From the Department of Cellular and
Molecular Pharmacology and the ¶ Endocrine Unit, Department of
Veterans Affairs, University of California, San
Francisco, California 94143
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ABSTRACT |
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The seven transmembrane helices of
serpentine receptors comprise a conserved switch that relays signals
from extracellular stimuli to heterotrimeric G proteins on the
cytoplasmic face of the membrane. By substituting histidines for
residues at the cytoplasmic ends of helices III and VI in retinal
rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II)
prevented the receptor from activating a retinal G protein,
Gt (Sheikh, S. P., Zvyaga, T. A.,
Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Now we report engineering of
metal-binding sites bridging the cytoplasmic ends of these two helices
in two other serpentine receptors, the Serpentine receptors are key signaling molecules that relay
extracellular signals from hormones and sensory stimuli to
heterotrimeric G proteins located on the cytoplasmic face of the plasma
membrane. Ligand-activated receptors activate G proteins by promoting
exchange of GTP for GDP bound to the Baldwin et al. (7) have proposed a three-dimensional
model of the transmembrane helices of receptors in the rhodopsin
family. Based on analysis of the amino acid sequences of ~500
rhodopsin-like receptors and guided by a projection density map of frog
rhodopsin (8), the model places each individual helix in the density map and specifies its position relative to the lipid bilayer, tilt in
the plane of the membrane, and position and orientation relative to
other helices. Thus experiments that define distance constraints
between specific positions in individual helices can test the model and
may enhance its precision. In addition, the model provides a starting
point for designing experiments to determine the molecular mechanism by
which the helix bundle transmits signals across the membrane from
ligand to G protein. In such a mechanism, it seems likely that
occupancy of the ligand-binding pocket induces a switch-like movement
in the relative positions of two or more helices.
At present we have only a fragmentary notion of how one part of
such a receptor switch may work. Site-directed spin labeling experiments with retinal rhodopsin suggest that activation causes the
cytoplasmic end of helix VI to move, as a rigid body, away from helix
III (9). In accord with this idea, activation of rhodopsin is blocked
by either of two kinds of biochemical constraints that prevent movement
of helices III and VI relative to one another. These constraints
include disulfide bonds (9) or a metal ion bridge (10), engineered by
substituting cysteines or histidines, respectively, at appropriate
positions in the two helices.
Does ligand-induced separation of helices III and VI play a key role in
activation of other serpentine receptors? To answer this question, and
to test the generality of the Baldwin-Schertler model, we have
constructed Zn(II) bridges connecting the cognate helices of two
additional serpentine receptors, the The results of our experiments indicate that both the
Materials--
Dulbecco's modified Eagle's medium H21, minimal
essential medium, and fetal bovine calf serum were obtained from the
UCSF Tissue Culture Facility. GTP Construction of Receptor Mutants--
Point mutations were
generated either by Kunkel mutagenesis, as described (12), with a
mutagenesis kit (CLONTECH), or by polymerase chain
reaction in two steps, using Pfu polymerase and wild type
receptor cDNA as probes. In the first polymerase chain reaction, we
generated overlapping fragments containing the desired mutation and a
diagnostic silent restriction site, using wild type cDNA as a
template. In the second polymerase chain reaction the generated
fragments were annealed by virtue of their overlapping sequence,
amplified in the absence of wild type cDNA, and subcloned into the
pcDNA1 expression vector containing wild type receptor cDNA.
Each mutation was verified by DNA sequencing.
Cell Culture and Transfection--
COS-7 and CHO cells were
maintained in Dulbecco's modified Eagle's medium H21 or modified
Eagle's (minimal essential medium) Membrane Preparation--
Membranes from COS-7 or CHO cells,
transfected with cDNAs encoding PTHRs or G Protein Purification--
Gs Activation--
Exchange of GTP Ligand Binding--
Binding of 125I-cyanopindolol
(CYP) was determined as described (17). Binding was initiated by
suspending membranes (5 µg of protein in a final volume of 500 µl)
in a mixture of 125I-CYP (75 pM), increasing
concentrations of isoproterenol, a buffer consisting of 25 mM NaHepes, pH 7.6, 0.05% (w/v) bovine serum albumin, and
0.1 mM ascorbic acid. Zn(II) (10 µM) and
GTP Alignments Using the Evolutionary Trace Method--
In the
absence of recognizable sequence identity, the evolutionary trace
method can be used to align positions so as to match their functional
importance during evolution.2
Here, 58 animal visual opsins, 56 adrenergic receptors, and 33 members
of the secretin-like family were gathered from Swiss-Prot version 34.0. These sequences are shown in Table I. The
seven transmembrane regions, recognizable by their hydrophobicity, were excised from the rhodopsin and adrenergic receptors. PILEUP (from the
GCG8.0 Wisconsin Sequence Analysis Package) then produced an alignment
and a sequence identity dendrogram of each helix. The evolutionary
trace computed for each position of each helix its evolutionary rank,
which is the minimum number of branches that span the dendrogram so
that this position is invariant in each branch. This ranking measures
the functional importance of this position during evolution relative to
the other positions in the multiple sequence alignment (18). The same
procedure was followed with sequences from the secretin-like receptor
family, except that, in the absence of a clearly defined alignment to the other receptors, nine possible alignments were considered for each
transmembrane helix, each shifted one residue further toward the C
terminus. Table II shows the alignments for PTHR helices III and VI
that showed, in comparison to the other eight alignments, the highest
non-parametric (Pearson) correlation of ranks between the secretin
family transmembrane helices and those from the opsin and adrenergic
receptors.
Receptor Activation Assay--
To assess activation of
Gs by wild type and mutant receptors, we measured
ligand-dependent binding of radioactive GTP
In the Choosing Sites for Histidine Substitutions--
In the
In the absence of obvious similarities of amino acid sequence, aligning
the PTHR and rhodopsin sequences (Table II) was more difficult. We
based this alignment on an evolutionary trace analysis (18, 21), as
described under "Experimental Procedures." This method has already
successfully identified G Zn(II) Sensitivity of
We next tested the abilities of histidines placed at successive
positions around helix VI to cooperate with the histidine substituted
at position 134 in helix III (Fig. 2 and Table III). Zn(II) blocked
receptor activation (IC50 ~1 µM) when the
histidine in helix III (red in Fig.
