We describe a general application of the nonsense suppression methodology for unnatural amino acid incorporation to probe drug–receptor interactions in functional G protein-coupled receptors (GPCRs), evaluating the binding sites of both the M2 muscarinic acetylcholine receptor and the D2 dopamine receptor. Receptors were expressed in Xenopus oocytes, and activation of a G protein-coupled, inward-rectifying K^+ channel (GIRK) provided, after optimization of conditions, a quantitative readout of receptor function. A number of aromatic amino acids thought to be near the agonist-binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at W6.48 of the D2 receptor establishes a cation–π interaction between the agonist dopamine and W6.48, suggesting a reorientation of W6.48 on agonist binding, consistent with proposed “rotamer switch” models. Interestingly, no comparable cation–π interaction was found at the aligning residue in the M2 receptor

Ballesteros

D. A. Dougherty

Dougherty

Doupnik

Gallivan

H. A. Lester

Hibert

Hopkins

Huang

K. S. Bower

Klabunde

Kofuji

Krapivinsky

Lagerstrom

M. M. Torrice

Mark

Nemeth

Nowak

Palczewski

Rasmussen

Schwartz

Warne

Zacharias

Zhong

English

PubMed

Torrice, Michael M.

Bower, Kiowa S.

Lester, Henry A.

Dougherty, Dennis A.

Caltech Authors - Main

Probing the role of the cation–π interaction in the binding sites of GPCRs using unnatural amino acids

