(Received for publication, April 30, 1997, and in revised form, May 30, 1997)
From the Center for Molecular Recognition and the
§ Departments of Psychiatry and Pharmacology, Columbia
University College of Physicians & Surgeons, New York, New York 10032 and the ¶ New York State Psychiatric Institute, New York, New York
10032
The binding site of the 2
adrenergic receptor, like that of other homologous G-protein-coupled
receptors, is contained within a water-accessible crevice formed among
its seven membrane-spanning segments. Methanethiosulfonate
ethylammonium (MTSEA), a charged, hydrophilic, lipophobic,
sulfhydryl-specific reagent, had no effect on the binding of agonist or
antagonist to wild-type
2 receptor expressed in HEK 293 cells. This suggested that no endogenous cysteines are accessible in
the binding site crevice. In contrast, in a constitutively active
2 receptor, MTSEA significantly inhibited antagonist
binding, and isoproterenol slowed the rate of reaction of MTSEA. This
implies that at least one endogenous cysteine becomes accessible in the
binding site crevice of the constitutively active
2
receptor. Cys-285, in the sixth membrane-spanning segment, is
responsible for the inhibitory effect of MTSEA on ligand binding to the
constitutively active mutant. The acquired accessibility of Cys-285 in
the constitutively active mutant may result from a rotation and/or
tilting of the sixth membrane-spanning segment associated with
activation of the receptor. This rearrangement could bring Cys-285 to
the margin of the binding site crevice where it becomes accessible to
MTSEA.
The interaction of a diverse array of signals, including neurotransmitters, peptides, hormones, light, and odorants, with G-protein-coupled receptors results in a conformational change which enhances their interaction with heterotrimeric G-proteins and thereby promotes GDP release and subsequent GTP binding and G-protein activation (1). In rhodopsin, for example, photoisomerization of retinal leads to rigid body movements of the third (M3)1 and sixth (M6) membrane-spanning segments relative to each other (2). Moreover, disulfide cross-linking of these membrane-spanning segments prevents the activation of transducin, the G-protein associated with rhodopsin, further supporting the relevance of their movement in the activation of rhodopsin. The generality of this movement to other G-protein-coupled receptors, however, is not known. Moreover, additional details of the structural mechanisms of receptor activation are necessary to better understand this process.
The binding sites of the 2 adrenergic receptor and of
the homologous receptors for other biogenic amines are formed among their seven, mostly hydrophobic, membrane-spanning segments (1, 3) and
are accessible to charged, water-soluble agonists like norepinephrine.
Thus, for each of these receptors, the binding site is contained within
a water-accessible crevice, the binding site crevice, extending from
the extracellular surface of the receptor into the plane of the
membrane. The surface of this crevice is formed by residues that can
contact specific agonists and/or antagonists and by other residues that
may play a structural role and affect binding indirectly.
To identify the residues that form the surface of the binding
site crevice in the dopamine D2 receptor and the 2
adrenergic receptor, we have used the substituted cysteine
accessibility method (4-10). Consecutive residues in the
membrane-spanning segments are mutated to cysteine, one at a time, and
the mutant receptors are expressed in heterologous cells. If ligand
binding to a cysteine substitution mutant is near-normal, we assume
that the structure of the mutant receptor, especially around the
binding site, is similar to that of wild type, and thus, that the
substituted cysteine lies in a similar orientation to that of the
wild-type residue. In the membrane-spanning segments, the sulfhydryl of
a cysteine can face either into the binding site crevice, into the
interior of the protein, or into the lipid bilayer; sulfhydryls
facing into the binding site crevice should react much faster with
charged, hydrophilic, lipophobic, sulfhydryl-specific reagents. For
such polar sulfhydryl-specific reagents, we use derivatives of
methanethiosulfonate (MTS). These reagents form mixed disulfides with
the cysteine sulfhydryl, covalently linking
-SCH2CH2X, where X is
NH3+ in the case of MTS ethylammonium
(MTSEA) (11). The reagents reportedly react with the ionized thiolate
(RS
) more than a billion times faster than with the
unionized thiol (RSH) (12), and only cysteines accessible to water are
likely to ionize. We use two criteria for identifying an engineered
cysteine as forming the surface of the binding site crevice: (i) the
reaction with an MTS reagent alters binding irreversibly and (ii) this reaction is retarded by the presence of agonists or antagonists.
