(Received for publication, January 22, 1997, and in revised form, April 7, 1997)
From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026
The G protein subunits,
s and
i2, have stimulatory and
inhibitory effects, respectively, on a common effector protein, adenylyl cyclase. These effects require a GTP-dependent
conformational change that involves three
subunit regions (Switches
I-III).
s residues in three adjacent loops, including
Switch II, specify activation of adenylyl cyclase. The adenylyl
cyclase-specifying region of
i2 is located within a
78-residue segment that includes two of these loops but none of the
conformational switch regions. We have used an alanine-scanning
mutagenesis approach within Switches I-III and the 78-residue segment
of
i2 to identify residues required for inhibition of
adenylyl cyclase. We found a cluster of conserved residues in Switch II
in which substitutions cause major losses in the abilities of both
i2 and
s to modulate adenylyl cyclase activity but do not affect
subunit expression or the GTP-induced conformational change. We also found two regions within the 78-residue segment of
i2 in which substitutions reduce the ability
of
i2 to inhibit adenylyl cyclase, one of which
corresponds to an effector-activating region of
s. Thus,
both
i2 and
s interact with adenylyl
cyclase using: 1) conserved Switch II residues that communicate the
conformational state of the
subunit and 2) divergent residues that
specify particular effectors and the nature of their modulation.
Upon activation by cell surface receptors, heterotrimeric G
proteins transmit signals to effector proteins that regulate a wide
variety of cellular processes (1-4). Receptors activate G proteins by
catalyzing the replacement of GDP bound to the subunit with GTP,
resulting in dissociation of
·GTP from the
subunits. The
GTPase activity of the
subunit regulates the timing of deactivation
and reassociation of the G protein subunits. The fidelity of cellular
signaling requires that
subunits modulate effector proteins only
when bound to GTP and that only the appropriate
subunit-effector
pairs interact. GTP-dependent effector interaction most
likely involves one or more of the three
subunit regions that
change conformation during the GTPase cycle (Switches I-III), identified by comparison of the x-ray crystal structures of the GTP
S-bound1 (active) and GDP-bound
(inactive) forms of
t (5, 6) and
i1 (7,
8). Differences in the amino acid sequences of the structurally
conserved
subunits (40% identity at the amino acid level, with
60-90% identity within subfamilies) determine the specificity and
nature of their interactions with effector proteins (9). However, the
relationship between the molecular determinants of effector specificity
and of GTP-dependent effector regulation is poorly
understood.
Regulation of adenylyl cyclase by the G protein subunits,
s and
i, raises issues specific for this
subunit-effector interaction.
s and
i, which are relatively poorly conserved among the
family of
subunits (~40% identical amino acids), both bind to
adenylyl cyclase but have opposite effects on activity. Inhibition of
adenylyl cyclase by
i requires prior activation by
s, forskolin, or calmodulin (10, 11). Since adenylyl
cyclase can be inhibited by
i in the absence of
s, inhibition does not appear to be due to competition
between
i and
s for binding to adenylyl
cyclase. Indeed, there is evidence that suggests that adenylyl cyclase
has distinct binding sites for
s and
i
(11). Key questions that arise are: why does
s activate
and
i inhibit, and why do only
s and
i, but not other
subunits, modulate adenylyl cyclase
activity?
The s residues that specify activation of adenylyl
cyclase are located in three adjacent loops, one of which includes
Switch II (12). The location of a conformational switch region within the effector-specifying surface of
s provides a simple
mechanism for the GTP-dependence of the
s-adenylyl
cyclase interaction. However, studies with chimeric
subunits
containing portions of
i2 and
q, which
does not interact with adenylyl cyclase (13), showed that an
q/
i2/
q chimera containing
only 78 residues of
i2 (residues 245-322) inhibits
adenylyl cyclase as well as
i2 does (14). This
78-residue effector-specifying segment includes residues homologous to
two of the three clusters of
s residues that specify
activation of adenylyl cyclase (12, 15) but does not include any of the
conformational switch regions. This was a surprise since the GTP-bound
form of
i is much more effective at inhibiting adenylyl
cyclase than the GDP-bound form is (11). However, the importance of the
conformational switch regions might have been missed using a chimeric
subunit approach due to the high degree of sequence similarity in
these regions between
q and
i2.
