From the Institute for Neuroscience, Northwestern
University, Chicago, Illinois 60611 and the
¶ Department of Pharmacology & Toxicology, West
Virginia University, Morgantown, West Virginia 26506
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ABSTRACT |
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We previously reported that residues 299-318 in
G The interaction of heptahelical receptors with their cognate
heterotrimeric guanine nucleotide-binding proteins (G proteins) represents an initial step in the transmission of extracellular signals
across the plasma membrane (2-4). The receptor-G protein interaction
modulates specific second messenger systems that result in a unique
physiologic response to the extracellular signal. The particular
downstream effect of G protein activation is not the result of an
explicit interaction between each heptahelical receptor and a unique
heterotrimeric G protein. On the contrary, G protein-coupled receptors
have repeatedly been demonstrated to couple to several related members
within the same family of G protein Numerous biochemical studies have suggested that several subregions of
G By using this approach, we previously demonstrated that substitution of
the In addition to providing a useful model for receptor-G protein
selectivity, the 5-HT1B receptor plays an important
modulatory role in the central nervous system. 5-HT1B
receptors are the primary terminal autoreceptors within the brain
serotonin system (26). Activation of these receptors inhibits the
release of 5-HT into the synaptic cleft (27, 28). In addition, drugs
that selectively interact with 5-HT1B receptors have proven
to be clinically useful for the treatment of migraine headache (29).
Thus, deducing the molecular events that are essential to
5-HT1B receptor-catalyzed G protein activation may aid our
understanding of both normal and pathologic processes in brain
serotonin systems.
By utilizing a series of G Materials--
Nucleotides and enzymes were purchased either
from Boehringer Mannheim or from Amersham Pharmacia Biotech. Serotonin
was obtained from Sigma. [35S]GTP Construction of G Expression and Purification of G Expression and Purification of G Protein Subunits--
For
affinity shift assays, the expression and purification of the G protein
Preparation of Sf9 Membranes Containing
5-HT1B-expressed Receptors--
Sf9 cells were
infected with recombinant baculovirus containing cDNA for the
5-HT1B receptor, cultured, and harvested as described (34).
Membranes were prepared according to a previously published protocol
(1). Briefly, harvested cells were thawed and resuspended in ice-cold
homogenization buffer (10 mM Tris-HCl, pH 8.0, 25 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml benzamidine
and 2 µg/ml each of aprotinin, leupeptin, and pepstatin A) and burst
by N2 cavitation. Cavitated cells were centrifuged at low
speed; the supernatant was removed and then centrifuged at 28,000 × g for 30 min at 4 °C. The resulting pellets were
resuspended in 5 mM NaHEPES, pH 7.5, containing 1 mM EDTA and supplemented with the aforementioned protease
inhibitors. The membranes were washed twice and resuspended in the same
buffer (1-3 mg of protein/ml).
Reconstitution of Receptors with Exogenous G
Proteins--
Frozen Sf9 membranes were reconstituted as
described (1). Briefly, membranes were pelleted and resuspended in
reconstitution buffer (5 mM NaHEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 500 nM GDP, 0.04% CHAPS). G protein
subunits were diluted in the same buffer, and the mixture was
incubated at 25 °C for 15 min and then held on ice. The
reconstitution mixture was diluted with binding assay buffer as
described (1).
[3H]5-Hydroxytryptamine Binding
Assay--
[3H]5-HT binding to 5-HT1B
receptors reconstituted with G 5-HT1B Receptor-stimulated GTP Fluorescence Assay--
To ensure that recombinant G Statistics--
Affinity shift activity data and the initial
rates of GDP/GTP exchange (Table I) were analyzed separately using a
one-way analysis of variance (ANOVA). Differences between groups were determined by a Newman Keuls' post hoc test only after the
ANOVA yielded a significant main effect.
The ability of 5-HT1B receptors to couple selectively
to heterotrimers containing members of the G The
Functional coupling between the 5-HT1B receptor and the
chimeric proteins was assessed by examining both the ability of the 5-HT receptor agonist, serotonin, to stimulate receptor-catalyzed GDP/[35S]GTP
As shown in Fig. 2A, 5-HT
stimulation of the 5-HT1B receptor results in a
time-dependent increase in [35S]GTP
The coupling ability of these same chimeras (Chi22, Chi24, and Chi25)
was also examined by agonist affinity shift activity. Consistent with
the [35S]GTP Amino Acid Residues Gln304 and Glu308 of
G
To confirm and further support the contention that G
This lack of correspondence between affinity shift activity and
[35S]GTP
Nonetheless, despite the variations between the affinity shift data and
the GDP/GTP exchange rates for the G Computer Simulation of Double Point Mutations Reveal Potential for
Direct Interaction of Gln304 and Glu308 with
the 5-HT1B Receptor--
Whereas the present study
demonstrates the critical role of two specific amino acids within the
Alternatively, rather than affecting direct contact sites with the
receptor, the amino acid substitutions within G By using G Previously published work has shown that there are several
receptor-binding regions present in heterotrimeric G proteins. The
primary receptor recognition region is believed to be localized to the
carboxyl-terminal domain of G Other studies implicating the According to the crystal structure of G Consistent with this idea, it has been suggested that the
