(Received for publication, October 19, 1995; and in revised form, December 20, 1995)
From the
A series of chimeras between a constitutively active mutant of
the -subunit of G
and the
-subunit of G
was constructed to identify the domains in
specifically involved in interaction with its effector
phosphoinositide phospholipase C (PLC). Transient expression of the
chimeric proteins and measurement of the production of inositol
phosphates and cAMP in HEK-293 cells revealed that the
Ile
-Lys
sequence of
contained the PLC interaction sites, whereas the residues for
activation of adenylyl cyclase were in the
Ile
-Leu
sequence of
.
Alanine scanning mutagenesis of the Ile
-Lys
region of
further identified two clusters of
amino acids (Asp
,Asn
,Glu
and
Arg
,Thr
) that were specifically required
for interaction with PLC.
Comparison of the sequences of
,
, and
showed that
the PLC-interacting residues identified in
are
different from the corresponding residues in
and
that are involved in effector activation. Alignment
of the sequences of
and
, based on
the crystal structure of
(Noel, J. P., Hamm, H. E.,
and Sigler, P. D.(1993) Nature 366, 654-663), indicated
that the PLC-activating residues of
are located in
-helix 3 and its linker to
-sheet 4, which are adjacent to a
switch region whose conformation changes with activation. It is
proposed that the selectivity of
for PLC involves
relatively few amino acids, but that the effector may interact with
other nonselective sequences in the
-subunit.
Heterotrimeric GTP-binding proteins (G-proteins) transduce
signals from certain cell surface receptors to various intracellular
effectors (1, 2, 3, 4) . Upon
agonist binding, the receptors promote the exchange of GTP for GDP on
the G-protein -subunit. As a result, the subunit undergoes
conformational changes which promote the dissociation of the
complex. The GTP-liganded
-subunit and the free
complex
can then modulate the activity of various intracellular enzymes and ion
channels(1, 2, 3, 4) .
Biochemical and mutational analyses of G-protein -subunits have
made it possible to assign specific functions to different domains in
the polypeptide. Regions involved in GTP binding and hydrolysis,
receptor and
recognition, and guanine nucleotide-induced
conformational changes have been identified by expression of mutant and
chimeric proteins(5) . By employing chimeric proteins in which
regions of
were replaced with cognate regions of
, Osawa et al.(6) and Berlot and
Bourne (7) revealed that 121 amino acids between Gln
and Arg
of
contained the adenylyl
cyclase activating region. Using the complementary approaches of
homolog and alanine scanning mutagenesis, Berlot and Bourne (7) further identified four noncontiguous stretches of amino
acids within this region that were critical for activation of adenylyl
cyclase. Studies by Hamm and associates (8) using synthetic
peptides also demonstrated that a peptide corresponding to
residues Glu
to Glu
stimulated cGMP
phosphodiesterase directly. Furthermore, site-directed mutagenesis of
Trp
of
abolishes the interaction with
the
-subunit of the phosphodiesterase(9) . However, until
very recently, there were no reports of the domains of
involved in the interactions with PLC. (
)Then,
Arkinstall et al.(10) mapped the regions of
that interact with recombinant PLC
1 using
multiple overlapping synthetic peptides. With this approach, they
identified peptides corresponding to two distinct regions of
(Ser
-Gln
and
Ala
-Asp
) that inhibited
G
-mediated PLC activation. Based on the crystal structure
of
(11) and findings with chimeras between
human and Xenopus
(12) , it has been
proposed that the helical domain of these
-subunits encodes yet
another effector-interacting domain.
In the present study, we
attempted to define more precisely the domain and amino acid residues
in that are responsible for regulating PLC activity.
Taking advantage of the similarity in the primary structures of
and
(43% identical) and the
completely different second messenger pathways that they regulate, we
constructed a series of chimeras between these
-subunits and
transiently expressed them in HEK-293 cells. The results indicated that
the sequence Ile
-Lys
of
encoded the PLC recognition site. Alanine scanning mutagenesis
further identified two clusters of amino acids
(Asp
,Asn
,Glu
) and
(Arg
,Thr
) as being specifically involved in
PLC interaction.
Figure 1:
Chimeric constructs.