To determine whether any of the conformational switch regions are
involved in inhibition of adenylyl cyclase by i2, we
substituted alanines for solvent-exposed residues in these regions. We
tested the effect of these mutations on both the inhibition of adenylyl cyclase and the ability of the mutant proteins to achieve the activated
conformation as measured by the acquisition of trypsin resistance upon
binding of GTP. We identified a part of Switch II that is conserved
among
subunits in which alanine substitutions blocked the
inhibition of adenylyl cyclase by
i2. We also found that
substitutions of alanines for the corresponding
s
residues specifically prevent activation of adenylyl cyclase. Thus it
appears that both
i2 and
s interact with
adenylyl cyclase using two types of residues: 1) conserved residues
within Switch II that signal that the
subunit is in the GTP-bound
active conformation and 2) divergent residues that specify activation
or inhibition of this effector enzyme.
To identify the i2 residues involved in specifying
inhibition of adenylyl cyclase, we substituted alanines for
solvent-exposed residues within the 78-residue segment. We found two
regions of sequence in which mutations impaired the ability of
i2 to inhibit adenylyl cyclase, the amino terminus of
3 and the
4/
6 loop. The
4/
6 loop is also important for
the effector interactions of
s (12) and
t
(16, 17). These substitutions did not cause as much of a decrease in
adenylyl cyclase inhibition as the Switch II mutations did, suggesting
that Switch II residues are the primary contributors to the interaction
between
i2 and adenylyl cyclase.
i2 mutants were
constructed from the mouse
i2 cDNA (18), and
s mutants were constructed from the rat
s
cDNA (19). Two modifications were made to each of the
subunits
to facilitate detection of their activities and expression levels. The
arginine at position 179 in
i2 and 201 in
s was mutated to cysteine to inhibit GTPase activity and
produce constitutive activation (20, 21). An epitope, referred to as
the EE epitope (22) was generated by mutating
i2
residues SDYIPTQ (166-172) to
EEYMPTE and
s residues DYVPSD (189-194) to EYMPTE (single
letter amino acid code, mutated residues are underlined). The resultant
constructs were designated
i2RCEE and
sRCEE respectively.
oRCEE was generated
from the rat
o cDNA (19) by mutating arginine 179 to
cysteine and residues DYQPTE (167-172) to
EYMPTE.
The i2RCEE cDNA (gift of Ann Pace and Henry Bourne,
University of California, San Francisco) was subcloned into pcDNA
I/Amp (Invitrogen) as an EcoRI fragment. The
sEE cDNA (gift of Paul Wilson and Henry Bourne,
University of California, San Francisco) was subcloned into pcDNA
I/Amp as a HindIII fragment. To produce the
sRC cDNA, the
sRCHA cDNA (12),
which contains the HA epitope from influenza virus (23), was digested
with XbaI and EcoRI to yield a fragment
containing the R201C mutation but not the HA epitope.
XbaI-EcoRI restriction of
sEEpcDNA I/Amp removed a fragment containing the EE
epitope, which was replaced by the XbaI-EcoRI
fragment from the
sRCHA cDNA to produce
sRCpcDNA I/Amp. To generate
sRCEEpcDNA I/Amp,
sRCpcDNA I/Amp
was digested with Alwn I to yield a fragment containing the R201C
mutation, which was ligated into
sEEpcDNA I/Amp in
place of the analogous fragment to produce an
s cDNA
containing both the R201C mutation and the EE epitope.