3A) was paired with a
histidine at position 268, 269, or 272 (yellow in the same
figure). Relative to a receptor lacking His-269 and the substituted
histidine in helix III, a histidine at position 268 or 272 in helix VI
produced a receptor with intermediate sensitivity to inhibition by
Zn(II) (IC50 ~10 µM; Table III); this
suggests that residues at all three positions (268, 269, and 272) may
cooperate with the same cryptic residue to produce a bidentate
metal-binding site with intermediate Zn(II) binding affinity. In
contrast, a histidine at each of the other positions (270, 271, or 273;
green in Fig. 3A) produced a receptor that was
quite insensitive to Zn(II) inhibition (IC50 ~30
µM; Table III), suggesting that these positions cannot
cooperate either with the cryptic residue or with the histidine at
position 134 of helix III to form a metal-binding site. According to
the Baldwin-Schertler model, positions 268, 269, and 272 are clustered
on one side of helix VI (Fig. 3A). Moreover, histidines at
positions 268 and 272 would occupy locations one turn apart in an
We have not identified the cryptic third member of this putative
tridentate binding site. A likely possibility is Asp-130, which forms
part of a highly conserved DRY/ERY motif in helix III; Asp-130 is one
turn (4 residues) away from the site of our histidine substitution
(position 134).
Zn(II) Sensitivity of PTH Receptors--
We first probed the PTHR
with histidines substituted at positions that correspond (according to
the evolutionary trace analysis) to those we mutated in the
From these results we infer that a histidine at position 301 in helix
III can partner with histidines at six different positions in helix VI
to form metal-binding sites that inhibit the ability of the receptor to
activate Gs. Table III shows that the Zn(II) sensitivity of
two of these putative metal-binding sites is much greater than those of
others: combinations of His-301 in helix III with histidines at
positions 401 or 402 in helix VI created receptors that were 20- or
80-fold more sensitive to Zn(II) than the wild type receptor (Table III
and Fig. 2).
Fig. 3B depicts the Baldwin-Schertler receptor model,
highlighting the predicted positions of histidines that create
metal-binding sites in the PTHR. In the model the key histidine
(red) in helix III of the PTHR, His-301, is located on the
opposite face of this helix from the position that creates a
Zn(II)-binding pocket in the
Fig. 3 highlights an apparent difference between the Agonist Binding Affinity of Wild Type and Mutant
It is worth noting that failure of Zn(II) to affect agonist binding
affinity of the In these experiments we engineered potential metal-binding sites
into two serpentine receptors as probes for elucidating the structure
and molecular mechanism of the receptor switch. This approach, which
has been applied to serpentine receptors (25-27) and many other
proteins (28), depends upon the ability of Zn(II) (or certain other
metals) to be chelated by side chains of two or more amino acids in a
protein. Because the imidazole group of histidine chelates metals
rather well, potential Zn(II) bridges are often constructed by
substituting histidines at appropriate positions in a mutant protein. A
Zn(II) bridge between appropriately oriented histidines in separate
structural elements of the protein will link the two elements together.
If Zn(II) inhibits a function of such a mutant protein, we infer that
normal function requires movement of one or both of the two structural
elements, relative to the other. For this inference to be valid, Zn(II)
must inhibit function of the mutant protein at a considerably lower
concentration than that required to inhibit function of the wild type
protein; moreover, neither histidine substitution should mediate the
Zn(II) effect on its own, and the histidine substitutions should not alter function of the mutant protein in the absence of Zn(II). Our
results meet these criteria.
From the effects of Zn(II) bridges in the A Test of the Baldwin-Schertler Model--
In known
three-dimensional structures,
These geometrical constraints constitute useful tests of the
Baldwin-Schertler model. This is because projection density maps obtained from electron cryomicroscopic studies of rhodopsin (8) indicate probable locations and tilts of
Other data can similarly be explained only if the helices are ordered
in a clockwise fashion. In the tachykinin NK-1 receptor, agonist
binding was blocked by either of two bidentate Zn(II) sites on the
extracellular side of the helix bundle (between helix III and helix II
or helix V) (26); formation of these Zn(II)-binding sites would have
been much less likely in a counterclockwise helix bundle. In addition,
functional folding of chimeric muscarinic receptors required alteration
of threonine residues in helices I and VII, suggesting that these
residues meet at an interface between these two helices (33).
The Zn(II) bridges we engineered into the
Although the geometric constraints imposed by histidine-histidine
Zn(II) bridges (30, 31) confirm the low resolution model (7) proposed
by Baldwin and colleagues, they do not enhance its precision. The helix
VI substitutions that did enhance sensitivity appear to be situated on
one face of the helix (Fig. 3), as the model would predict. The low
"resolution" of these experiments, however, is evident from
comparing the effects of Zn(II) on mutant The PTHR Versus Rhodopsin and the
The shared seven-helix topology suggests the hypothesis that all three
receptor families evolved from a common precursor and share a common
three-dimensional architecture and mechanism for transducing signals
from the agonist-binding site to the G protein. Based on this
hypothesis we used the evolutionary trace approach (18) to align
sequences in the secretin-like receptor family with apparently cognate
positions in helices III and VI of the rhodopsin family (for details,
see "Experimental Procedures" and "Results"). As an initial
test of the hypothesis, we compared effects of metal-binding sites
engineered into a member of each family, the PTHR and the
The effects of Zn(II) on the PTHR mutants indicate that the cytoplasmic
ends of helices III and VI are close to one another in this receptor,
much like the corresponding helices of the
Overall, Zn(II) sensitivities of the PTHR mutants generally agree with
the Baldwin-Schertler model, which would predict distances between the
appropriate positions ranging from 7.3 to 12.5 Å (see Table III).