To identify activation-induced structural changes in the residues
forming the surface of the binding site crevice, we sought to determine
the rates of reaction of MTSEA with a series of engineered cysteines in
the resting and activated receptor. Agonist cannot be used to activate
receptor, however, because the presence of a ligand within the binding
site would interfere with access of the MTSEA to the engineered
cysteines. Alternatively, the activated state of the receptor can be
achieved by using a constitutively active mutant (CAM) 2
adrenergic receptor as a background for further cysteine substitution.
The CAM used here, L266S/K267R/H269K/L272A, has been well characterized
previously (13, 14) and is intrinsically active and has a higher
affinity for agonist than does the wild-type receptor. The high
affinity state for agonist is typically associated with the activated
receptor-G-protein complex. That agonist affinity is higher in the CAM
even in the absence of G-protein suggests that the structure of the
binding site of the CAM is likely to be similar to that of the
agonist-activated wild-type receptor binding site. Thus, using
wild-type
2 receptor and the CAM, we can compare
the resting and active forms of the receptor by determining the
accessibility of residues in the binding site crevice in these two
states.
To determine the accessibilities of substituted cysteines, the
background receptor into which we substitute cysteines must be
unaffected by our sulfhydryl reagents. Although we find here that
ligand binding to the wild-type 2 adrenergic receptor
was unaffected by MTSEA, surprisingly, MTSEA inhibited antagonist binding to the CAM form of the
2 receptor. We identify
Cys-285 in M6 as the sole cysteine responsible for this susceptibility of CAM. Because this Cys is protected by ligand, it is likely accessible within the binding site crevice in the CAM, but not in
wild-type
2 receptor.
The DNA sequence encoding the
human 2 adrenergic receptor, epitope-tagged at the amino
terminus with the cleavable influenza-hemagglutinin signal sequence
followed by the "FLAG"-epitope (IBI, New Haven, CT) and tagged with
six histidines at the carboxyl terminus, was a gift from Dr. B. Kobilka
(Stanford, CA) (15). This DNA was subcloned into the bicistronic
expression vector pcin4 (16), a gift from Dr. S. Rees (Glaxo, UK),
thereby creating the vector pcin4-SF
2H. The CAM
2
receptor (13) (L266S/K267R/H269K/L272A) was a gift from Dr. R. J. Lefkowitz (Durham, NC). A fragment encoding the CAM mutation was
subcloned into pcin4-SF
2H to create the vector pcin4-SFCAM
2H.
Mutations of endogenous cysteines were generated using the Chameleon
mutagenesis kit (Stratagene). Mutations were confirmed by DNA
sequencing. Mutants are named as (wild-type residue) (residue number)
(mutant residue), where the residues are given in the single-letter
code. Plasmids containing the mutations C77V and C116V were a gift from
Dr. B. Kobilka (Stanford, CA), and fragments containing these mutations
were subcloned into the pcin4-SFCAM2H plasmid as were fragments
encoding the mutations C125V, C285S, and C327S.
HEK 293 cells were grown
in Dulbecco's modified Eagle's medium/F-12 (1:1) containing 3.15 g/liter glucose in 10% bovine calf serum at 37 °C and 5%
CO2. Thirty-five mm dishes of 293 cells at 60-80%
confluence were transfected with 2 µg of wild-type or mutant
pcin4-SF2H using 9 µl of LipofectAMINETM (Life
Technologies, Inc.) and 1 ml of Opti-MEMTM (Life
Technologies, Inc.). Five hours after transfection, the medium was
changed. For transient transfection, the medium was changed after
24 h, and the cells were harvested after 48 h. For stable
transfection, 24 h after transfection the cells were split to a
100-mm dish, and 700 µg/ml Geneticin (Life Technologies, Inc.) was
added to select for a stably transfected pool of cells.
Cells were washed with phosphate-buffered saline (PBS) (8.1 mM NaH2PO4, 1.5 mM KH2PO4, 138 mM NaCl, 2.7 mM KCl, pH 7.2), briefly treated with PBS containing 5 mM EDTA (no trypsin), and then dissociated in PBS. Cells were pelleted at 1000 × g for 5 min at 4 °C and resuspended for binding or treatment with MTS reagents.