All mutations were generated by oligonucleotide-directed in
vitro mutagenesis (24) using the Bio-Rad Muta-Gene kit except for
those in the i2RCEE derivatives, Constructs 2 and 3, which were produced by polymerase chain reactions that generated DNA fragments with overlapping ends that were subsequently combined in a
fusion polymerase chain reaction (25). All mutagenesis procedures were
verified by restriction enzyme analysis and DNA sequencing.
Recombinant subunits were
transiently expressed in the human embryonic kidney fibroblast line,
HEK-293 (American Type Culture Collection CRL-1573), using DEAE-dextran
(26) under the control of the cytomegalovirus promoter in the
expression vector, pcDNA pcDNA I/Amp. To measure inhibition of
adenylyl cyclase, 106 cells/60-mm dish were co-transfected
with 0.1 µg of vector containing
sRC and 0.3 µg of
vector containing
i2RCEE,
oRCEE, or
mutant derivatives of
i2RCEE. To measure activation of
adenylyl cyclase, 106 cells/60-mm dish were transfected
with 1.5 µg of vector containing
sRCEE or mutant
derivatives of this construct or with vector alone. Intracellular cAMP
levels in cells labeled with [3H]adenine were determined
as described (14).
HEK-293 cells were
transiently transfected with recombinant subunit constructs using
DEAE dextran (26). Membranes were prepared 48 h after transfection
as described (14). For the trypsin resistance assay (12), membrane
proteins (70 µg) were diluted to a concentration of 6 mg/ml in a
buffer containing 20 mM HEPES (pH 8.0), 10 mM
MgCl2, 1 mM EDTA, 2 mM
-mercaptoethanol, and 0.64% (w/v) of the detergent lubrol PX.
Solubilized proteins were collected after centrifugation for 10 min at
4 °C in a microcentrifuge and incubated for 30 min at 30 °C in
the presence or absence of 125 µM GTP
S.
Tosylphenylalanyl chloromethyl ketone-treated trypsin (Sigma T-8642)
was added to a final concentration of 5 µg/ml, and the mixture was
incubated for 5 min at 30 °C. The digestion was terminated by adding
soybean trypsin inhibitor to a final concentration of 1 mg/ml. The
samples were then resolved by SDS-polyacrylamide electrophoresis
(10%), transferred to nitrocellulose, and probed with the anti-EE
monoclonal antibody (22), which was purified from hybridoma
supernatants using E-Z-SEP reagents (Middlesex Sciences, Inc). The
antigen-antibody complexes were detected using an anti-mouse
horseradish peroxidase-linked antibody according to the ECL Western
blotting protocol (Amersham Life Science, Inc.).
To characterize mutant i2 subunits after
transient expression in HEK-293 cells, two features were included, as
in a previous study (14), to enable measurement of their functions
without interference from the activities of the
i
proteins endogenous to these cells. First, a conserved arginine (R179C)
was replaced by cysteine. This mutation constitutively activates
i2 by inhibiting its GTPase activity (20) and made it
possible to measure inhibition of adenylyl cyclase without requiring
receptor-mediated activation of the mutant
i2 subunits.
Second, the
i2 constructs include an epitope from an
internal region of polyoma virus medium T antigen, referred to as the
EE epitope (22), which does not interfere with the
i2-adenylyl cyclase interaction (27).
We measured the ability of recombinant subunits to inhibit adenylyl
cyclase in HEK-293 cells by co-expressing them with the constitutively
activated
s mutant,
sRC, in which
arginine 201 is mutated to cysteine (21). As in a previous study (14), transfection with 0.1 µg of vector containing
sRC
resulted in an approximately 18-fold increase in cAMP production
compared with cells transfected with vector alone. Co-transfection with 0.3 µg of vector containing
i2RCEE resulted in ~60%
inhibition of the cAMP response to
sRC, while
co-transfection with the same amount of vector containing
oRCEE inhibited the response to
sRC by
only ~15% (Fig. 1). We used
oRCEE as a
negative control because
o has been shown to have little
or no ability to inhibit adenylyl cyclase (10, 11).