Diagrammatic representation of chimeras between and
. The regions corresponding to
are
the open segments, whereas those corresponding to
are the dark-striped segments. The numerical scale
at the bottom is relative to the
sequence. The numbers inside the chimeras indicate the residues at the
beginning and end of segments derived from
; the numbers on the outside refer to residues of
. The mutations Q209L and R201C in
and
subunits, respectively, render the
-subunits constitutively active.
The used
in the construction of the chimeras was mutated
(
Q209L). This mutation renders the polypeptide
constitutively active by inhibiting its intrinsic GTPase(18) .
Transfection of HEK-293 cells with vector containing no insert,
wild-type and constitutively active
(
R201C) had very little effect on IP production (Fig. 2). Expression of
Q209L resulted in a
very large elevation of IP compared with wild-type
,
as expected. Expression of chimeras 1, 2, and 3 also resulted in
elevated levels of IP, comparable to that obtained with
Q209L. Chimeras 4 and 5, however, failed to increase
IP production above the basal level seen with vector alone. Fig. 3shows the accumulation of cAMP in HEK-293 cells
overexpressing
R201C,
Q209L, and the
various
/
chimeras. The results
indicate that, besides
R201C, only chimera 4 behaved
as a functional
. Expression of this chimera produced,
in fact, a higher level of cAMP than did
R201C. These
data are consistent with a previous report showing that the amino acids
required for activation of adenylyl cyclase are discontinuously spaced
in a domain from Gln
to Arg
of
(7) . The only chimera that contained this
entire domain was chimera 4 (Fig. 1). The data of Fig. 2and Fig. 3also demonstrate that chimeras 1-4
were all functional. From the results obtained with chimeras 3 and 4,
it can be concluded that the sequence Ile
-Lys
in
encodes a domain that is required for
interaction of
with PLC.
Figure 2:
Accumulation of inositol phosphates in
HEK-293 cell transfectants. Cells were transfected with 2.5 µg/ml
pCMV4 or pCMV4 containing ,
Q209L,
R201C, or the chimeric cDNA. Cells were labeled for 24
h with myo-[
H]inositol (2 µCi/ml),
and the IP formation was measured as described under ``Materials
and Methods.'' Each value represents the mean ± S.E. of 3
to 6 independent experiments.
Figure 3:
Accumulation of cAMP in HEK-293 cell
transfectants. Cells were transfected with 2.5 µg/ml pCMV4 or pCMV4
containing ,
Q209L,
R201C, or the chimeric cDNA. Cells were labeled for 24
h with [
H]adenine (5 µCi/ml), and cAMP
synthesis was measured 24 h later after incubating with
1-methyl-3-isobutylxanthine for 25 min at 37 °C, as described under
``Materials and Methods.'' Each value represented the mean
± S.E. from 3 separate experiments.
Although the data of Fig. 2and Fig. 3indicated that the full-length forms of
and
and chimeras 1-4 were
functionally expressed in the HEK-293 cells, this was not evident for
chimera 5. Fig. 4shows Western blots of membrane preparations
from cells transfected with full-length and chimeric forms of
using an
-specific antiserum. In
cells transfected with vector (pCMV4) alone, a low level of a 42-kDa
protein was detected which was presumably endogenous
.
Transfection of the cDNAs resulted in varying levels of expression of
recombinant protein. The molecular masses of chimeras 1 and 2 were
similar to that of
, as expected (Fig. 1).
However, the relative mobility of chimeras 3 and 4 was slower because
the
segment contains additional amino acids in its
sequence (Fig. 1). The level of 42-kDa protein corresponding to
chimera 5 that was detected on the immunoblot was low, and there was a
band of lower molecular mass, which probably represents a proteolytic
fragment (Fig. 4).
Figure 4:
Autoradiography of immunoblots showing
transient expression of G and chimeric
-subunits.
HEK-293 cells were transiently transfected with pCMV4 alone or pCMV
carrying
Q209L or chimeric cDNA. Membrane proteins (20
µg/lane) were resolved on SDS-14% polyacrylamide gels, transferred
to Immobilon, incubated with the
-specific antiserum
E973, and developed as described under ``Materials and
Methods.''
It is clear from Fig. 4that the
extent of expression of the proteins was very different, but these
differences were seen consistently. Since transfection of twice the
amount of cDNA did not alter the pattern of IP production seen in Fig. 2(data not shown), it appears that sufficient protein was
expressed with chimeras 1-4 to provide the same large stimulation
of PLC or adenylyl cyclase as seen with Q209L or
R201C.