Accordingly, we propose that receptors in the secretin receptor-like
family share the overall three-dimensional architecture of receptors in
the rhodopsin family. Extension of the model to this second family of
receptors was not anticipated (7) but is in keeping with the basic
topology shared by the two families and with their apparently similar
signaling functions.
Donnelly (4) has proposed a molecular model for the transmembrane
helices of another member of the secretin-like receptor family, the
glucagon-like peptide 1 receptor. In this model, as compared with the
Baldwin-Schertler model, helix III is less buried within the helix
bundle; helix VII, lying closer to the receptor core, is located
between helices III and VI. Donnelly's tentative model, which includes
loops and tilts of helices, predicts much longer Functional Role of Helices III and VI in Activating the G
Protein--
How does Zn(II) prevent receptors with the appropriate
histidine substitutions from activating the G protein? A
straightforward interpretation, previously applied to the inhibitory
effect of a cognate Zn(II) bridge in rhodopsin (10), is that the Zn(II) ion prevents movement of helix VI relative to helix III; the relative motion of the two helices results from activation of the serpentine receptor switch and is necessary for effective catalysis of GDP-GTP exchange on the G protein. This notion is strongly supported by results
of site-directed spin labeling experiments (9), which were interpreted
as showing that photo-excitation of rhodopsin causes the cytoplasmic
end of helix VI to move 10-15 Å away from helix III and to rotate on
its own axis, in a clockwise
direction.4
Our speculative extension of this scenario (29) includes two additional
inferences: the stimulus-induced separation of helix VI from helix III
opens a cleft or pocket in the cytoplasmic surface of the receptor, and
occupancy of the cleft by the C-terminal tail of the G protein's Agonist Binding Affinities of Mutant
Instead, Zn(II) had little or no effect on agonist binding affinity
(Fig. 4); wild type and mutant receptors showed nearly identical
affinities for binding isoproterenol, whether or not Zn(II) was
present. Moreover, a GTP analog reduced isoproterenol binding
affinity to the same degree in both wild type and mutant receptors,
again in a fashion that was unaffected by Zn(II). Thus transmission of
conformational change from the G protein to the ligand-binding site is
unaffected by the same Zn(II) bridges that inhibit transmission of
conformational change in the other direction, from the ligand-binding
site to the G protein. Moreover, the Zn(II) bridges prevent the G
protein from activating the receptor but not from
interacting with the receptor.
Can these asymmetric effects of Zn(II) tell us something useful about
how the receptor switch works? If Zn(II) bridges prevent movement of
helix VI relative to helix III, as described above, then this movement
does not mediate the effect of G protein on agonist binding affinity.
For example, Zn(II)-induced immobilization of helices III and VI may
prevent the receptor from promoting release of GDP from the G protein
trimer, whereas the GDP-bound form of the trimer interacts with a
separate site on the cytoplasmic face of the receptor to initiate the
conformational change that enhances agonist binding affinity. In this
regard, several rhodopsin mutants furnish an instructive precedent (37,
38); these mutations prevent the receptor from promoting GDP release
from the trimer (38), but their association with the trimer nonetheless
stabilizes the metarhodopsin II spectral form of rhodopsin. The
locations of these mutations suggest that, like the engineered Zn(II)
bridges, they affect a function mediated by the cytoplasmic ends of
helices III and VI. One of the mutations substituted a different
sequence (which included the cytoplasmic end of helix III) for a part
of the second intracellular loop of rhodopsin; the other deleted most
of the third intracellular loop, including the cytoplasmic end of helix VI.
Peptides representing the 11 C-terminal amino acids of two different
G
Finally, the failure of Zn(II) to alter high agonist affinity of the
A second, more difficult question relates to the fact that we assessed
2-adrenoreceptor
and the parathyroid hormone receptor; occupancy of the metal-binding
site by Zn(II) markedly impairs the ability of each receptor to mediate
ligand-dependent activation of Gs, the
stimulatory regulator of adenylyl cyclase. We infer that these two
receptors share with rhodopsin a common three-dimensional architecture
and an activation switch that requires movement, relative to one
another, of helices III and VI; these inferences are surprising in the
case of the parathyroid hormone receptor, a receptor that contains
seven stretches of hydrophobic sequence but whose amino acid sequence
otherwise shows no apparent similarity to those of receptors in the
rhodopsin family. These findings highlight the evolutionary
conservation of the switch mechanism of serpentine receptors and help
to constrain models of how the switch works.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit (G
) of the
heterotrimer, causing liberation of both
-GTP and free
complexes, which in turn activate effector enzymes and ion channels (1,
2). Patterns of conserved amino acid sequence distinguish three
separate families of serpentine receptors in mammals; these include the rhodopsin-like receptors, with more than 1000 members, and two smaller
families, related to the secretin receptor or to metabotropic glutamate
receptors, respectively (3-6). Although the three families share no
similarities of primary structure, all members of each family activate
heterotrimeric G proteins, and all contain seven stretches of
hydrophobic amino acids, which are thought to be folded into a bundle
of transmembrane
-helices.
2 adrenoreceptor (
2AR)1 and the
parathyroid hormone receptor (PTHR). The
2AR, one of the
best studied serpentine receptors, belongs to the rhodopsin family but
is stimulated by different ligands (norepinephrine or epinephrine,
rather than light-activated retinal) and activates a different G
protein (Gs rather than Gt). A member of the
secretin-like receptor family, the PTHR regulates calcium homeostasis,
is stimulated by a polypeptide hormone, and activates both
Gs and Gq; its primary structure shows no
resemblance to that of either rhodopsin or the
2AR (11),
whereas amino acid sequences of the latter two receptors are 16%
identical. Thus the PTHR furnishes an opportunity to probe a distinct
family of serpentine receptors, whose evolutionary relation to the
rhodopsin family is unknown.
