also directly interact with a site on the
effector molecules.
We thank Dr. P. Gierschik for the PLC-2
cDNA and Dr. R. Iyengar for communicating results prior to
publication.