The ability of an -subunit to activate
its effector is dependent on its capability to bind GTP and assume an
active conformation. It has been shown for other
-subunits, e.g.
(19) ,
and
(20) , and
(21) ,
that the conformational change induced by GTP binding results in
decreased susceptibility to trypsin degradation. Although the data of Fig. 2and Fig. 3indicated that
Q209L
and chimeras 1-4 were capable of binding GTP, it was necessary to
demonstrate this for chimera 5. Fig. 5shows the effect of
GTP
S on trypsin digestion of the in vitro translated
proteins in a reticulocyte lysate. In vitro translation of
Q209L and chimeras 1, 2, and 5 generated a major
42-kDa labeled band. Incubation with 25 µg/ml trypsin resulted in
complete or almost complete degradation of the translated products.
However, the extent of protection by GTP
S was slight, reflecting
the very low affinity of
for GTP analogs in the
absence of
subunits and receptors(22) . Data are
absent for chimeras 3 and 4 since transcription/translation of these
was negligible.
Figure 5:
Tryptic digestion of in vitro translated GQ209L and chimeric polypeptides. The in vitro translation mixture (5 µl) (see ``Materials
and Methods'') was incubated at 30 °C for 30 min in the
absence(-) or presence (+) of 125 µM GTP
S
prior to digestion with 25 µg/ml trypsin at 30 °C for 30 min.
The control without trypsin is shown in the first lane of each
group. The reaction was terminated by the addition of 2
SDS-PAGE sample loading buffer. The
S-labeled proteins
were separated by SDS-PAGE and autoradiographed as described under
``Materials and Methods.''
Figure 6:
Alanine scanning mutagenesis. The sequence
at the top is that of residues
215-276. The sequence below it is that of
, with
residues identical with
being represented by dashes. The numbered sequences below this represent
the 14 separate mutant constructs in which nonidentical residues were
mutated to alanine. All mutants were constructed in the
context with the
Q209L
mutation.
Figure 10:
Alignment of partial sequences of
,
, and
. Shown are
the putative effector interaction sequences (underlined
sequences) and the locations of the
-helices,
-sheets,
and switch region determined from structural studies of
(23, 24) . This figure is modified from Fig. 1e in (24) . The residues identified in
the present study and (7, 8, 9, 10) and 12 as being
involved in effector activation are underlined beneath each
sequence. The double-underlined sequences represent residues
identified in the present study. Linker sequences between
-helices
and
-sheets have been omitted for
clarity.
Figure 7:
Effects of mutations in G on inositol phosphate accumulation in HEK-293 cell transfectants.
Cells were transfected with 2.5 µg/ml pCMV4 alone or pCMV4 carrying
mutant
cDNA. Cells were labeled for 24 h with myo-[
H]inositol (2 µCi/ml), and the
IP accumulation was measured as described under ``Materials and
Methods.'' Each value represents the mean ± S.E. of three
independent experiments.
Figure 8:
Autoradiography of the immunoblots showing
transient expression of mutant G polypeptides. HEK-293
cells were transiently transfected with pCMV4 alone or pCMV4 carrying
mutant
cDNA. The other procedures were as described
in the legend to Fig. 4.
Figure 9:
Tryptic digestion of in vitro translated G mutant polypeptides. The procedures
were the same as those described in the legend to Fig. 5.
The data of Fig. 8and Fig. 9indicate that the failure of mutant 4 to stimulate PLC was
probably due to the effect of the mutation on expression of the protein
and its capacity to bind GTP. The inability of mutant 8 to activate the
enzyme may likewise be due to poor GTP binding. For these reasons, the
only residues that are clearly implicated by the present study as being
involved in the selective interaction of with PLC are
Asp
, Asn
, Glu
,
Arg
, and Thr
.
The present studies with /
chimeras indicated that a stretch of amino acids between
Ile
and Lys
in
was
required for PLC activation. The data (Fig. 2) were very clear
cut. Chimera 3, having the entire C terminus from Asp
to
Val
replaced by the corresponding sequence from
, was fully active, whereas chimera 4, with the
C-terminal sequence from Ile
to Val
being
replaced, was completely inactive. This chimera was still capable of
fully activating adenylyl cyclase (Fig. 3), indicating that it
was adequately expressed and inserted into the membrane, and assumed a
configuration that permitted GTP binding.