agonist-activated receptor interacts with the GDP-bound form of the
heterotrimeric G protein, and the release of GDP from the G Sequence alignment and comparison of the primary structure between
Gi1 participate in the selective interaction
between G
i1 and the 5-hydroxytryptamine1B (5-HT1B) receptor (Bae, H., Anderson, K., Flood, L. A., Skiba, N. P., Hamm, H. E., and Graber, S. G. (1997)
J. Biol. Chem. 272, 32071-32077). The present study
more precisely defines which residues within this domain are critical
for 5-HT1B receptor-mediated G protein activation. A series
of G
i1/G
t chimeras and point mutations were reconstituted with G
and Sf9 cell membranes
containing the 5-HT1B receptor. Functional coupling to
5-HT1B receptors was assessed by 1)
[35S]GTP
S binding and 2) agonist affinity shift
assays. Replacement of the
4 helix of G
i1 (residues
299-308) with the corresponding sequence from G
t
produced a chimera (Chi22) that only weakly coupled to the
5-HT1B receptor. In contrast, substitution of residues within the
4-
6 loop region of G
i1 (residues
309-318) with the corresponding sequence in G
t either
permitted full 5-HT1B receptor coupling to the chimera
(Chi24) or only minimally reduced coupling to the chimeric
protein (Chi25). Two mutations within the
4 helix of
G
i1 (Q304K and E308L) reduced agonist-stimulated
[35S]GTP
S binding, and the effects of these mutations
were additive. The opposite substitutions within Chi22 (K300Q and
L304E) restored 5-HT1B receptor coupling, and again the
effects of the two mutations were additive. Mutations of other residues
within the
4 helix of G
i1 had minimal to no effect on
5-HT1B coupling behavior. These data provide evidence that
4 helix residues in G
i participate in directing
specific receptor interactions and suggest that Gln304 and
Glu308 of G
i1 act in concert to mediate the
ability of the 5-HT1B receptor to couple specifically to
inhibitory G proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits, albeit with differing
levels of efficiency (5-11). Clawges et al. (12)
demonstrated that the serotonin (5-HT)1 1B receptor couples
to heterotrimers containing either G
i1, G
i2, G
i3, or G
o.
Nevertheless, this receptor does not couple to heterotrimers containing
another member of this same family, the G
t subunit (12).
Therefore, the 5-HT1B receptor represents one receptor
system that can be exploited to investigate the precise molecular
determinants governing selective receptor-G protein interactions.
(13-21) in addition to regions on G
(20, 21) and G
(22-25)
may act in concert to determine selective receptor-G protein
interactions. The carboxyl-terminal domain of G
subunits, in
particular, has been demonstrated to play a key role in eliciting several specific receptor-G protein interactions. However, the carboxyl-terminal regions of G
i1, G
i2,
G
i3, and G
t are highly homologous, and
therefore, the carboxyl-terminal domain is not likely to be the primary
determinant of 5-HT1B receptor-G protein selectivity
between G
i/o and G
t. The selectivity
profile of the 5-HT1B receptor has facilitated the use of
G
i/G
t chimeras to map the residues that
play a role in determining the specific interaction of the
5-HT1B receptor to inhibitory G proteins.
4 helix and
4-
6 loop (amino acids 299-318) regions of
G
i with the respective sequence from G
t
markedly reduced the ability of this chimera to couple to the
5-HT1B receptor (1). These studies determined that the
region corresponding to amino acids 299-318 in G
i1
plays a key role in determining the selective interaction between
G
i1 and the 5-HT1B receptor (1). The intent
of the present study was to define more precisely which amino acids
within this domain are critical for selective 5-HT1B
receptor coupling to inhibitory G proteins.
i/G
t chimeras
coupled with site-directed mutagenesis, the present study reveals that
4 helical residues Gln304 and Glu308 of
G
i1 are critical determinants of 5-HT1B
receptor coupling to inhibitory G proteins. This conclusion is
supported by the observed marked reduction in receptor-catalyzed
GDP/GTP exchange on G
i1 Q304K, E308L, and Q304K-E308L
mutants. Moreover, whereas Gln304 and Glu308
are absolutely conserved among all G
i/o isoforms, they
are divergent between G
i/o and G
t
subunits. The crystal structure of G
i reveals that
Gln304 and Glu308 are surface-exposed (30, 31),
and mutation of these residues (Q304K and E308L) may alter the surface
potential of the
subunit. Hence, these residues may interact
directly with the 5-HT1B receptor to mediate receptor
coupling. Mutation of these residues may also indirectly influence the
secondary structure of neighboring domains resulting in an inability of
the 5-HT1B receptor to couple to the mutant inhibitory
subunits.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S (1250 Ci/mmol) and
[3H]5-HT (22-30 Ci/mmol) were purchased from NEN Life
Science Products.
Mutant Genes--
The present study used
the Escherichia coli expression vectors
pHis6G
i1 or pHis6Chi3 that
contain a nucleotide sequence encoding a hexa-histidine tag under the
control of a T7 promoter (32). BamHI and HindIII
digestion of pHis6Chi3 released a 450-base pair DNA
fragment corresponding to amino acid residues 214-354 of Chi3. This
fragment was subcloned into pUC19 (pUC19-Chi3). To construct Chi22,
Chi24, and Chi25, duplex oligonucleotides containing the desired
mutations were ligated into the NaeI- and
BglII-digested pUC19-Chi3. Sequences were confirmed, and the
correct pUC19-based chimeras were cloned into the
pHis6G
i1 as a BamHI and
HindIII fragment. Generation of the BamHI site in
pHis6G
i1 did not mutate any residues.