2AR and the PTHR share with rhodopsin a conserved
structure and activation mechanism. Thus it is likely that the
secretin-like and rhodopsin families evolved from a common serpentine precursor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, GDP, leupeptin, and
phenylmethylsulfonyl fluoride were purchased from Roche Molecular
Biochemicals, isoproterenol, cholic acid, and Lubrol from Sigma, and
synthetic bovine PTH-(1-34) from Bachem (Irvine, CA). Nitrocellulose
filters and the vacuum manifold used for GTP
S binding assays were
from Millipore. The
2AR antagonist 125I-CYP
was obtained from Amersham Pharmacia Biotech and
[35S]GTP
S from NEN Life Science Products.
HitrapChelate and HitrapQ columns were obtained from Amersham Pharmacia
Biotech. ZnCl2 and other materials were of reagent grade
and purchased from Sigma or Fisher.
medium, containing 10% fetal
calf serum, Fungizone, and 10 µg/ml gentamycin. Transient
transfections with wild type and mutant receptors were performed using
a DEAE-dextran/adenovirus method as described (12).
2ARs,
respectively, were prepared by a modification of a previously described
method (12). Briefly, cells were harvested, lysed in a buffer
containing 20 mM NaHepes, pH 7.4, with protease inhibitors
(phenylmethylsulfonyl fluoride, bacitracin, pepstatin, and leupeptin),
and homogenized by passing 12 times through a 27-gauge needle. Membrane
fractions were obtained by centrifugation at 4 °C, first at 900 × g for 10 min and then at 100,000 × g for 30 min. The membranes were stripped of GTP-binding proteins essentially as described (13), by incubation in 6 M urea buffered by 25 mM NaHepes, pH 7.4, for 30 min on ice, followed by
sedimentation at 100,000 × g for 30 min at 4 °C.
After a second urea wash and centrifugation, the membranes were
reconstituted in 250 mM sucrose, 5 mM Tris/HCl,
pH 7.4, frozen in liquid nitrogen, and stored at
70 °C.
s was purified from
cytosol of Sf9 cells infected with baculovirus encoding the wild
type protein, exactly as described (14). In some experiments we used
His6-tagged
s, purified without detergents in two steps at 4 °C. Sf9 cell cytosol, prepared by nitrogen
cavitation, was passed over a nickel-charged HiTrapChelate column (5-ml
bed volume), and
s was eluted with 0.5 M
imidazole, followed by chromatography on a HitrapQ column (5 ml bed
volume) with a NaCl gradient. G
was purified from Sf9
cells using His6-tagged
i2, as described (15).
S for GDP bound
to Gs was measured by a modification of a previously
described procedure (14, 16). Briefly, membranes containing receptors
(~5 nM) were preincubated with purified
s
(50 nM) and
(100 nM) for 15 min on ice
in a buffer containing 20 mM NaHepes, pH 7.6, 1 mM Tris/HCl, pH 7.6, 100 mM NaCl, 0.1 mM ascorbic acid, 2 mM MgCl2, 1 µM GDP, and 1 mM
-mercaptoethanol. Assays
were initiated by addition of agonist and 1 µM
[35S]GTP
S (105 cpm per tube), in a total
volume of 20 µl. After incubation for the indicated times at
30 °C, reactions were terminated by adding 400 µl of ice-cold stop
solution containing 20 mM Tris/HCl, pH 8, 100 mM NaCl, and 10 mM MgCl2, and
filtered over nitrocellulose membranes on a vacuum manifold; filters
were then washed 5 times with 250 µl of stop solution. Radioactivity
was quantitated by liquid scintillation in a
-counter. Nonspecific
binding (binding to the filter in the absence of membranes) was less
than 10% of total binding. Specific binding was defined as the
difference between total binding and nonspecific binding.
S (30 µM) were present or absent, as indicated.
Nonspecific binding was assessed in the presence of 10 µM
isoprotenerol. Reactions were conducted for 45 min at 30 °C, stopped
by adding 2 ml of ice-cold binding buffer, and filtered over Whatman
GF/C filters. Membranes used for binding assays were not subjected to
washes with urea.
Receptors used in the evolutionary trace alignments
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S in a
mixture containing pure
s, pure
, and urea-washed
membranes from cells expressing the recombinant receptor. Washing the
membranes with urea, a procedure modified from previous assays (13, 19, 20), removes contaminating GTP-binding proteins from the membrane preparation without inactivating the recombinant serpentine receptors. We expressed wild type and mutant versions of the PTHR and the
2AR in COS-7 cells and CHO K1 cells (which lack the
endogenous
-adrenoreceptors present in COS-7 membranes), respectively.
2AR assay, GTP
S binding required the presence
of receptor,
s,
, and the agonist, isoproterenol
(Fig. 1A). The effect of
isoproterenol was rapid (complete within 3 min; Fig. 1B),
saturable by increasing concentrations of
s (Fig.
1C) or
(not shown), and dependent on concentration
(Fig. 1D); the EC50 for isoproterenol, 68 nM, is comparable to values reported previously
(e.g. Ref. 17). Isoproterenol increased GTP
S binding 5-20-fold in different experiments; at maximal stimulation,
radioactive GTP
S bound to 10-30% of the total
s
present in the assay. Concentrations of
s and
required in the PTHR assay (not shown) were similar to those in the
2AR assay, but a longer time (10 min) was required for
maximal PTH-dependent binding of GTP
S.
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Fig. 1.
2AR-dependent activation of
Gs in a reconstituted system. Urea-washed membranes
from CHO cells expressing the recombinant
2AR were
treated with or without isoproterenol in the presence of Gs
subunits, and binding of [35S]GTP
S to
s
was measured, as described under "Experimental Procedures."
A, binding of [35S]GTP
S after incubating
membranes for 4 min at 30 °C in the presence or absence (as
indicated) of isoproterenol (1 µM),
s (50 nM), and
(100 nM); the concentration of
2AR (assessed by binding radioactive antagonist) was 5 nM, except for the two columns on the right,
which represent incubations of membranes from cells not expressing the
2AR. The total volume of each incubation was 20 µl.