Alanine Substitutions within Conformational Switch Regions
Since the GTP-bound form of i2 inhibits
adenylyl cyclase much more effectively than the GDP-bound form does
(11), it was surprising that the effector-specifying region of
i2, as defined by the 78-residue segment, residues
245-322 (14), did not include any of the three regions, Switches I-III
(6, 8), that undergo GTP-dependent conformational changes.
However, the sequences of these regions are highly conserved in
i2 and
q. 7 of the 11 Switch I residues,
18 of the 21 Switch II residues, and 6 of the 12 Switch III residues
are identical in the sequences of
i2 and
q. Therefore, the importance of these regions as
effector binding sites could have been missed using homologous sequence
substitutions.
To directly test the importance of Switches I-III as effector contact
sites, we mutated solvent-exposed residues within each of these regions
to alanine residues. Substitutions using alanine residues eliminate the
side chain beyond the carbon but generally do not alter the main
chain conformation and do not impose significant electrostatic or
steric effects (28). We identified clusters of solvent-exposed residues
by inspection of the x-ray crystal structures of the GTP
S-bound
forms of
i1 (7) and
t (5) and
calculations of fractional accessibility values (29) from the
coordinates. As shown in Fig. 1, we mutated three clusters of residues
in Switch I (6 residues), five clusters of residues in Switch II (8 residues), and four clusters of residues in Switch III (7 residues).
We found that alanine substitutions of three residues in Switch II,
Arg-209, Lys-210, and Ile-213, blocked i2RCEE from
inhibiting adenylyl cyclase (Fig. 1). These residues are located in the
middle of the
2 helix and are highly conserved among
subunits
(see Fig. 7). We previously found that substituting
i2
homologs for three
s Switch II residues located at the
carboxyl terminus of
2 and in the
2/
4 loop, Gln-236, Asn-239,
and Asp-240 (see Fig. 7), specifically prevents
s from
activating adenylyl cyclase (12). Although not conserved between
s and
i2, these residues are identical in
the sequences of
i2 and
q and therefore
were not tested in our previous
i2/
q
chimera studies (14). The
t and
i1
homologs of the first two of these residues are solvent-exposed in the
structures. Alanine substitutions of the corresponding
i2 residues, His-214 and Glu-217, did not block the
ability of
i2 to inhibit adenylyl cyclase (Fig. 1).
We did not obtain evidence that either the Switch I or Switch III
regions of i2 are specifically involved in inhibition of adenylyl cyclase (Fig. 1). The only substitutions that caused a partial
loss of function were in residues Ile-185 and Glu-187 in Switch I (Fig.
1). However, these substitutions also greatly reduced the expression
level of
i2RCEE (see below).
Mutations that prevent
i2RCEE from inhibiting adenylyl cyclase could do so for
reasons other than disruption of residues that interact with this
effector. Therefore, we subjected constructs with these mutations to
the following criteria for specificity. The first criterion was that
the mutants should be expressed at wild-type levels in HEK-293 cell
membranes. This criterion was tested by performing immunoblots on
membranes prepared from cells expressing the mutants. The second
criterion was that the mutants should be able to bind GTP and undergo
the GTP-dependent conformational change that is detected as
the acquisition of resistance to trypsin cleavage (30-32). This second
criterion is quite stringent because it requires not only proper GTP
binding but also the ability to respond to this binding with an
activating conformational change. Under the conditions of this assay,
in the presence of GTP
S, trypsin removes a short segment from the
amino terminus but leaves most of the protein intact (Fig.
2). However, in the absence of GTP
S, trypsin degrades
i2RCEE to small fragments not seen on SDS-polyacrylamide
gels.
We found that the Switch II mutant constructs,
(R209A)i2RCEE and
(K210A,I213A)
i2RCEE, were expressed as well as
i2RCEE and achieved the GTP-dependent
activated conformation, as measured by the trypsin assay (Fig.
2). Therefore, by our criteria, residues Arg-209, Lys-210, and Ile-213
are specifically required for interaction with adenylyl cyclase.