Site-directed mutagenesis of either Chi22 or G
i1 was
carried out using the QuikChangeTM Site-directed
Mutagenesis Kit (Stratagene, La Jolla, CA) according to the
manufacturer's instructions. Either
pHis6G
i1 or pHis6Chi22 served as
the template for polymerase chain reaction-based mutagenesis.
i1 and G
Mutants in E. coli--
Hexa-histidine-tagged G
i1 or
chimeric G
subunits were expressed in E. coli BL21(DE3)
cells and purified as described (32) with minor modification. Briefly,
cell pellets were resuspended in buffer A (50 mM Tris-HCl,
pH 8.0, 50 mM NaCl, 5 mM MgCl2)
supplemented with 50 µM GDP, 0.1 mM
phenylmethylsulfonyl fluoride, and 5 mM
-mercaptoethanol, sonicated, and then centrifuged at 100,000 × g for 60 min. The supernatant was loaded onto a
Ni2+-nitrilotriacetic acid-agarose resin column (His-Bond,
Novagen). Eluted samples were dialyzed overnight against buffer A in
the presence of 20% glycerol, 0.1 mM phenylmethylsulfonyl
fluoride, and 2 mM
-mercaptoethanol and then further
purified by high performance liquid chromatography (Waters Protein-Pak
QHR-15, Waters Chromatography). Protein concentrations were determined
using the Coomassie Blue method (33) with bovine serum albumin (Pierce)
as the standard.
subunits in Sf9 cells was performed as described (34, 35)
except that the final chromatography step was performed on 15-micron
Waters Protein-Pak QHR (Waters Chromatography). Recombinant
1
2 subunits were purified from Sf9
cells using a His6-
2 as described by Kozasa
and Gilman (36). The native retinal
subunits used for the
GTP
S binding experiment were purified as described (37).
and G
1
2
was determined as described previously (1). To estimate receptor number
in individual membrane preparations, the membranes were reconstituted
with a large excess (5 µM) of G protein heterotrimers containing G
i1 and the 5-HT1B receptors
labeled with 1, 10, and 40 nM [3H]5-HT. It
has been shown that such reconstitution effectively converts all of the
expressed receptors to a coupled, high affinity for agonist state (12).
Bmax values were estimated by nonlinear regression analysis with GraphPad PRISM of the specific binding data
using a fixed value (0.62 nM) for the high affinity
KD (12). For affinity shift assays, high affinity
binding to 5-HT1B receptors was determined using a single
concentration of [3H]5-HT near the KD
value for the radioligand (0.8-1.3 nM).
S Binding
Assay--
A GTP
S binding assay was used to quantitate
receptor-catalyzed GDP/GTP exchange on G
subunits as described (1).
Briefly, membranes were incubated with 1 mM
AMP-PNP at 37 °C for 1 h, and receptor coupling was
reconstituted with G
and G
subunits on ice in 70 µl of
reaction buffer A (25 mM Hepes, pH 7.4, 5 mM
MgCl2, 1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol) for 30 min and then diluted with 200 µl of reaction buffer A containing 150 nM GDP and 60 nM GTP
S. The addition of 30 µl of
[35S]GTP
S (~7 × 106 cpm) initiated
the reaction which was incubated at 25 °C. The following final
concentrations were used for the GTP
S assay: 1.28 nM
5-HT1B, 40 nM G
, and 40 nM
retinal
. For agonist activation, 1 µM of 5-HT was
included. Aliquots (20 µl) were withdrawn at various times, and the
reaction was terminated by filtration. Radioactivity retained on the
filters was quantitated with a liquid scintillation counter. As the
actual receptor densities and protein concentrations varied in
different experiments, some degree of variation in the absolute level
of GTP
S binding was observed. As rhodopsin can couple to either
Gt or Gi heterotrimers with equal efficiency
(32), GTP
S binding data were normalized to the percent of maximal
binding that occurred after incubating samples for 30 min in the
presence of an excess amount (500 nM) of light-activated rhodopsin.
proteins
are properly folded, instrinsic fluorescence of the subunits was
measured as described (1, 32). Briefly, the
AlF4
-dependent
conformational changes of activated G
subunits were monitored by
intrinsic tryptophan fluorescence changes with excitation at 280 nm and
emission at 340 nm. The relative increase in fluorescence of 200 nM G
subunits was determined from absorbance readings before and after the addition of 10 mM NaF and 20 µM AlCl3.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i/o family
of G proteins (12) facilitated the use of a series of
G
i/G
t chimeras to determine which amino
acids within G
i mediate this specific interaction. By
using this approach, we previously demonstrated that G
i1
amino acid residues 299-318 (corresponding to the
4 helix and
4-
6 loop regions of G
i1) play a major role in
determining the selective interaction with the 5-HT1B
receptor (1). Additional residues in the amino-terminal domain of
G
i1 were also determined to play a secondary role in
5-HT1B receptor coupling (1).
4 Helical Domain of G
i1 Mediates the Ability
of the 5-HT1B Receptor to Couple to Inhibitory G
Proteins--
The present study initially used the same approach
described above to identify which subdomain within this stretch of
amino acids (G
i1 299-318) is critical for functional
5-HT1B receptor coupling to inhibitory G proteins. As shown
schematically in Fig. 1, three chimeric
G
i/G
t proteins were generated in which
either the
4 helix (Chi22) or portions of the
4-
6 loop region
(Chi24 and Chi25) of G
i1 were replaced with the
corresponding sequence from G
t. Prior to the assessment
of coupling activity, the ability of each chimeric G
subunit to bind
GDP and undergo conformational change upon binding to GTP was tested by
determining if the proteins undergo an
AlF4
-dependent increase in
tryptophan fluorescence (32). This assay is based on the ability of
AlF4
, which mimics the
-phosphate
of GTP, to induce the active conformation, which then results in an
increase in intrinsic fluorescence of Trp211 in
G
i1. Tryptophan fluorescence of all mutants and
G
i1 used in this study increased 40-45% upon the
addition of AlF4
(Fig. 1), consistent
with our previous results (1, 32).