[3H]CGP-12177 BindingWhole cells from a 35-mm dish were suspended in 400 µl of buffer (140 mM NaCl, 5.4 mM KCl, 1 mM EDTA, 0.006% bovine serum albumin, 25 mM HEPES, pH 7.4). [3H]CGP-12177 (NEN Life Science Products) binding was performed as described previously (17) with minor modifications. For isoproterenol competition, triplicate polypropylene minitubes contained five different concentrations of isoproterenol between 10 nM and 100 µM, 0.4 nM [3H]CGP-12177, and 300 µl of a 20-fold dilution of cell suspension in a final volume of 0.5 ml. The mixture was incubated at room temperature for 60 min and then filtered using a Brandel cell harvester through Whatman 934AH glass fiber filters (Brandel). The filter was washed 3 times with 1 ml of 120 mM NaCl, 10 mM Tris-HCl, pH 7.4 at 4 °C. Specific [3H]CGP-12177 binding was defined as total binding less nonspecific binding in the presence of 1 µM alprenolol (Research Biochemicals).
Reactions with MTS ReagentsWhole cells from a 35-mm dish
were resuspended in 400 µl of buffer A. Aliquots (50 µl) of cell
suspension were incubated with freshly prepared MTSEA (Toronto Research
Biochemicals) at the stated concentrations at room temperature for 2 min. Cell suspensions were then diluted 20-fold, and 300-µl aliquots
were used to assay for [3H]CGP-12177 (400-800
pM) binding in triplicate as described above. The
fractional inhibition was calculated as 1 [(specific binding after MTS reagent)/(specific binding without reagent)].
We used the SPSS for Windows (SPSS, Inc.) statistical software to analyze the effects of MTSEA reagents by one-way ANOVA according to Student-Neuman-Keuls post hoc test (p < 0.05).
MTSEA, at concentrations as high as 100 mM for 2 min,
had no significant effect on [3H]CGP-12177 binding to
wild-type 2 receptor (Fig.
1A). This finding is consistent with previous
observations that even the less polar N-ethylmaleimide had
no effect on antagonist binding to the
2 receptor (18).
MTSEA also had no effect on the affinity of isoproterenol in
competition with [3H]CGP-12177 binding (data not shown).
Thus, it is likely that none of the 13 endogenous cysteine residues is
accessible for reaction in the binding site crevice of wild-type
2 receptor.
In contrast, MTSEA did inhibit [3H]CGP-12177 binding in
the CAM 2 receptor up to 46 ± 8% (mean ± S.E., n = 6) at the highest concentration of MTSEA
tested (Fig. 1A). Because the CAM mutation does not add
cysteines, an endogenous cysteine likely became accessible for reaction
with MTSEA in the CAM.2 Isoproterenol
retarded the reaction of MTSEA with the CAM (Fig. 1B),
consistent with access to the endogenous cysteine being through the
binding site crevice.
The 2 receptor contains five cysteines in the putative
membrane-spanning segments. These include Cys-77 in M2, Cys-116 and Cys-125 in M3, Cys-285 in M6, and Cys-327 in M7. To determine which
cysteine was responsible for the MTSEA inhibition in the CAM, we
created a series of mutations in the CAM background in which we
substituted the endogenous cysteines, one at a time or in combination.
Mutation of Cys-285 to serine decreased the inhibitory effect of MTSEA
on binding to the CAM receptor to a level indistinguishable from that
observed in wild-type receptor (Fig. 2). In contrast, substitution of Cys-77, Cys-116, Cys-125, or Cys-327 had no significant effect on the inhibition of binding caused by MTSEA (Fig. 2).
There are two ways in which the mutation of Cys-285 to serine could
abolish the inhibitory effect of MTSEA on binding to the CAM receptor.
If MTSEA reacts with Cys-285 to inhibit binding, then mutation of this
residue to serine would abolish the inhibition. Another possibility,
however, is that mutation of Cys-285 to serine might act as a second
site revertant and eliminate the CAM phenotype. In this case, Cys-285
need not be the reactive cysteine. We investigated the phenotype of the
CAM/C285S mutant by determining the affinity of isoproterenol in
competition with [3H]CGP-12177 binding. As described
previously (13), the affinity of isoproterenol for the CAM is
approximately 20-fold higher than for the wild-type receptor (Fig.
3). This affinity was not altered significantly in the
CAM/C285S mutant (Fig. 3); thus the phenotype of this receptor was
still CAM-like. Cys-285, therefore, was responsible for the inhibitory
effect of MTSEA on ligand binding to the CAM, and its substitution by
serine abolished this inhibition.