In contrast, although the Switch I mutant construct,
(I185A,E187A)
i2RCEE, exhibited resistance to trypsin in the presence of GTP
S, it was expressed very poorly (Fig. 2). The
role of residues Ile-185 and Glu-187 in effector interaction is,
therefore, uncertain.
In the course of these studies, we mutated the i2
residue, Arg-209, that corresponds to the GTP
S-protected trypsin
site determined by amino-terminal sequencing of tryptic peptides from
t and
o (31). Elimination of this
cleavage site would be expected to result in an
subunit that was
resistant to trypsin cleavage in both the presence and absence of
GTP
S. However, (R209A)
i2RCEE was resistant to trypsin
cleavage in the presence but not the absence of GTP
S (Fig. 2).
Similar results were obtained upon mutation of each of the other
potential trypsin sites in Switch II,
Arg-206,2 Lys-210 (Fig. 2), and
Lys-211,2 as well as mutation of all four residues
simultaneously.2 These results suggest that, although
Switch II may contain cleavage sites that change conformation upon GTP
binding, there are also other sites outside of this region that are
preferentially cleaved by trypsin in the absence compared with the
presence of GTP
S. Nevertheless, the ability of the trypsin assay to
detect GTP-dependent conformational changes in Switch II is
demonstrated by the fact that the Switch II
s mutant,
G226A
s, which is unable to undergo the activating
conformational change required for dissociation from
,
does not acquire trypsin resistance in the presence of GTP
S
(32, 33).
To determine whether the highly conserved
middle region of Switch II (see Fig. 7) is required for the activation
of adenylyl cyclase by s, we tested the effects of
substituting alanines for the
s residues (Arg-232 and
Ile-235) that correspond to Lys-210 and Ile-213 in
i2.
We introduced these substitutions into
sRCEE, which
contains the EE epitope, previously shown to have no effect on the
interaction between
s and adenylyl cyclase (34). The substitutions almost entirely prevented
sRCEE from
activating adenylyl cyclase without affecting the
GTP-dependent conformational change measured by the trypsin
assay (Fig. 3). Thus, the same region of Switch II is
required for the interaction of both
s and
i2 with adenylyl cyclase.
Alanine Substitutions within the 78-Residue Segment
Since
i2, but not
q, inhibits adenylyl cyclase
(10, 13) and an
q/
i2/
q
chimera containing only 78
i2 residues (245-322) inhibits adenylyl cyclase as well as
i2 does (14), the
i2 residues that specify inhibition of adenylyl cyclase
must be located within this 78-residue segment. To identify these
effector-specifying residues, we tested the effects of mutating nine
clusters of solvent-exposed residues (22 residues total) to alanine
residues (Fig. 4). Within the 78-residue segment of
i2, 65 residues are identical among the three
i isoforms, which have equal abilities to inhibit
adenylyl cyclase (11). Of these 65 residues, 28 are different in
q and therefore might account for the ability of
i, but not
q, to inhibit adenylyl
cyclase. 20 of the substitutions were in residues that are identical
among the three
i subunits, and 18 were in residues that
differ between
i2 and
q. The thoroughness
of our mutational analysis is illustrated in Fig. 6A.
Mapping of effector-interacting residues of
i2 and
s onto the x-ray crystal structure
of the GTP
S-bound form of
i1. A,
space-filling model showing
i2 residues required for
inhibition of adenylyl cyclase. Residues that were mutated are shown in
red, magenta, orange, and dark
blue, as follows. Residues in Switch II specifically required for
inhibition of adenylyl cyclase are red. Residues in Switch I
in which mutations reduce both inhibition of adenylyl cyclase and
expression level are magenta. Residues within the 78-residue
segment in which mutations cause a partial loss of adenylyl cyclase
inhibition are orange. Residues in which mutations do not
affect inhibition of adenylyl cyclase are dark blue.