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Fig. 1.
Secondary structure and
AlF4 -dependent tryptophan
fluorescence change of G
subunits. Numbers above the
wild type forms of G
i1 and G
t represent
the corresponding residues in each respective
subunit.
Numbers above chimeric structures indicate the junction
points of G
t and G
i1 sequences and refer
to amino acid positions in G
t. *, diagram of secondary
structural domains common to G
subunits. SI, SII, and
SIII refer to the switch regions of G
. a Percent
increase in tryptophan fluorescence in the presence of 10 mM NaF and 20 µM AlCl3 compared
with basal (see "Experimental Procedures" for detail). The increase
in tryptophan fluorescence indicates that all constructs were capable
of undergoing conformational change in the presence of
AlF4
. b As full-length
G
t is not easily expressed or purified, this
G
i1/G
t chimera was generated previously,
crystallized, and shown to exhibit
t-like coupling
activity (32, 51).
S exchange on the
subunit, and the
ability of the chimeric protein to induce the high affinity agonist
binding state of the receptor (affinity shift activity). The underlying
principle of the affinity shift assay is that activated receptors can
be converted to a high affinity agonist binding state by the
appropriate G protein heterotrimers (12). By using a low concentration
of agonist near the KD for the high affinity state
and well below the KD for the low affinity state,
the formation of a functional agonist-receptor-G protein complex is
readily detected as an enhanced level of ligand binding. This assay can detect changes in coupling with either native or recombinant receptor and G protein preparations (12, 38-40).
S
binding to recombinant G
i1. Substitution of the
4
helix of G
i1 with the corresponding sequence from
G
t (Chi22) dramatically reduces (
90%) the ability of
the 5-HT1B receptor to couple to the chimeric G protein, as
indicated by a marked reduction in the ability of 5-HT to stimulate
[35S]GTP
S binding to the Chi22
subunit (Fig.
2B and Table I). In contrast,
substitution of the
4-
6 loop G
i1 residues
308-314 with the corresponding region from G
t (Chi24)
failed to alter serotonin-stimulated receptor-catalyzed incorporation
of [35S]GTP
S into the chimeric
subunits (Fig.
2C and Table I). Interestingly, an overlapping chimera,
Chi25, with G
i residues 305-314 substituted with the
corresponding G
t residues shows a modest (28%) but
significant (p < 0.05) reduction in the initial rate
of GDP/GTP exchange (Table I).
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Fig. 2.
Time-dependent 5-HT1B
receptor-catalyzed GDP/GTP exchange on
G i1 and
G
i1/G
t
chimeras. Membranes expressing the 5-HT1B receptor
were reconstituted with either 40 nM G
i1
(A), Chi22 (B), Chi24 (C), or Chi25
(D) in the presence of 40 nM
1
1. Data are expressed as the percentage
of maximal GTP
S binding obtained in the presence of excess (500 nM) light-activated rhodopsin. Squares indicate
GTP
S binding in the presence of 1 µM 5-HT (added at
the 8.5-min time point), and triangles indicate the binding
in the absence of agonist. Data represent the mean ± S.E. from
three independent experiments.
Affinity shift activity and initial rates of GDP/GTP exchange
S experiments, the affinity shift activity
of Chi22 was significantly reduced (
53%) in comparison to
recombinant G
i1 (Fig. 3
and Table I). Likewise, as expected from the GTP
S data, the affinity
shift activity of Chi24 did not significantly differ from
G
i1. In contrast, although the affinity shift activity
of Chi25 was reduced by 22%, this reduction did not reach statistical significance (Table I, Fig. 3). Taken together, these results localized
the major molecular elements underlying the selective interaction of
the 5-HT1B receptor with G
i1 to the
4
helical domain.
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Fig. 3.
Affinity shift activity of
G i1/G
t
chimeras with 5-HT1B receptors. Affinity shift
activities refer to the -fold enhancement above buffer controls of high
affinity [3H]5-HT binding to 5-HT1B receptors
reconstituted with G protein heterotrimers containing the indicated
subunits. Data represent the mean ± S.E. from 5 to 9 independent
determinations using three separate membrane preparations where
5-HT1B receptors were expressed between 5.2 and 11.7 pmol/mg membrane protein. Exogenous G proteins were 1.3-3.3
µM during reconstitution and 45-112 nM
during the binding assays which was a 35-50-fold molar excess over
receptors. The concentration of [3H]5-HT was 0.8-1.3
nM in all experiments. Data were analyzed using a one-way
ANOVA followed by a Newman Keuls' post hoc test as
described under "Experimental Procedures." *, significantly lower
than G
i1 (p < 0.05).
i Are Critical Determinants of Coupling to
5-HT1B Receptors--
In order to map more precisely the
specific amino acids that are critical for 5-HT1B receptor
coupling to G
i1, systematic site-directed mutagenesis
studies were conducted based on either Chi22 or G
i1
(Fig. 4). Within Chi22 there are 5 residues that are divergent between G
i1 and
G
t: G
i1-A300G, A301N, Q304K, C305V, and
E308L (Fig. 4). Previous studies demonstrated that mutation of
G
i1 residue Ala300 to glycine did not impair
coupling to the 5-HT1B receptor (1). Therefore, the present
studies focused only on divergent residues between
G
i1301 and G
i1308. The aim of the
substitutions within Chi22 was to determine whether coupling to the
5-HT1B receptor could be restored by replacing
G
t residues individually or in combination with the
corresponding residues from G
i1. Mutants based on Chi22
(Chi22-N297A, Chi22-K300Q, Chi22-V301C, Chi22-L304E, and
Chi22-K300Q-L304E) demonstrated varying degrees of receptor-catalyzed GDP/GTP exchange (Fig. 5A and
Table I) and affinity shift activity (Fig. 5B and Table I).