The conclusion that the
sequence between Ile and Lys
was required
for
activation of PLC was consistent with the data
with chimera 5. However, Western blotting (Fig. 4) indicated
that this chimera was poorly expressed in the membranes and underwent
proteolytic degradation. The trypsin proteolysis data also indicated
that the protection by GTP
S was minimal (Fig. 5). Thus, the
data with chimera 5 are not conclusive.
The alanine mutagenesis
studies applied to the Ile-Lys
sequence
indicated that four mutants had a drastically impaired capacity for
activating PLC (Fig. 7). However, one of these was poorly
expressed (Fig. 8) and another did not bind GTP
S when
expressed in a reticulocyte lysate (Fig. 9). Thus, the data only
unequivocally support the specific involvement of five residues
(Asp
, Asn
, Glu
,
Arg
, Thr
). In assessing these data, it has
to be emphasized that the overall purpose of the study was to identify
residues specifically involved in PLC activation. Thus, the 25
conserved residues and the 3 other residues that are common to
and
were not mutated. A
priori, these residues cannot be specific determinants,
and any loss of function due to their mutation could be due to changes
in the overall structure of the
-subunit. (
)
It should be noted that the possibility cannot be rigorously excluded that the specific loss-of-function mutations identified in the present study prevent an activating conformational change rather than interfere with effector interaction. Although alanine substitution is reputed to produce minimal changes in the conformation of polypeptides, structural studies will be required to prove definitively that the mutations did not interfere with the conformational changes resulting from activation.
Arkinstall et al.(10) used multiple
overlapping synthetic peptides to block the interaction of in a mixture of homogenates of yeast and Sf9 cells expressing
PLC
1 and
and refined their data to show that the
amino acids required for PLC activation were confined to sequences
Ser
-Gln
and Ala
-Asp
in
(underlined in Fig. 10). The
first sequence contains two of the residues (Arg
,
Thr
) identified in the present study (doubly
underlined in Fig. 10), but the second sequence is
contained entirely within the domain of
that was
replaced by
in chimera 3 (Fig. 1). Since this
chimera was fully active in stimulating PLC (Fig. 2), there is a
discrepancy between the two sets of data. Since the two studies used
entirely different methodologies, there may be many technical reasons
for the discrepancy. However, it should be noted that the specificity
of the peptides used by Arkinstall et al.(10) was
not established, i.e. they could have blocked the interactions
of other
-subunits with their effectors. (
)Thus, if, in
fact, a PLC interaction site is present in the
Ala
-Asp
sequence, it may not be specific.
It is interesting to compare the present data with the similar study
by Berlot and Bourne (7) who extended the investigation of
Osawa et al.(6) using /
chimeras to define the domains in
required for
interaction with adenylyl cyclase. These studies revealed that the
sequence Gln
-Arg
was sufficient to activate
the enzyme. Homolog and alanine mutagenesis (7) further
identified the following clusters of residues as specifically required
for adenylyl cyclase activation: Gln
-Asp
,
Trp
-Ile
, and
Ser
-Arg
. The
sequence
(His
-Asn
) corresponding to
Gln
-Asp
is not involved in PLC
activation ( Fig. 7and Fig. 10and (10) ), and
the
sequence (Asn
-Ile
)
corresponding to
Ser
-Arg
(Fig. 10) was not identified for PLC activation in the
present study and only partly overlaps the
Ala
-Asp
sequence reported by Arkinstall et al.(10) . Thus, the individual residues specifying
PLC and adenylyl cyclase activation occupy different positions in the
aligned sequences of
and
(Fig. 10) and hence are in different locations in their
three-dimensional structures (see below).
In another study of the
domains in required for adenylyl cyclase activation,
Antonelli et al.(12) employed human-Xenopus chimeras of
. This approach indicated an
additional requirement for activating residues in a sequence between
Gly
and Lys
, a conclusion reached for
on structural grounds by Coleman et
al.(11) . Antonelli et al.(12) proposed
that previous chimeric studies (6, 7) failed to
recognize the need for this sequence because the
segment used for the constructs preserves the structures needed
for activation of adenylyl cyclase. The possibility that the
Gly
-Lys
sequence can be
substituted from
would mean that it cannot be
exclusive for
. (
)
In the study of Rarick et al.(8) who used synthetic peptides to mimic the
ability of to activate cGMP phosphodiesterase,
activation was observed with a peptide corresponding to the
Glu
-Glu
sequence. Fig. 10shows that
this sequence overlaps corresponding sequences in
and
identified by Arkinstall et al.(10) and Berlot and Bourne (7) as being partly involved
in the activation of PLC and adenylyl cyclase, respectively. The
failure of Rarick et al.(8) to identify the residue
(Trp
) recognized by Faurobert et al.(9) could be due to the approach used. Thus, it is
possible that a combination of the Glu
-Glu
peptide with one containing Trp
may have provided a
greater stimulation than seen with Glu
-Glu
alone.