The bars represent means of duplicate determinations, shown
in circles. This experiment is representative of four
separate experiments using two different membrane preparations.
B-D, binding of [35S]GTP
S under conditions
identical to those described for A, except that incubations
were conducted for different times (B), at different
concentrations of
s (C), or at different
concentrations of isoproterenol (D). Values in B
represent determinations in the presence or absence of 1 µM isoproterenol (filled and open
symbols, respectively). C, the concentrations of
isoproterenol (1 µM),
2 adrenoreceptors (5 nM), and G
(250 nM) were fixed.
2AR and PTHR we sought to reproduce metal-binding sites
cognate to the site we had constructed in rhodopsin (10). To do so, we
used the alignments of helix III and VI amino acid sequences shown in
Table II. Similar primary structures made
it straightforward to align these segments of the
2AR
with those of rhodopsin. In TM III of the
2AR we
substituted a histidine for Ala-134, which is cognate in sequence to
Val-138 of rhodopsin, a position that participated in the helix
III-helix VI metal ion-binding site we created in that receptor (10).
In TM VI, we substituted histidine, in separate mutant receptors, for
each of six consecutive amino acids. These six residues (Table II)
cover more than a full turn of the putative
helix; they include
Leu-272, which is cognate to the position in helix VI of rhodopsin
(residue 251) at which a substituted histidine participated in the
engineered metal ion-binding site (10).
Alignments of bovine rhodopsin, the human 2AR, and the
opossum PTHR
surfaces that interact with
and
with serpentine receptors (21, 22). Briefly, the analysis assumes that
shared structures and molecular mechanisms dictate similarly located
interfaces between helices and therefore similar patterns of
functionally important residues in each helix; this should be true even
if the sequences themselves show no identical amino acids. The analysis
(to be described in detail elsewhere)2 identified apparent
functionally important positions (as indicated by patterns of sequence
conservation) in serpentine receptors related to rhodopsin and compared
distributions of these positions to those of similarly important
positions in receptors related to the PTHR. The evolutionary trace
approach revealed putative structural and functional similarities
between the rhodopsin and secretin-like receptor families. Based on the
analysis, we substituted histidines for Leu-303 in helix III and at
each of six consecutive positions in helix VI of the PTHR (Table II).
In addition, we tested the Zn(II) sensitivity of mutants containing the
histidine at position 301 in helix III of the PTHR (Table II).
2AR Receptors--
To our
surprise, a relatively low concentration of Zn(II) (IC50
~10 µM) inhibited the ability of the wild type
2AR to activate Gs (Fig.
2). This suggested that the wild type
receptor contains a cryptic endogenous site where Zn(II) can bind and
block activation. One partner in such a site may be a naturally
occurring histidine (His-269) in helix VI (see Table II). Several
observations suggest that this residue participates in a metal
ion-binding site. Replacement of His-269 by an alanine reduced the
sensitivity of the receptor to inhibition by Zn(II); moreover,
substitution of a histidine for Ala-134 in helix III increased the
Zn(II) sensitivity of the receptor containing His-269 ~10-fold (Fig.
2 and Table III). A 30-fold higher Zn(II)
concentration was required for half-maximal inhibition of the control
receptor, containing the histidine substituted in helix III but lacking
histidine at position 269 in helix VI (Table III). Taken together,
these observations indicate that histidines in helices III and VI can
form a Zn(II) bridge that inhibits activation by the
2AR
and that the metal-binding site is in fact tridentate, involving an
unidentified third amino acid (see below) somewhere nearby.
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Fig. 2.
Effect of different concentrations of Zn(II)
on Gs activation (assessed by binding of
[35S]GTP S) stimulated by wild
type and mutant
2AR
(top) and PTHR (bottom).
Concentration of [35S]GTP
S, receptors,
s, and
are described under "Experimental
Procedures." The concentrations of both isoproterenol and PTH were 1 µM. Circles and bars represent
means ± S.E. of 3-5 separate experiments.
Effects of Zn(II) on activation of Gs by wild type and mutant
receptors
S to recombinant
s, as described under "Experimental Procedures." Results
indicate the maximal ligand-induced binding of [35S]GTP
S
(fmol per tube) and the Zn(II) concentration that inhibited activation
by 50% (IC50, µM). Data for each receptor
construct represent mean values from 3 to 12 separate experiments,
conducted on 2-6 different membrane preparations. Distances between
the
-carbons of mutated residues are those specified in the
Baldwin-Schertler model (7).
-helix, in keeping with evidence (23) that the cytoplasmic end of
helix VI in rhodopsin projects as an
-helix beyond the sequence that
is buried in the lipid core of the membrane.
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Fig. 3.
Mutated positions and Zn(II) bridges.
Ribbons represent the seven helices of serpentine receptors
in the rhodopsin family, according to the Baldwin-Schertler model (7).
Balls represent -carbons of the positions where histidine
substitutions were tested for their ability to participate in Zn(II)
bridges. A,
2AR, in which a histidine at
position 134 (red) in helix III formed a Zn(II) bridge with
histidines at each of three positions (yellow) in helix VI,
but did not form bridges with three other substituted histidines
(green) in helix VI. B, PTHR, in which a
histidine at position 301 (red) in helix III formed a Zn(II)
bridge with histidines at each of six positions in helix VI; two helix
VI positions (yellow) formed bridges at especially low
Zn(II) concentrations, whereas four (green) formed bridges
at intermediate Zn(II) concentrations.
2AR, that is Leu-303 in helix III and six positions in
helix VI. Each of the helix III-helix VI double histidine mutants
activated Gs poorly, however, even in the absence of Zn(II)
(results not shown). To our surprise, each of the helix VI
substitutions by itself (in the absence of a substitution at position
303 in helix III) produced a functioning receptor that was inhibited by
Zn(II). The susceptibility to Zn(II) turned out to depend on a
histidine residue naturally present in helix III of the PTHR, at
position 301. Replacement of the histidine at position 301 by alanine
produced receptors whose sensitivity to Zn(II) was unaffected by
histidines substituted at any of the six positions in helix VI (Table
III). The Zn(II) sensitivity of this H301A mutant (lacking histidines
substituted into helix VI) was identical to that of the wild type PTHR,
that is, to that of a receptor with a histidine at position 301 but no
histidine at any of the six positions in helix VI (Table III).