The region in M6 which contains Cys-285 is highly conserved in the
2 and dopamine D2 receptors. In the D2 receptor, Cys-385 (the cysteine which aligns with
2 Cys-285) is
inaccessible to reaction with MTSEA (6). On a helical wheel plot,
however, Cys-385 is within about 40 degrees of Phe-389 (also
conserved), which is accessible to MTSEA when substituted with
cysteine.3 Because we have not yet
determined the accessibility of multiple cysteine substitutions in M6,
we cannot yet define the specific conformational change responsible for
the increased accessibility of Cys-285 in the
2 CAM.
Based on our findings in the D2 receptor, however, a rotation of less
than 40 degrees and/or a tilting of M6 might be sufficient to make
Cys-285 accessible in the binding site crevice (Fig.
4).
It is not clear whether the CAM represents the active form of the
receptor or a form of the receptor that can more readily assume the
active conformation. The change in MTSEA accessibility in the CAM may
thus reflect the increased frequency with which the receptor attains
the active state. The rate of reaction of Cys-285 in the CAM with MTSEA
was 0.18 ± 0.02 M1 s
1
(mean ± S.E., n = 3). In contrast, the rate of
reaction of the endogenous Cys-118 accessible in the binding site
crevice of the dopamine D2 receptor was approximately 40 M
1 s
1
(7).4 Thus, while Cys-285 is much more
reactive in the CAM than in the wild-type receptor, it is not likely to
be freely accessible in the binding site crevice but rather
intermittently accessible at the margin of the crevice.
A possible mechanism for the movement of Cys-285 might involve the
highly conserved Pro-288 in M6 of the 2 receptor.
Mobility about proline kinks (9, 20-22) has been implicated in the
mechanism of activation of a number of membrane proteins (23, 24).
Interestingly, highly conserved Pro residues are found in M2, M5, M6,
and M7 of G-protein-coupled receptors. A possible mechanism of receptor activation utilizing proline kink flexibility in M6 of a
G-protein-coupled receptor has been demonstrated (25, 26). Flexibility
about the proline kink in M6 might allow rotation and/or tilting of Cys-285 toward the binding site crevice in the
CAM.5
Other findings are consistent with such a conformational change being
part of the normal mechanism of receptor activation. Fluorescence
spectroscopy has been used to monitor structural changes in the
2 receptor in response to agonist binding (27). Reaction
of the cysteine-reactive, fluorescent probe
N,N
dimethyl-N-(iodoacetyl)-N
-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) with purified
2 receptor did not affect
ligand binding, consistent with the absence of endogenous cysteines in
the binding site crevice. Addition of agonist elicited a
dose-dependent decrease in fluorescence in IANBD-labeled
2 receptor, consistent with movement of the probe toward
a more polar environment. Cys-125 and Cys-285 are responsible for this
agonist-induced fluorescence decrease.5 Increased polarity
could come about by movement of the probe closer to the binding site
crevice and/or to another membrane spanning segment. Our results
suggest that Cys-285 moves closer to the binding site crevice in the
CAM than in the wild-type receptor. Thus, the conformational change
observed in the CAM is consistent with the rearrangement induced by
agonist in wild-type receptor. In contrast, Cys-125 was not accessible
to reaction with MTSEA.
A rotation and/or tilting of M6 that would make Cys-285 accessible
(Fig. 4) is also consistent with the rotational-translational motion
predicted by spin labeling studies in rhodopsin (2). These studies
indicated that the distance between a particular pair of cysteines at
the cytoplasmic ends of M3 and M6 decreased with activation, while the
distances between other pairs increased. To explain these findings, the
authors proposed that M6 tilts away from M3 and rotates in an
anti-clockwise direction (when viewed from the extracellular face). As
illustrated in Fig. 4, the proposed rotation would reposition Cys-285
toward the water-accessible surface of the binding site crevice where
it could react with MTSEA, thus accounting for our observations in the
CAM 2 receptor. Moreover, flexibility around the proline
kink, as discussed above, could facilitate the observed rotation and
tilting in the cytoplasmic half of the membrane-spanning segment (20)
without requiring movement of the entire segment.
The systematic application of the substituted cysteine accessibility
method with both the wild-type receptor and CAM/C285S mutant as
backgrounds should allow us to detect other conformational changes
which accompany 2 receptor activation.
We are grateful to Drs. Brian Kobilka, Robert
Lefkowitz, and Stephen Rees for gifts of the epitope-tagged
2 receptor DNA, the CAM
2 DNA, and the
pcin4 plasmid, respectively. We thank Myles Akabas, Juan Ballesteros,
Arthur Karlin, and Harel Weinstein for valuable discussion and comments
on this manuscript.