Residues that were not mutated in this study are shown in green, light blue, and white, as
follows. Residues outside of the 78-residue segment and Switches I-III
are green. Also green are the residues within the
78-residue
i2 segment that are conserved between
i2 and
q. The
i2 residues
within this segment that differ from
q residues but are
not identical among the three
i isoforms are light
blue. Residues in Switches I-III that were not mutated and
residues within the 78-residue segment that differ from
q residues and are conserved among the three
i isoforms, but were not mutated, are white.
Main chain backbone atoms are gray. The GTP is
yellow. The numbers on the model refer to
i2 residues. The model on the right is
rotated approximately 90° about the vertical axis relative to the
model on the left. B, ribbon diagram showing
comparison of effector-interacting surfaces of
i2 and
s. Residues important for the effector interactions of
i2 are red, of
s are
blue, and of both
subunits are magenta. Switches I-III are orange. The model is in the same
orientation as the model on the left in panel A. Coordinates
of the GTP
S-bound form of
i1 are from Coleman
et al. (7). The figures were drawn using MidasPlus,
developed by the Computer Graphics Laboratory at University of
California, San Francisco.
As shown in Fig. 4, substitutions of three sets of residues: His-245
(Construct 1), Lys-313, Asp-316, and Thr-317 (Construct 8), and
Arg-314, Lys-315, and Glu-319 (Construct 9), significantly reduced
inhibition of adenylyl cyclase. However, in contrast to the Switch II
mutations, which entirely blocked the ability of i2RCEE
to inhibit adenylyl cyclase, the mutations in Constructs 1, 8, and 9 had only partial effects. The other six clusters of mutations (15 residues) did not significantly impair the ability of
i2RCEE to inhibit adenylyl cyclase.
All of the constructs that inhibited adenylyl cyclase to a similar or
decreased extent compared with i2RCEE were expressed in
HEK-293 cell membranes and were able to undergo the
GTP-dependent conformational change that results in
increased resistance to trypsin digestion (Fig. 5).
However, since scanning densitometry of immunoblots showed that
Constructs 1, 8, and 9 were expressed at lower levels than
i2RCEE was, their decreased abilities to inhibit
adenylyl cyclase may be due to effects of the mutations on protein
folding and/or stability. Nevertheless, since we have substituted
alanines for the majority of solvent-exposed residues within the
effector-specifying 78-residue segment (see Fig.
6A) and the other substitutions did not
significantly reduce adenylyl cyclase inhibition, the residues in
Constructs 1, 8, and 9 are, by default, the most likely candidates for
specifying inhibition of adenylyl cyclase.
Comparison of the Effector-Interacting Surfaces of
We used the x-ray crystal
structure of the GTPS-bound form of
i1 (7) to map the
results of our mutagenesis studies. 88% of the residues in
i2 can be aligned with identical residues in
i1, while 67% of the
i1 residues can be
aligned with identical residues in
t. Since the
structures of the active (GTP
S-bound) forms of
i1 (7)
and
t (5) are virtually identical, the structure of
i1 is an excellent model for that of
i2.
Our mutagenesis analysis of Switches I-III in
i2 and the
78-residue effector-specifying
i2 segment, residues
245-322, focused on solvent-exposed residues. In addition, most of the
alanine substitutions in the 78-residue segment were of residues that
are: 1) different from the homologous
q residues and 2)
conserved among the
i isoforms. The thoroughness of this
study is demonstrated by the fact that the residues in Switches I-III
that were not mutated and the residues in the 78-residue segment that
meet criteria 1 and 2 but were not mutated represent a very small
fraction of the available surface area (shown in white in
Fig. 6A).
The alanine substitutions that caused the largest decrease in the
ability of i2RCEE to inhibit adenylyl cyclase were in
the middle of the
2 helix in Switch II (red in Fig.
6A). The effector-interacting surfaces of
s
and
i2 overlap exactly in this region
(magenta in Fig. 6B) where the sequences of the
two
subunits are highly conserved (Fig. 7). However,
the
2/
4 loop at the carboxyl-terminal end of Switch II is
important for the interaction of
s (12) but not
i2 (Fig. 1) with adenylyl cyclase (blue in
Fig. 6B).