2AR (position 134 in Fig.
3A); as noted above, substitution at a different site was
necessary because histidine at position 303 inactivated the PTHR.
Nonetheless, the ability of His-301 to form Zn(II)-binding sites in
cooperation with histidines in helix VI agrees both with the
Baldwin-Schertler model and with the alignment of PTHR sequence with
those of receptors in the rhodopsin family. This is because the
-carbon of each histidine substituted in helix VI would be almost
equidistant in the model from
-carbons at either position 301 or 303 of helix III.
2AR
and the PTHR. All six histidines in helix VI of the PTHR enhanced its
sensitivity to inhibition by Zn(II) (Fig. 3B), whereas in the
2AR only three of the six histidine residues tested
did so (Fig. 3A). This may indicate that the cytoplasmic end
of helix VI in the PTHR is more mobile than its counterpart in the
2AR. It should be pointed out, however, that
substitutions at two of the helix VI sites in the PTHR (indicated by
yellow balls in Fig. 3B) produce receptors that
are much more sensitive to inhibition by Zn(II). One of these, at
position 401, is precisely cognate to the position in the
2AR where histidine substitution (L272H) induced the
greatest sensitivity to Zn(II); in rhodopsin a histidine substituted
(T251H) at the corresponding position also produced a Zn(II)-sensitive
receptor (10).
2ARs--
Interaction of Gs with the
2AR is known to enhance the receptor's affinity for
agonists (for review, see Ref. 24), presumably via a conformational
change transmitted to the ligand-binding pocket from the G
protein-binding cytoplasmic surface of the receptor. The effect of
Gs on agonist binding affinity can be reversed by adding a
GTP analog, such as GTP
S, as shown for the
2AR(H269A) mutant in Fig. 4; this reversal is
thought to reflect GTP-induced dissociation of
s from
and of both
s-GTP and
from the receptor.
If occupancy of the Zn(II) metal-binding site in appropriate
2ARs prevents them from interacting with Gs,
Zn(II) should partially or completely mimic the effect of GTP
S. This
prediction was not fulfilled (Fig. 4). In the absence of GTP
S,
addition of Zn(II) (10 µM) caused small increases
(~2-fold) in the apparent agonist binding affinities of both the
2AR(H269A) mutant and the
2AR(H134/H269) double mutant; addition of GTP
S caused equivalent decreases in agonist binding affinities of both mutant receptors, measured either in
the absence or presence of 10 µM Zn(II), despite the ~30-fold difference in sensitivity of the same receptors to
inhibition by Zn(II) (Table III). From these results we infer that
occupancy by Zn(II) of the metal-binding site does not prevent the
receptor from associating with Gs, although it does block
agonist-dependent activation of Gs by the
receptor.
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Fig. 4.
Competition between isoproterenol and
125I-cyanopindolol (CYP) for binding to
the 2AR(H269A) (A
and B) or the
2AR(A134H/269H) mutant (C
and D). Membranes of CHO cells expressing
the wild type or mutant receptor were incubated, as described (14),
with 125I-CYP (75 pM) and the indicated
concentrations of isoproterenol for 45 min, in the presence
(circles, B and D) or absence
(triangles, A and C) of 10 µM Zn(II) and in the presence or absence of 30 µM GTP
S (filled and open
symbols, respectively).
2AR(H134/H269) double mutant rules out the possibility that the cation inhibits stimulation of Gs
by denaturing the receptor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR and the
PTHR, we infer that helices III and VI of each receptor lie close to one another and also that the two helices must move, relative to one
another, for the receptor to activate the G protein. These inferences
extend our understanding of the serpentine receptor switch in four
ways. First, experiments with the
2AR confirm that the
Baldwin-Schertler structural model, based on density maps made from
rhodopsin, applies to other receptors in the rhodopsin family of
serpentine receptors. Second, experiments with the PTHR show that a
member of the secretin-like family of serpentine receptors shares a
common three-dimensional structure and activation mechanism with
receptors in the rhodopsin family. Third, our results confirm the
notion (10, 29) that ligand-induced movement of helix VI relative to
helix III is necessary for the receptor to catalyze efficient
replacement by GTP of GDP bound to the G protein. Finally, however, the
surprising failure of Zn(II) bridges to alter the agonist binding
affinity of
2AR mutants (Fig. 4) indicates that relative
movement of helix VI versus helix III is not required for
the G protein to associate with the receptor and to regulate the
affinity of its ligand-binding site.
-carbons of histidines that form
Zn(II) bridges are found no more than 13 Å apart (30, 31). The
Baldwin-Schertler model (7) predicts that the
-carbon of the amino
acid (Ala-134) replaced by histidine in helix III of the
2AR (Fig. 3) lies within 8-10.3 Å of the
-carbons
of helix VI positions at which a second histidine substitution makes
2AR(A134H) sensitive to inhibition by Zn(II).
Although details of the coordination of the metal ion remain to be
elucidated, these results confirm a key prediction of the model, that
the cytoplasmic ends of helices III and VI are located relatively close
to one another. Abundant evidence has already indicated proximity of
the cytoplasmic ends of helices III and VI in retinal rhodopsin; this
evidence includes the Zn(II) bridge we engineered into rhodopsin (10),
distances between the helices as assessed with site-directed spin
labels (9), and formation of disulfide bonds between cysteines
substituted into the same regions of these helices (9).