Similar to Chi22, Chi22-N297A exhibited only very weak coupling to the
5-HT1B receptor as exemplified both by the low levels of
agonist-stimulated GTP
S binding (Fig. 5A, Table I) and by
the low affinity shift activity of the mutant chimeras (Fig.
5B). Mutation of valine 301 to cysteine in Chi22 clearly
failed to elevate the initial rate of GTP
S binding above Chi22
(Table I and Fig. 5A). Likewise, the affinity shift activity of this mutant did not significantly differ from Chi22. Mutants Chi22-K300Q and Chi22-L304E exhibited significant (p < 0.05) 2-4-fold increases in agonist-stimulated GDP/GTP exchange in
comparison to the parent Chi22 (Table I). Consistent with these data,
enhanced 5-HT1B receptor coupling was also observed with
Chi22-L304E as measured by a significant 72% increase in the affinity
shift activity over Chi22 (Fig. 5B). Although Chi22-K300Q
caused a 42% increase in affinity shift activity over that observed
for Chi22, this increase did not reach statistical significance. The
critical nature of residues Gln304 and Glu308
is supported by the observation that the double mutant
Chi22-K300Q-L304E completely restored GDP/GTP exchange and
affinity shift activity to wild type G
i1 levels (Fig. 5
and Table I).
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Fig. 4.
Primary sequence alignment of the
4-
4/
6
loop region of bovine G
i1
(residues 296-318) and G
t
(residues 292-314). Numbers immediately above the
primary sequence of G
i1 correspond to residues in
G
i1. Numbers immediately below the primary
sequence of G
t correspond to residues in
G
t. The box indicates the region of
G
i1 that was substituted with the corresponding sequence
from G
t to generate Chi22. Boldface
letters indicate residues within G
t which
diverge from G
i1 residues. Single or double mutations of
G
i1 or Chi22 are listed below the sequence
alignment.
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Fig. 5.
Functional coupling of the 5-HT1B
receptor to Chi22 mutants. Membranes expressing the
5-HT1B receptor were reconstituted with the indicated Chi22
mutants and subunits. A illustrates
5-HT1B receptor-catalyzed GDP/GTP exchange on Chi22 point
mutants. Data are expressed as the percentage of maximal GTP
S
binding obtained in the presence of excess (500 nM)
light-activated rhodopsin. Curves depict the difference
between the rates of GTP
S binding in the presence and absence of 1 µM 5-HT. Data shown represent the mean ± S.E. of
three independent experiments. The initial rates of nucleotide exchange
calculated from these data are shown in Table I. B depicts
the affinity shift activity of Chi22 mutants with 5-HT1B
receptors. Affinity shift activities refer to the -fold enhancement
above buffer controls of high affinity [3H]5-HT binding
to 5-HT1B receptors reconstituted with G protein
heterotrimers containing the indicated
subunits. Data represent the
mean ± S.E. from 3 to 9 independent determinations using three
separate membrane preparations where 5-HT1B receptors were
expressed between 5.2 and 11.7 pmol/mg membrane protein. Exogenous G
proteins were 1.3-3.3 µM during reconstitution and
45-112 nM during the binding assays which was a
35-50-fold molar excess over receptors. The concentration of
[3H]5-HT was between 0.8 and 1.3 nM in all
experiments. Data were analyzed using a one-way ANOVA followed by a
Newman Keuls' post hoc test as described under
"Experimental Procedures." *, significantly greater than Chi22
(p < 0.05); #, significantly greater than
Chi22-K300Q.
i
residues Gln304 and Glu308 are particularly
important determinants of 5-HT1B receptor coupling, single
or double mutations were constructed in G
i1 (A301N,
Q304K, C305V, and E308L). The aim of these experiments was to determine whether coupling to the 5-HT1B receptor could be reduced or
eliminated by replacing these specific G
i1 residues with
the corresponding residues from G
t. As illustrated in
Fig. 6A and Table I, the single amino acid substitution G
i1-E308L markedly
(
70%) and significantly (p < 0.05) reduced
agonist-mediated stimulation of GTP
S binding to the mutant
subunit. Likewise, mutation G
i1-Q304K resulted in a
moderate (>40%; p < 0.05) reduction in
agonist-mediated GTP
S binding in comparison to recombinant
G
i1. Consistent with these data, the double mutant
G
i1-Q304K-E308L exhibited an even greater reduction in
agonist-mediated GTP
S binding than either single mutation alone
(Fig. 6A and Table I). G
i1-C305V did not significantly alter coupling to the 5-HT1B receptor as
evidenced by the lack of effect on agonist-stimulated GDP/GTP exchange
with this mutant (Table I). Quite unexpectedly,
G
i1-A301N resulted in a small (+12%) but statistically
significant (p < 0.05) increase in the initial rate of
GDP/GTP exchange in comparison to G
i1 (Table I). In
contrast to the marked reductions in [35S]GTP
S binding
observed with G
i1 mutants E308L, Q304K, and Q304K-E308L, no significant differences in affinity shift activities were observed between these mutants and G
i1 (Table I and Fig.