Supporting Information
Torrice et al. 10.1073/pnas.0903260106
SI Text
Conventional expression of the M2–GIRK1/GIRK4 system with
both RGS4 and GoA produced consistent and accurate wild-
type dose–response relationships. We next proceeded to evalu-
ate the nonsense suppression protocols by using the wild-type
recovery experiment, in which a stop codon was placed at a
specific site within the M2 receptor gene, and THG73 tRNA
ligated with the wild-type amino acid was injected into the cell
to reestablish the wild-type protein. In these nonsense suppres-
sion experiments, we injected GoA and RGS4 mRNA, along
with the GIRK1/GIRK4 and mutant M2 receptor mRNA.
Analysis of the nonsense suppression data revealed a significant
increase in EC50 for W7.40Trp (440 nM) compared with the
conventional wild-type experiment (240 nM). In addition, there
was considerable variability among different batches of Xenopus
oocytes.
To remedy the failed wild-type recovery experiment and,
hopefully, the batch-to-batch data variability, we began to search
for trends between individual cell data and other properties of
our M2/GIRK1/GIRK4 signaling system. In our normal EC50
measurements, the responses to each drug dose are averaged
across all cells, and these averaged responses are fit to the Hill
equation. For individual cell analyses, we used the measurement
cEC50 to refer to the EC50 obtained when each cell’s dose–
response relationship data were individually fit to the Hill.
When we analyzed the relationship between IK,Agonist and
cEC50, we found that the nonsense suppression and conventional
expression cEC50 datasets diverged only when comparing cells
with low IK,Agonist (Fig. S1). By separating cells from both
conventional and W7.40Trp experiments into 2-A bins, we
showed that in the lowest-current bin (0–2 A), cells from our
nonsense suppression experiments had significantly higher
cEC50 values on average than cells in the conventional expression
experiments (conventional, 380 nM; nonsense suppression, 680
nM; t test P  0.001). If cells with IK,Agonist less than 2 A were
removed from both datasets, the difference between the 2 EC50
values narrowed (230 nM and 300 nM for the conventional and
nonsense suppression experiments, respectively). We concluded
that low levels of M2 receptor expression in the nonsense
suppression experiments—the source of low IK,Agonist levels—
produced abnormally shifted ACh dose–response relationships.
One possible explanation for this connection between expres-
sion levels and dose–response relationships is the injection of
GoA mRNA. Nonsense suppression produces lower levels of
receptor expression than conventional expression methods, and
this would produce a lower flux of free G subunits in response
to ACh application. Injection of GoA mRNA increases the level
of free G inside the cell, which could bind not only to the
endogenous Xenopus oocyte G subunits (the desired out-
come), but also to G subunits liberated by activation of the
GPCR. This would prematurely terminate M2–GIRK signaling.
Under such conditions, a higher dose of ACh would be needed
in low-expressing cells to produce the equivalent degree of signal
saturation compared with normally expressing cells. Such a
phenomenon would shift the dose–response relationship to
higher cEC50 values.
To test this hypothesis, we performed a series of wild-type
recovery experiments in which we reduced the amount of GoA
mRNA injected and monitored the change in mean cEC50. Cells
with 0 ng of injected GoA mRNA had cEC50s that were not
significantly different from the conventional expression exper-
iments (suppressed, 260  30 nM; conventional, 380  40 nM;
t test P  0.1) and were significantly lower than cells with 2 ng
(680  60 nM; t test P  0.002) or 1 ng (530  120 nM; t test
P  0.02) of GoA. When all of the cells from each condition
were pooled together, the EC50 for W7.40Trp without GoA was
290 nM, similar to the conventional wild-type EC50 of 240 nM.
As further confirmation of our hypothesis, we observed that
GoA injection does not change the dose–response relationship
of the conventionally expressedM2 receptor: the EC50 measured in
the conventional expression experiment with GoA was 250 nm,
whereas the EC50 recorded without the G protein was 240 nM.
Torrice et al. www.pnas.org/cgi/content/short/0903260106 1 of 4
Fig. S1. Suppression M2AChR experiments exhibit higher cEC50 values in cells with low IK,Agonist. When IK,Agonist is plotted with cEC50, conventional and
suppressed wild-type data diverge most for cells with IK,Agonist 2 A.
Torrice et al. www.pnas.org/cgi/content/short/0903260106 2 of 4
Fig. S2. Alignment of2AR, M2AChR, and D2DR binding-site sequences. Residues within 5 Å of the ligand in the2AR crystal structure are underlined. Aromatic
residues examined in this study are highlighted. We have adopted the numbering system, in which the most highly conserved residue of transmembrane helix
X is designated X.50. A residue 5 to the N terminus would be X.45; 5 to the C terminus would be X.55. Note that the X.50 residue does not necessarily lie in the
middle of the transmembrane helix; it is simply the most conserved residue [see: Ballesteros JA, Shi L, Javitch JA (2001) Mol Pharmacol 60:1–19].
Torrice et al. www.pnas.org/cgi/content/short/0903260106 3 of 4
Table S1. Data for M2 AChR at W7.40, W6.48, and W3.28 and for the D2 receptor at F3.28, F5.47, W6.48, F6.51, and F6.52*
EC50 nH† n
M2 AChR
WT
W3.28
dCA 1,900  80 0.8  0.02 12
W6.48
Trp 310  6 0.8  0.01 17
F2Trp 1,100  70 0.8  0.04 12
F3Trp 420  30 1.1  0.06 14
W7.40
Trp 190  20 0.9  0.1 41
F1Trp 240  9 0.9  0.03 26
F2Trp 1,000  80 0.8  0.04 20
F3Trp 170  10 0.9  0.05 12
D2 receptor
WT 59  3 1.06  0.02 44
F3.28
Phe (WT) 55  1 1.13  0.04 5
F1Phe 140  10 0.84  0.03 4
F2Phe 36  1 1.0  0.1 3
F3Phe 140  10 0.89  0.14 3
Cha 97  2 1.03  0.04 5
F5.47
Cha 78  1 1.30  0.14 7
W6.48
Trp (WT) 42  4 0.96  0.05 15
F1Trp 120  10 0.98  0.05 14
F2Trp 290  30 0.95  0.06 11
F3Trp 840  60 0.85  0.05 13
F4Trp 1,800  300 0.84  0.06 16
Nap 190  20 1.12  0.06 8
F6.51
Phe (WT) 65  4 1.03  0.04 6
F1Phe 76  6 0.98  0.03 12
F2Phe 4,200  350 0.97  0.04 7
F3Phe 6,200  400 0.095  0.03 18
Cha 55,000  4,000 0.87  0.06 4
4-CNPhe 1340  160 0.94  0.06 4
4-MePhe 690  40 1.01  0.02 6
3,5-Me2Phe 75,000  5000 0.89  0.05 6
F6.52
Phe (WT) 45  3 1.04  0.11 6
F1Phe 41  2 1.01  0.11 7
F2Phe 1,700  100 1.14  0.03 8
F3Phe 5,500  400 1.09  0.03 7
4-CNPhe 240  30 1.06  0.05 5
4-BrPhe 1,500  100 1.02  0.04 4
4-MePhe 91  6 1.00  0.05 5
3,5-Me2Phe 33,000  3,000 1.11  0.10 7
Conventional
T7.39V 100  20 0.89  0.12 4
D3.32E 50,000  4000 1.07  0.08 9
D3.32N 140,000  10,000 1.12  0.03 6
D3.32S 730,000  60,000 1.13  0.09 5
D3.32A 2,000,000  300,000 0.83  0.07 2
*EC50 (nM) and nH values are  SEM.
†Hill coefficient.
Torrice et al. www.pnas.org/cgi/content/short/0903260106 4 of 4


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Probing the role of the cation-  interaction in the binding sites of GPCRs using unnatural amino acids

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Probing the role of the cation–π interaction in the binding sites of GPCRs using unnatural amino acids

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