The alanine substitutions within the 78-residue effector-specifying
segment that caused a moderate reduction in the ability of
i2RCEE to inhibit adenylyl cyclase (orange in
Fig. 6A) were in the amino terminus of
3 (Construct 1)
and in the
4/
6 loop (Constructs 8 and 9) (Fig. 6B).
The amino terminus of
3 (red in Fig. 6B) is
important for the effector interactions of
i2 (Fig. 4),
but not
s (12), while mutations in the
3/
5 loop (blue in Fig. 6B) disrupt interaction between
s and adenylyl cyclase (12) but do not have a
significant effect on the
i2-adenylyl cyclase
interaction (Fig. 4). Residues in the
4/
6 loop found to be
important for specifying the effector interactions of both
i2 and
s are magenta in Fig.
6B.
The studies reported here investigated two key aspects of subunit-effector interactions, GTP-dependence and specificity. We found
that in the case of
i2, these two components of effector interaction are mediated by distinct regions of surface residues. GTP-dependent effector interaction is mediated by Switch II
residues that are conserved among
subunits (Fig. 1) while
specificity (inhibition of adenylyl cyclase) is mediated by
nonconserved residues (the amino terminus of
3 and the
4/
6
loop) outside of the conformational switch regions (Fig. 4). In
contrast, in the case of
s, Switch II plays a role in
regulating both the GTP dependence of effector interaction as well as
effector specificity. The conserved Switch II region is required for
GTP-dependent activation of adenylyl cyclase (Fig. 3) while
nonconserved Switch II residues, as well as residues outside of the
conformational switch regions (the
3/
5 and
4/
6 loops), are
involved in regulating effector specificity (12). In the case of
t, the conformational switch regions and regions that
don't switch conformation (
3 and the
3/
5 loop) interact with
distinct regions of the effector molecule, PDE (35).
Taken together, our results and those of others indicate that two subunit regions, Switch II and the
4/
6 loop, may be important for
effector interactions in general (Fig. 7). The conserved middle region
of Switch II has been shown to be important for the interaction between
t and PDE. Mutation of a conserved tryptophan in
t reduces binding to PDE (36) while mutation of a
conserved glutamate causes constitutive activation of PDE by the
GDP-bound form of
t (37). The
4/
6 loop is involved
in specifying the effector interactions of at least three
subunits
(Fig. 7). We previously found that replacement of
s
residues in this region by their
i2 homologs prevents
s from activating adenylyl cyclase without preventing
the mutant protein from attaining the GTP-dependent active
conformation (12). Rarick et al. (16) found that a 22-amino
acid peptide (
t residues 293-314) activates PDE. Within this region, Spickofsky et al. (17) identified five residues in which substitutions of homologs from other
subunits block PDE
activation by peptides. Three of these residues are in the
4 helix
and two are in the
4/
6 loop. Mutations in the
4/
6 loop of
s and
i2, but not in
4 cause decreases
in effector modulation. In the case of
q,
4 and the
4/
6 loop have been implicated in PLC activation in studies using
peptides (38). However, chimera studies showed this region could be
replaced with
s sequence without affecting PLC
activation (39).
Since s and
i2 have opposite effects on
adenylyl cyclase activity, the conserved region of Switch II required
for the effector interactions of both
subunits is most likely
involved in regulating GTP-dependent effector binding. Of
the three residues found to be important for inhibition of adenylyl
cyclase by
i2, Arg-209 and Ile-213 are identical in the
sequences of
s and
i2 (see Fig. 7). The
third residue is conserved but not identical between the two
subunits (Lys-210 in
i2, Arg-232 in
s).
