Article
10% fetal bovine serum (Gibco), glutamine, penicillin and streptomy-
cin) 1 day before transfection. Transfection solution was prepared by
combining 4 μl (per well in a 6-well plate, hereafter) of polyethylenimine
solution (Polysciences; 1 mg ml−1) and a plasmid mixture consisting of
100 ng Lg–βarr1 and 500 ng NTSR1–Sm in 200 μl of Opti-MEM (Thermo
Fisher Scientific). After incubation for 1 day, transfected cells were
collected with 0.5 mM EDTA-containing Dulbecco’s PBS, centrifuged
and suspended in 2 ml of HBSS containing 0.01% bovine serum albumin
(BSA; fatty-acid-free grade; SERVA) and 5 mM HEPES (pH 7.4) (assay
buffer). The cell suspension was dispensed in a white 96-well plate at
a volume of 80 μl per well and loaded with 20 μl of 50 μM coelentera-
zine (Carbosynth) diluted in the assay buffer. After 2-h incubation at
room temperature, the plate was measured for baseline luminescence
(Spectramax L, Molecular Devices) and 20 μl of 6× neurotensin (Peptide
Institute; final concentrations ranging from 0.1 nM to 10 μM) diluted
in the assay buffer or the assay buffer alone (vehicle) were manually
added. The plate was read for 15 min with an interval of 20 s at room
temperature. Luminescence counts were normalized to the initial
count and arrestin association kinetics were calculated by fitting the
normalized luminescence data to a one-phase association model built
in Prism 8 software (GraphPad Prism). In cases in which luminescent
kinetics were bell-shaped, data points over increasing luminescence
were used for the fitting. Association speed was calculated by a formula
of (plateau – Y 0 )*K in which ‘plateau’ and Y 0 represent saturated and
initial luminescent counts, respectively, and K denotes a rate constant
in units that are the reciprocal of time (min). The resulting association
speed data were fitted to a four-parameter sigmoidal concentration–
response curve, from which pEC 50 values (negative logarithmic values
of EC 50 values, in which EC 50 is the half-maximal effective concentration)
and Emax values (‘top’ − ‘bottom’) were used to calculate the mean and
standard deviation.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
The cryo-EM density map for the NTSR1–βarr1(ΔCT) complex has been
deposited in the Electron Microscopy Data Bank under accession code
EMD-20836. The coordinates for the model of NTSR1–βarr1(ΔCT) have
been deposited in the PDB under accession number 6UP7. Proteom-
ics data have been deposited in the ProteomeXchange Consortium
via the PRIDE partner repository and can be accessed at https://doi.
org/10.6019/PXD016224. The data supporting the findings of this
study are available as Supplementary Information (Supplementary
Tables 1–39 and Supplementary Figs. 2–5). All other data are available
upon request to the corresponding authors.
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Acknowledgements We thank M. Krawitzky and C. Adams for instrument and software access;
R. Leib, F. Liu and M. Bern for discussions pertaining to mass spectrometry data analysis; G.
Lam and B. Fitch for assistance with lipidomics analysis; K. Sato, Y. Sugamura and A. Inoue for
plasmid construction and cell-based GPCR assays; and D. Mayer for suggestions on arrestin
purification. This work was supported in part by National Institutes of Health grants
R01NS028471 (B.K.K.), 1U19AI109662 (J.S.G.) and P30 CA124435 (for using the Stanford Cancer
Institute Proteomics/Mass Spectrometry Shared Resource). Additional support to G.S. and
B.K.K. was provided by the Mathers Foundation. B.K.K. is a Chan-Zuckerberg Biohub
investigator. M.M. was supported by an American Heart Association postdoctoral fellowship
(17POST33410958). J.J. is a Damon Runyon Fellow supported by the Damon Runyon Cancer
Research Foundation (DRG-2318-18). A.I. was funded by the PRIME 18gm5910013 and the LEAP
18gm0010004 from the Japan Agency for Medical Research and Development (AMED) and
KAKENHI 17K08264 from the Japan Society for the Promotion of Science (JSPS). H.E.K. was
funded by KAKENHI 19H03163 from JSPS, The Naito Foundation, The Kurata Grants from The
Hitachi Global Foundation, and Grant-in-Aid from the Tokyo Biochemical Research Foundation.Author contributions W.H. initiated the project and performed GPCR screening to identify
strong arrestin couplers with H.E.K. W.H. expressed and purified NTSR1, screened NTSR1
constructs and performed initial NTSR1–βarr1 complexing. M.M. established arrestin
expression and purification, performed fluorescence polarization measurements, engineered
pre-activated arrestin constructs and developed fluorescence and electron paramagnetic
resonance reporters to guide the complex formation strategy. Q.Q. screened samples by
negative-stain electron microscopy and cryo-EM, prepared grids, collected and processed
cryo-EM data and generated the cryo-EM maps. J.J. expressed and purified GRK5, screened
and optimized NTSR1 phosphorylation conditions, phosphorylated NTSR1 for cryo-EM studies,
performed mass spectrometry experiments and analysed all mass spectrometry data. M.M.
and J.J. established GRK5 expression and purification conditions and screened and optimized
NTSR1–βarr1(ΔCT) complexing conditions. W.H. and J.J. chose crosslinkers to screen. W.H.,
M.M. and J.J. screened crosslinkers and optimized sample preparation for cryo-EM. W.H. and
Q.Q. build the NTSR1–βarr1(ΔCT) model, with contributions from J.J. and M.J.R. K.C.N
performed lipidomics measurements, which were overseen by J.J. and J.S.G. A.I. performed
NanoBiT experiments. M.M., J.J., G.S. and B.K.K. wrote the paper with input from W.H. and Q.Q.
B.K.K. and G.S. supervised the project.Competing interests B.K.K. is a co-founder of and consultant for ConfometRx.Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
1953-1.
Correspondence and requests for materials should be addressed to G.S. or B.K.K.
Peer review information Nature thanks Oliver Clarke, Martin Lohse and John Tesmer for their
contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/reprints.