-helices, but the low resolution of these maps does not make it possible to identify a
specific density with a specific helix or to know whether the density
map is being viewed from the cytoplasmic or the extracellular side. The
Baldwin-Schertler model, however, does identify the helices and
predicts that they are arranged in a clockwise fashion, as viewed from
the cytoplasm (7, 32). Effects of Zn(II) sites on the
2AR mutants, along with earlier biochemical studies of rhodopsin (9, 10), support the predicted proximity of helices III and
VI as well as the predicted clockwise arrangement. Baldwin identified
helix III with the centrally located density of the rhodopsin map (32),
based on its amino acid sequence; among all the helices, the
hydrophilic and conserved residues of helix III show the least tendency
to be distributed on one face; this is the pattern expected of a helix
in the center of the helix bundle, where it must interact with other
helices on all sides. If the central density corresponds to helix III,
the Zn(II) bridges indicate a clockwise arrangement of the helices;
this is because in the opposite (counterclockwise) arrangement, helix
VI would be too far (25 Å) from helix III for formation of a
Zn(II) bridge (not shown).
2AR involve
positions in helices III and VI cognate to positions that participated in function-inhibiting Zn(II) bridges (10) engineered into retinal rhodopsin. Thus it is likely that these two receptors share both a
similar three-dimensional architecture and a highly conserved activation switch, even though they stimulate different G proteins, Gs and Gt, respectively. By extension, this
architecture and switch mechanism are probably common to all receptors
in the rhodopsin family. The same overall inference can be drawn from
the effects of engineered metal-binding sites on the extracellular
sides of two other receptors in this family (25-27); the engineered
metal ion-binding site between helices V and VI on the extracellular side of the tachykinin NK-1 receptor (25, 26) was duplicated by
mutations at cognate positions in the same helices of the
-opioid receptor (27).
2ARs
versus the predicted
-carbon distances between the
positions substituted in helix III and helix VI. The model shows short
-carbon distances (~8.7 Å) for two of the mutants that were
strongly inhibited by Zn(II) and considerably longer distances (~12.5
Å) for two of the mutants without enhanced sensitivity to inhibition
by Zn(II); the model predicted an intermediate distance (10.3 Å) for
the other two mutants, only one of which showed enhanced sensitivity to Zn(II).
2AR--
In
mammals, the receptors coupled to G proteins can be grouped into three
families, which resemble, respectively, rhodopsin, the secretin
receptor, and "metabotropic" receptors for glutamate (4-6, 32).
Within each of these families, deduced amino acid sequences of
individual receptors show clear-cut patterns of conserved and identical
amino acids. No such sequence conservation or identity has been found
in comparisons of receptors in any one of the three families
versus receptors in another. Receptors in all three families do, however, exhibit seven stretches of hydrophobic amino acids, each
of which is thought to constitute an
-helix that crosses the plasma
membrane (4-6, 32).
2AR, respectively. The results suggest that these two
receptors and, by extension, the two receptor families do share a
common origin in evolution, as well as similar three-dimensional architectures and switching mechanisms.
2AR and other
receptors in the rhodopsin family. Indeed, the "strongest" Zn(II)
bridges (those at which the lowest Zn(II) concentrations inhibit G
protein activation) link positions that would be quite close to one
another in a PTHR that conforms to the Baldwin-Schertler model and the
alignment based on the evolutionary trace approach, that is,
-carbon
distances between position 301 in helix III and positions 401 or 402 in
helix VI are 7.3 or 10.3 Å, respectively (Table III); these are within
the range of distances (30, 31) that allow histidine residues to form
metal-binding sites. In comparison to the wild type PTHR, histidine
substitutions at these two positions in helix VI markedly enhanced
Zn(II) sensitivity (20- and 83-fold, respectively). Substitutions at
the other four positions tested in helix VI also increased Zn(II)
sensitivity, although to a lower degree (3.5-5.3-fold; Table III). We
do not know how histidines at six positions around the entire
circumference of helix VI can form Zn(II) bridges with a single
histidine in helix III. Indeed, residues 397-402 in the PTHR may not
belong to an
-helix at all; alternatively, the putative helix may be unusually flexible. The latter possibility is consistent with a
rhodopsin experiment (9), in which a cysteine at each of five positions
in helix VI could form a disulfide bond with a cysteine in helix III.
-carbon distances
between positions 301 (helix III) and positions 401 or 402 (helix VI)
of the PTHR, 20.73 and 20.71 Å, respectively. These distances
substantially exceed those that allow histidines to form effective
metal ion-binding sites (28). Consequently, our results are much more
congruent with the Baldwin-Schertler model than with that of Donnelly.
subunit is required for the receptor to catalyze GDP-GTP exchange. The
first of these additional inferences implies that the Baldwin-Schertler
model represents the inactive conformation of serpentine receptors, and
is in keeping with both the spin labeling results and our experiments
with metal-binding sites. The second inference, that helix VI interacts
specifically with the C terminus of G
, was suggested by results of
an experiment (34) that tested the ability of chimeric muscarinic
receptors to interact with chimeric G
subunits; a 4-residue
epitope in helix VI functionally complemented a similarly short
sequence in the G
C terminus. One of the complementing receptor
residues in that study (34) is cognate to residue 272 of the
2AR, a position at which substitution of a second
histidine made the
2AR(A134H) mutant susceptible to
inhibition by Zn(II) (Table III); this raises the possibility that a
Zn(II) bridge may inhibit G protein activation not only by immobilizing
helix VI relative to helix III but also by steric hindrance.
2ARs--
Surprisingly, Zn(II) failed to reduce the
agonist binding affinity of the histidine-substituted
2AR mutants whose ability to activate Gs was
sensitive to inhibition by Zn(II) (Fig. 4). We had expected the
contrary result, based on the scenario for G protein activation
described above, in combination with a number of observations in many
laboratories. Thus in the absence of added guanine nucleotide the
2AR (like many other receptors) exhibits an enhanced
affinity for binding agonists (reviewed in Ref. 24). This high affinity
is thought to result from association of the receptor with the
appropriate G protein, because addition of GTP analogs reduces agonist
binding affinity and at the same time causes G protein
and
subunits to dissociate from each other and from the receptor. The
affinity of the
2AR for agonists is similarly low in
cells genetically lacking the
subunit of Gs (35, 36).