However,
i2/
s chimera studies showed that
substitution of lysine for arginine at position 232 in
s
has no effect on activation of adenylyl cyclase (12). Furthermore, the
q residue corresponding to Lys-210 is an arginine
residue and
q/
i2 chimera studies showed
that substitution of arginine at this position does not affect
inhibition of adenylyl cyclase (14). Therefore, these Switch II
residues do not determine the nature of adenylyl cyclase modulation by
s and
i2.
Although all subunits are conserved in this Switch II region, other
subunits do not modulate adenylyl cyclase, with the exception of a
weak inhibition of type I adenylyl cyclase by
o (11). A
possible explanation for this selectivity is that other
subunits
contain residues that preclude a productive adenylyl cyclase
interaction. If so, then replacing
i2 residues in the amino terminus of
3 and in the
4/
6 loop with the homologous residues from
q or other
subunits might cause a
larger reduction in ability to inhibit adenylyl cyclase than was
observed for alanine substitutions.
Our studies show that the effector-specifying regions of
s and
i2 overlap but are not identical
(see Fig. 6B). Studies using
subunit chimeras localized
the region of
i2 that specifies inhibition of adenylyl
cyclase to a 78-residue segment (amino acids 245-322) that extends
from
3 to
6 (14). Residues corresponding to two of the three
s regions that specify activation of adenylyl cyclase
(12, 15), the
3/
5 and
4/
6 loops, are included in this
segment. The only region of overlap that we have found among the
effector-specifying regions of
s and
i2
is in the
4/
6 loop. Effector-specifying regions unique for
s are located in the
3/
5 loop and in the
carboxyl-terminal part of Switch II (12). Similarly, mutation of a
single residue in the amino terminus of
3 reduces the ability of
i2 to inhibit adenylyl cyclase but is not required for
the activation of adenylyl cyclase by
s (12).
Since both s and
i2 interact with
adenylyl cyclase, the effector-specifying residues of each
subunit
presumably determine whether activation or inhibition will result from
subunit binding. However, the effector-specifying residues of
s appear to contribute more to the interaction with
adenylyl cyclase than do those of
i2. Substitutions in
the effector-specifying segment of
i2 do not cause as
large of a decrease in the ability to inhibit adenylyl cyclase as do
substitutions in the conserved middle part of Switch II. However,
mutations in two of the effector-specifying regions of
s, the nonconserved carboxyl-terminal part of Switch II
and the
3/
5 loop, decrease effector activation to the same extent as do mutations in the conserved Switch II region.2
Consistent with our results, Taussig et al. (11) found that replacing
i1 residues with
s homologs in
the
3/
5 loop results in an
subunit that weakly activates
certain adenylyl cyclase isoforms. Thus, the effector-specifying
regions of
s appear to be dominant over those of
i.
Mutagenesis studies of hGH and its receptor, for which a structure of the hormone-receptor complex is available (40), have characterized the functional importance of residues in the binding interface. Individual replacements of residues in hGH (41) and its receptor (42) demonstrated that only a small subset of the residues at the center of the contact region contribute substantially to binding affinity. However, hGH residues in the periphery of the interface, which do not contribute much to the affinity of binding (41), are important for the specificity of binding (43).
In a similar manner, our studies of the interaction between
i2 and adenylyl cyclase implicate Switch II residues as
being the major contributors to this binding interaction. Substitutions in the effector-specifying segment of
i2 have a more
modest effect on the ability of
i2RCEE to inhibit
adenylyl cyclase. In the absence of any structures of
subunit-effector complexes, we predict that interactions between these
proteins will include the conserved Switch II region as well as
nonconserved specificity regions but that, as seen in the case of hGH
and its receptor (41, 42), the contact surfaces may be larger than the
"functional epitopes" defined by our mutagenesis studies.
We thank Gernot Walter (UCSD) for the EE
hybridoma; Ann Pace, Paul Wilson, and Henry Bourne (UCSF) for the
i2RCEE and
sEE constructs; and Thomas
Hynes (Pfizer, Inc.) and Rolando Medina for helpful discussions and
critical reading of the text.