6B). Affinity shift activity also did not significantly vary
between G
i1 and G
i1-A301N, C305V, or
C305V/E308L (Table I).
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Fig. 6.
Functional coupling of the 5-HT1B
receptor to G i1 mutants.
Membranes expressing the 5-HT1B receptor were reconstituted
with the indicated G
i1 mutants and
subunits.
A illustrates 5-HT1B receptor-catalyzed GDP/GTP
exchange on G
i1 point mutants. Data are expressed as the
percentage of maximal GTP
S binding obtained in the presence of
excess (500 nM) light-activated rhodopsin.
Curves depict the difference between the rates of GTP
S
binding in the presence and absence of 1 µM 5-HT. Data
shown represent the mean ± S.E. of three independent experiments.
The initial rates of nucleotide exchange calculated from these data are
shown in Table I. B depicts the affinity shift activity of
G
i1 mutants with 5-HT1B receptors. Affinity
shift activities refer to the -fold enhancement above buffer controls
of high affinity [3H]5-HT binding to 5-HT1B
receptors reconstituted with G protein heterotrimers containing the
indicated
subunits. Data represent the mean ± S.E. from 3 to
6 independent determinations using 2 separate membrane preparations
where 5-HT1B receptors were expressed between 5.2 and 11.7 pmol/mg membrane protein. Exogenous G proteins were 1.3-3.3
µM during reconstitution and 45-112 nM
during the binding assays which was a 35-50-fold molar excess over
receptors. The concentration of [3H]5-HT was between 0.8 and 1.3 nM in all experiments. Data were analyzed using a
one-way ANOVA followed by a Newman Keuls' post hoc test as
described under "Experimental Procedures." There were no
significant differences between the affinity shift activity of
G
i1 and those of the mutant G
subunits.
S binding data for the G
i1
mutants is likely to result from differences in the sensitivity of
these assays stemming from technical aspects involved in these
measures. For example, the initial exchange rates (Table I) are
determined from linear regression analysis of the
[35S]GTP
S binding data generated over the course of 10 min following the introduction of agonist to the assay (see Figs.
5A and 6A) with saturation of
[35S]GTP
S binding occurring by 30 min (Fig.
2A). In contrast, as is required for radioligand binding
assays, the affinity of [3H]5-HT for the
5-HT1B receptor is determined only after equilibrium is
reached (i.e. following a 1.5-h incubation with the agonist) and in the absence of GTP. Therefore, if GDP release is
impaired in the G
i1 mutants (as would be
suggested by alterations in [35S]GTP
S binding) but not
prevented, sufficient GDP could be released over the course
of the experiment (1.5 h) such that at equilibrium the amount of high
affinity receptors present in the preparation is similar for both
G
i1 and the G
i1 mutants. Alternatively, the divergence between affinity shift activity and
[35S]GTP
S binding for the G
i1 mutants
may be indicative of a change in GTP
S binding in the absence of a
change in GDP release. This situation could only arise if GTP
S
binding (rather than GDP release) has become the rate-limiting step as
a result of the G
i1-Q304K and G
i1-E308L
mutations. If this were the case, we might expect to observe this same
phenomenon with the Chi22-K300Q, Chi22-L304E, and Chi22-K300Q-L304E
mutants, which we do not. Therefore, although the studies herein do not
preclude the possibility of changes in GTP
S binding in the absence
of a change in GDP release, further studies will be required to assess
the likelihood of this potential outcome.
i1 mutants, the affinity shift activity appeared to show overall trends in the same
direction as the [35S]GTP
S binding data. We,
therefore, determined whether there was a correlation between affinity
shift activity and the initial rates of GDP/GTP exchange as measured by
[35S]GTP
S binding. Fig.
7 illustrates the correlative comparison between these two data sets. Correlation analysis yielded a Pearson correlation coefficient of 0.80, indicating a significant correlation between the GDP/GTP exchange rate and the affinity shift activity.
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Fig. 7.
Correlation between GDP/GTP exchange and
affinity shift activity. The data plotted represent the means of
each data set as reported in Table I. Filled circle
represents the G i1 control value; open circle
represents Chi22; open squares represent Chi22 mutants;
filled squares represent G
i1 mutants. Chi24
is represented by ×, and Chi25 is represented by an
asterisk. The Pearson correlation coefficient was 0.80, representing a significant correlation between both data sets.
4 helical domain of G
i1, this mutagenesis approach
could not assess whether the 5-HT1B receptor directly
interacts with Gln304 and Glu308 or whether
these residues have an indirect effect on receptor coupling as a result
of structural changes that occur secondary to amino acid substitution.
As shown in Fig. 8, both of these residues are potentially available for direct interaction with the
5-HT1B receptor. In fact, the residues might represent one contact point within a receptor-binding surface which also includes the
carboxyl-terminal
-helix of G
i1 and perhaps the
6
strand (Fig. 8). To determine whether the Q304K-E308L mutation could alter the surface electrostatic potential of G
i1, we
utilized the GRASP program (developed by A. Nicholls and B. Honig,
Columbia University) to compare the mutant
subunit
(G
i1-Q304K-E308L) to native G
i1. The net
molecular charge of G
i1 is
3, whereas the net charge
of the mutant is
1. As shown in Fig.
9A, G
i1 residues Gln304, Glu308, and Thr321
produce a pocket of negative charge at the surface. The Q304K-E308L mutation of G
i1 changes the surface potential near these
residues to a net positive charge. In addition, the surfaces
immediately surrounding these residues are now more neutral than in
native G
i1. The marked change in the surface potential
as a result of these mutations may alter the strength of receptor
contact with the
subunit.