In contrast, neither GTP analogs nor genetic absence of the G protein
affect the receptor's affinities for binding pharmacological
antagonists. The ability of the G protein to enhance binding of
agonists, but not antagonists, suggests a reciprocal interaction
between the receptor-G protein interface and the agonist-binding pocket. A similar reciprocity is thought to account for the ability of
retinal transducin (Gt) to stabilize a spectral form of
photorhodopsin called metarhodopsin II (see references in Ref. 10);
that is, hormone and light induce a conformational change that enhances affinity of their respective receptors for binding G proteins, and G
proteins reciprocate by enhancing stability of this agonist-bound "activated" conformation of the receptors. For this reason, we had
expected Zn(II) bridges to prevent Gs from increasing the affinity of the
2AR for binding agonist, just as the
same bridges inhibited agonist-induced activation of
Gs.
subunits are reported (39, 40) to reproduce the effects of G
proteins on ligand-binding sites of the corresponding receptors. Such a
peptide from
t stabilized the metarhodopsin II state of
rhodopsin (39), and the cognate peptide from
s enhanced
affinity of the
2AR for binding isoproterenol (40). These results conflict with the proposal (29), described above, that
the C termini of G
subunits associate with a cleft between helices
III and VI on the cytoplasmic face of the receptor: if Zn(II) bridges
prevent formation of such a cleft, how can a G protein use the C
terminus of G
to regulate the ligand-binding site? One possibility
is that our interpretation (29) of the receptor-G
complementation
experiments (34) is wrong, i.e. the G
C terminus does not
in fact associate with this region of the receptor but regulates the
ligand-binding site by contacting the receptor at a different site.
Regardless of whether the G
C terminus interacts with the putative
cleft between helices III and VI, it is likely that G proteins can
exert their effects on ligand-binding sites by contacting either of two
(or more) separate sites on the receptor. This is suggested by the
observation (39) that the metarhodopsin II state of rhodopsin is
stabilized by a peptide representing residues 311-328 of
t. Because these residues contribute to a surface of the
G protein near but quite distinct from the C-terminal tail (41), it is
likely that the receptor makes multiple contacts with the G protein
trimer; some of these may suffice to transmit conformational change to
the ligand-binding site of the receptor, whereas a different (but
overlapping) subset of these contacts mediates receptor-induced release
of GDP.
2AR raises two questions. The first question relates to
the ability of GTP
S to shift the agonist binding curve to the right,
even in the presence of Zn(II). Does this suggest that the receptor is
promoting GDP/GTP exchange, contrary to our demonstration that Zn(II)
prevents the receptor from activating Gs? We think not,
because it is well established that Gs releases GDP
spontaneously and binds GTP
S even in the absence of receptor
stimulation. Although this basal exchange process is slower (minutes
rather than seconds) than that catalyzed by receptors, it could
certainly reach completion during the 45-min ligand binding assay. Thus
the rightward shift does not necessarily reflect receptor activation of
Gs.
2AR binding affinity and Gs activation under
quite different conditions. In the first case we added ligands to
Gs-containing particulate fractions, while we assessed
activation by adding pure G proteins to urea-washed membranes that lack
endogenous Gs. Thus it is possible that endogenous
Gs, "pre-coupled" to
2ARs in the
particulate extracts, could prevent entry of Zn(II) into the
metal-binding site and thereby prevent Zn(II) from altering any effect
of the G protein on agonist binding affinity. We cannot rule out this
possibility at present, although the extent of pre-coupling in these
(or any) membranes is unknown. Resolution of this question will require
that the binding and activation assays be performed under similar
conditions. This has proved difficult because (a) the high
background of guanine nucleotide-binding proteins in intact membranes
can obscure receptor-catalyzed binding of GTP
S, and (b)
we have found it extremely difficult to reconstitute high affinity
receptor binding by adding pure G protein subunits to receptors
in vitro.
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ACKNOWLEDGEMENTS |
---|
We thank Helen Czerwonka for excellent
secretarial assistance and members of the Bourne laboratory for advice
and encouragement. We thank Brian Kobilka (Stanford University) for
pointing out that pre-coupling of Gs to the
2AR in membranes might prevent Zn(II) from entering the site.
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FOOTNOTES |
---|
* This work was supported in part by grants from the National Institutes of Health (to H. R. B. and R. N.).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.
§ Supported by the Danish Medical Research Council and a fellowship from the Danish Heart Association. Present address: Copenhagen Cardiovascular Research Institute, and Dept. of Cardiology B, Rigshospitalet 9312, Juliane Mariesvej 20, DK-2100 Copenhagen Ø, Denmark.
Present address: Dept. of Molecular and Human Genetics, Baylor
College of Medicine, 1 Baylor Plaza, Rm. T921, Houston, TX 77030.
** Present address: Fourth Department of Internal Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan.
To whom correspondence should be addressed: S1212, Box 0450, 513 Parnassus Ave., San Francisco, CA 94143-0450. Tel.: 415-476-8161; Fax: 415-476-5292; E-mail: bourne{at}cmp.ucsf.edu.
2 O. Lichtarge, A. Philippi, R. L. Dunbrack, S. R. Coughlin, H. R. Bourne, and F. E. Cohen, manuscript in preparation.
4
The spin labels were attached to a cysteine
substituted at position 139 in helix III and to individual cysteines
engineered into positions cognate to those where we substituted
histidines in helix VI of the 2AR and the PTHR.
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ABBREVIATIONS |
---|
The abbreviations used are:
2AR,
2-adrenoreceptor;
PTH, parathyroid
hormone;
PTHR, parathyroid hormone receptor;
Gs, trimeric G
protein that stimulates adenylyl cyclase;
Gt, trimeric G
protein that mediates vision in rod cells (also called transducin);
CYP, cyanopindolol;
GTP
S, guanosine
5'-3-O-(thio)triphosphate;
TM, transmembrane;
CHO, Chinese
hamster ovary.
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REFERENCES |
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