For full understanding of the above results, it is
necessary to relate the sequences to their location in the
three-dimensional structures of the various -subunits. Although
the crystal structures of
and
liganded to GDP, GTP
S, or GDP-AlF
are known(11, 23, 24, 25) ,
those of
and
have not been
reported. However, the three-dimensional structure of
has been modeled, based on the structure of GTP
S
(10) . (
)
The main point of the present
study was to identify the residues specifically involved in the
interaction of with PLC, and the data suggest that
the selectivity is encoded by residues in
-helix 3 and the linker
between this helix and
-sheet 4, based on sequence alignment (Fig. 10) and computer modeling(10) .
This
is different from
where the activating residues are
confined to
-helix 4 and the linker to
-sheet
6(8, 23) . The situation with adenylyl cyclase is more
complex, with the involvement of residues in
-helices 2, 3, and 4
and the linkers between
-helix 2 and
-sheet 4 and between
-helix 3 and
-sheet 5(7, 23) . As pointed
out by Hamm, Sigler and associates(23, 24) , the
residues in the four linkers and three
-helices that are involved
in effector activation define a contiguous surface on the
-subunit.
Whereas the present study and that of Berlot and
Bourne (7) were selective in that they compared with
and
with
, the other two reports (8, 10) did
not test the peptides on other effector systems. Thus, it is possible
that some of the domains identified in the latter
studies(8, 10) , especially those in
-helix 4 and
the adjacent linker to
-sheet 6 may contain sequences that are
required, but are not selective, for the activation of given effectors.
Because of such a lack of selectivity, it is possible that the
sequences could be substituted from one
-subunit to another in
chimeric studies without apparent loss of effector activation. This
explanation does not, of course, exclude the possibility that the
domains could also include other sequences that designate
selectivity for some effectors, e.g. residues in the linker
between
-helix 4 and
-sheet 6 that are specifically required
for adenylyl cyclase or cGMP phosphodiesterase. In short, it is
proposed that the interaction of
-subunits with their effectors
could involve both highly selective sequences and nonselective
sequences.
Finally, it is of interest to relate the domains involved
in effector interaction with those whose conformation changes when the
-subunit is converted to the active state by GTP
S binding.
Lambright et al.(24) , in their crystallographic
studies of
, have demonstrated that the largest
conformational changes resulting from activation occur in three domains
designated switches I-III. Switch I is located in a linker between the
-helical and GTPase components of the
-subunit, whereas
Switch II includes part of
-sheet 3, all of
-helix 2, and its
linkers to
-sheet 3 and 4 (24) . Switch III includes most
of the linker between
-sheet 4 and
-helix 3(24) .
Since all of the critical residues for the conformational switches
in are present in the other
-subunits(24) , it is anticipated that similar switching
mechanisms hold for these. Accordingly, Switches II and III would
correspond to sequences Val
-Thr
and
Asp
-Arg
in
( (24) and Fig. 10). Switch III would thus include or be
adjacent to the sequences (Asp
-Glu
and
Arg
,Thr
) in
-helix 3 and the adjacent
linker that have been implicated in the activation of PLC by the
present study. Thus, it is not difficult to conceive that the
conformational changes resulting from activation of
would be transmitted to the sites that specifically interact with
PLC via Switch III. (
)Understanding how this leads to
activation of the enzyme will require a more precise definition of the
residues in PLC that interact with
and knowledge of
the three-dimensional structure of the mammalian enzyme. (
)
Note
Added in Proof-In a recent study, Skiba et al. (28)
showed that the most important site for interaction of with the
-subunit of cGMP phosphodiesterase involves a
sequence encompassing
-helix 3 and the
/
loop, which corresponds closely to
the PLC interaction sequence identified in
in the
present study.