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Fig. 8.
Space filling model of the GTP bound form of
G i1 highlighting both known and
hypothetical receptor contact sites on the G
subunit. Residues within the
4 helical domain
(purple), which were determined to be critical for
5-HT1B receptor coupling (Gln304 and
Glu308), are colored orange to illustrate that
these residues are surface-exposed and could potentially directly
interact with the 5-HT1B receptor. A hypothetical
receptor-binding site may also include portions of the
6 strand
(green) as well as the
5 helical-carboxyl-terminal domain
(fuchsia). The guanine nucleotide (orange) is
shown buried within the core of the
subunit. The model was
generated using INSIGHT II (Biosym Technologies, San Diego, CA) with
the crystal structure coordinates from (30).
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Fig. 9.
Solvent-accessible surface of
subunits colored according to the electrostatic
potential. The solvent-accessible surface of GTP
S-bound
G
i1 (30) and GDP-bound Chi6 (51) are shown in
A and C, respectively. Using INSIGHT II (version
2.3.0 Viewer Module), computer-simulated models of the crystal
structures of G
i1-Q304K-E308L and Chi6-K300Q-L304E were
generated. The solvent-accessible surfaces of these mutants were then
calculated using the GRASP program (developed by A. Nicholls and B. Honig, Columbia University). These models simulate the impact of the
double mutation on the surface electrostatic potential of the
subunit. The electrostatic potential is contoured in the range from
10kBT (red) to +10
kBT (blue) where
kB is Boltzmann's constant and T is the
absolute temperature (K). Amino acids are labeled based on their
relative sequence positions (30, 31, 51, 52). C, carboxyl
terminus.
i1 may have resulted in secondary structural changes in neighboring domains. The
4-
6 loop region of G
i1 and the
6 strand
are possible candidate domains for secondary disruptions consequent to
amino acid mutation. The three-dimensional crystal structures of both
Gt and G
i1 show that the conformations of
the
4 helix and
4-
6 loop are almost identical between these
subunits (Fig. 10A).
Therefore, as the amino acid substitutions that were generated in the
present study were substitutions of G
t residues for
G
i1 residues, it is unlikely that these substitutions
would have a marked effect on the secondary structure in this domain.
This contention is supported by the fact that all mutant G
subunits
examined in the current study remain capable of full activation by
light-activated rhodopsin (data not shown). However, as illustrated in
Fig. 10B, Gln304 of G
i1 forms a
hydrogen bond with both the side chain carboxyl groups of
Glu308 and with the
hydroxyl group of
Thr321. In contrast, G
t residue
Lys300 forms a van der Waals interaction with the
carbon on Leu304. Substitution of G
i1
residues Gln304 and Glu308 with the
corresponding amino acids from G
t (Lys300
and Leu304) results in the loss of strong side chain
interactions (compare G
i and G
t contacts
in Fig. 10B). This suggests that these mutations might
weaken the interaction of the
4 helical domain with the
6 strand
resulting in an
subunit structure that is less responsive to
5-HT1B receptor-mediated conformational change.
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Fig. 10.
Secondary structure and residue contact
sites in G i1 and
G
t. Superimposed C
traces
of
4 helix through carboxyl-terminal residues (A) of free
G
i-GTP (green) and G
t-GTP
(blue). This overlay demonstrates that there is little
difference between the secondary structure of G
i and
G
t within this domain. In addition, the TCAT motif is
"linked" to the carboxyl-terminal domain via the
5 helix and to
the
4 helix via the
6 strand followed by the
4-
6 loop.
Ribbon representation of portions of the
4 helix and
6
strand of free G
i1-GTP (B) and
G
t-GTP (C). The side chains from
G
i1 residues Gln304, Glu308, and
Thr321 in addition to G
t residues
Lys300, Lys304, and Ser317 are
drawn as stick models to illustrate physical contacts of critical
residues. Fuchsia residues represent oxygen atoms, and
green residues represent nitrogen atoms. Dashed
lines indicate intramolecular contacts between residues. Amino
acids are labeled based on their respective sequence positions (30,
52). The GTP-bound
subunits are illustrated because the GDP-bound
form of G
i contains a microdomain that changes the
conformation of the carboxyl terminus.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i/G
t chimeras and
site-directed mutagenesis, the present study determined that two
residues (Gln304 and Glu308) within the
4
helical domain of G
i1 are required for
5-HT1B receptor coupling to G
i1. These
results are consistent with our previous work implicating the
4
helix and
4
6 loop region of G
i1 in
5-HT1B receptor coupling to inhibitory G proteins. Taken together, these studies provide evidence for a previously unappreciated role for the
4 helix of
subunits in directing G protein-coupled receptor interactions.
subunits (13-18), although at least
three other regions in G
are involved in receptor interaction: the
amino-terminal domain (18, 20, 41); the
2 helix and
2-
4 loop
regions (16, 19); and the
4 helix and
4-
6 loop domain (1, 16,
42). In addition, segments of the
and
subunits may contribute
to the receptor interacting surface of heterotrimers (20-25). Whether
individual G protein-coupled receptors interact simultaneously with
several regions on heterotrimers and/or whether the profile of physical
contacts for a particular receptor may direct more subtle features of
specificity such as the efficiency of receptor coupling remains to be determined.
4 helix and/or
4-
6 loop regions
of G
in receptor interactions include studies from alanine scanning
mutagenesis on G
t (16), patterns of evolutionary
conservation (43), and tryptic digestion of G
t bound to
rhodopsin (42). Interestingly, the recently resolved crystal structure
of G
s by Sunahara et al. (44) indicates that
although the overall structures of G
s and
G
i are quite similar, the
4 helix and
4-
6 loop
region varies between these subunits both in the length of the helical
domain and the positioning of this region within the molecule itself
(44). Thus the sequence divergence and structural differences between
these G
's is consistent with an important role in specific receptor recognition.
i (30), in
three-dimensional space Gln304 and Glu308 are
situated on the same molecular surface as the carboxyl-terminal tail of
the G
subunit which has been well established as a receptor-binding site (Fig. 8). The
4 helix is connected to the carboxyl-terminal domain via the
4-
6 loop, followed by the
6 strand and the
6-
5 loop which contains a conserved guanine nucleotide binding
motif TCAT (Fig. 10A). Several biochemical studies have
reported that mutations within this TCAT motif dramatically decrease
the affinity of the
subunit for GDP (16, 45, 46). Therefore, one
could hypothesize that the 5-HT1B receptor interaction with
both the
4 helical domain and the carboxyl-terminal region might
trigger changes in the
6-
5 loop. Upon agonist activation of the
receptor, both domains may translate a conformational change to the
TCAT motif resulting in a lowered affinity of GDP for the nucleotide binding pocket. Key mutations within either one of these domains (i.e. the
4 helix or carboxyl terminus) may alter the
transmission of the receptor-induced conformational signal to the TCAT
domain and result in an inability to release GDP upon agonist binding.
subunit
is the rate-limiting step in G protein activation (47-49). This
guanine nucleotide free form of the G protein exists in a highly stable
complex with the agonist-bound receptor in the absence of guanine
nucleotides in the medium (50). Therefore, mutations of
G
i that generate defects in agonist-activated
receptor-catalyzed GDP release from the G
subunit should also result
in a failure to establish the high affinity ternary complex of agonist,
receptor, and G protein. In fact, as shown in Fig. 7, a good overall
correlation does exist between the two measurements of GTP
S binding
and affinity shift activity.
i/o family members reveals that
4 helical residues
Gln304 and Glu308 are absolutely conserved
among the members of the Gi/o family of
subunits shown
in Fig. 11. In contrast, residues in
the homologous position on G
t are different. The
conservation of these critical residues across G
i1,
G
i2, G
i3, and G
o members
of the Gi/o family is consistent with the ability of the
5-HT1B receptor to couple selectively to heterotrimers
containing any one of these members within this family of
subunits
(12). These data are also consistent with published studies indicating
that the 5-HT1B receptor is incapable of coupling to
heterotrimers containing G
t (1, 12). Clawges et
al. (12) demonstrated that whereas Gi1,
Gi2, Gi3, or Go can couple to the
5-HT1B receptor, these subunits exhibited a rank order
profile of coupling efficiency (Gi3
Gi1 > Go > Gi2) to the receptor. Together, the
current data and previously published work suggest that residues other than those identified within the
4 helical domain may mediate more
subtle differences in coupling efficiency between G proteins within the
same subfamily.
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Fig. 11.
Primary sequence alignment of
4 helical region of G
subunits. Numbers above the sequences refer to
amino acid positions in the context of G
i1.
Boldface letters represent identical amino acid
residues among G
i/o subunits. The circled
amino acid residues in G
i1 are those that were
determined to be critical for 5-HT1B receptor coupling.
Notice that these amino acids are conserved among several members of
the G
i/o family of
subunits but diverge from the
corresponding residues in G
t. bov., bovine;
hum., human; cavpo., guinea pig.
In summary, 2 amino acids within the 4 helix of the G
subunit
play a key role in directing the specificity of 5-HT1B
receptor coupling. These residues are essential for ensuring both the
formation of the high affinity state of the receptor in the presence of agonist and receptor-catalyzed GDP/GTP exchange. Conservation of these
residues across several members of the Gi/o family of
subunits strengthens the importance of these residues in agonist G
protein activation and suggests that other receptors that distinguish between G
i and G
t may utilize these
residues as well. It remains to be determined whether these residues
interact directly with the receptor or act indirectly by affecting the
secondary structure of G
or the transmission of conformational
changes to the GDP-binding pocket. Future work on the generalizability
of these results to other Gi-coupled receptors will
contribute to the understanding of the mechanism of receptor-catalyzed
G protein activation and the nature of selectivity governing various
cellular responses elicited by different biological stimuli.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Dr. Maria Mazzoni
and Tarita Thomas for critically reading the manuscript and for
providing editorial assistance. We also thank Drs. Tohru Kozasa and
Alfred G. Gilman for kindly providing the baculovirus expressing
His6-2.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant EY10291 (to H. E. H), American Heart Association Ohio-West Virginia Affiliate grant-in-aid (to S. G. G) and Research Fellowship Award 5T32CA70085-02 (to T. M. C.-V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1TND) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
To whom correspondence should be addressed: Institute for
Neuroscience, Northwestern University, 320 E. Superior, Searle Bldg., Rm. 5-555, Chicago, IL 60611. Tel.: 312-503-1109; Fax: 312-503-7345; E-mail: h-hamm{at}nwu.edu.
§ Current address: Dept. of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115.
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ABBREVIATIONS |
---|
The abbreviations used are:
5-HT, 5-hydroxytryptamine;
GTPS, guanosine
5'-3-O-(thio)triphosphate;
AMP-PNP, adenosine 5'-(
,
imino)triphosphate;
ANOVA, analysis of variance;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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