1

Supporting information for:

The luminescent HiBiT peptide enables selective quantitation of GPCR ligand

engagement and internalization in living cells

Michelle E. Boursier1#, Sergiy Levin2, Kris Zimmerman1, Thomas Machleidt1, Robin Hurst1, Braeden L.

Butler1, Christopher T. Eggers1, Thomas A. Kirkland2, Keith V. Wood1†, and Rachel Friedman Ohana1

From the 1Promega Corporation, 2800 Woods Hollow, Fitchburg, Wisconsin 53711, USA, 2Promega

Biosciences LLC, 277 Granada Drive, San Luis Obispo, California 93401, USA

#Present address: Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320 †Present address: Light Bio, Inc, Madison WI 53711

2

Contents

• Supporting Methods:

o Sanger sequence analysis of endogenously-tagged PC3 clones

o Droplet digital PCR (ddPCR) for detection of HiBiT incorporation

o CRE reporter gene assay

o Assessing the relative fraction of total NanoLuc donor signal that is intracellular

o Biochemical analysis of pH influence on NanoLuc and HiBiT/LgBiT luminescence signal

o Propranolol-NB590 synthesis and characterization

• Supporting Figures and Tables

o Figure S1. Characterizing relationships between DNA titration used for transfection, resulting

donor signal, and BRET saturation ligand binding assay.

o Figure S2. Influence of transient expression level on saturation binding of propranolol-NB590 to

β2-AR.

o Figure S3. Inhibition of CRE signaling in PC3 cells by propranolol derivatives.

o Table S1. Literature pKi values of unmodified ligands for β2-AR.

o Figure S4. Filtered luminescence for HEK293 cells transiently transfected with varying

concentrations of NanoLuc and HiBiT DNA constructs.

o Figure S5. Correlation between transient donor signals and corresponding equilibrium

dissociation constants (KD) of propranolol-NB590 for NanoLuc and HiBiT β-AR

fusions.

o Figure S6. Correlation between transient donor signals for NanoLuc-β-ARs and the fraction of

those signals that is originating from intracellular compartments.

o Figure S7. Competitive displacement of propranolol-NB590 by unmodified ligands for β1-AR

and β3-AR.

o Table S2. Unmodified ligand affinities (pKi) for β1-AR and β3-AR.

o Figure S8. Binding kinetics of unmodified ligands to β1-AR.

o Figure S9. Binding kinetics of unmodified ligands to β2-AR.

o Figure S10. Binding kinetics of unmodified ligands to β3-AR.

o Table S3. Kinetic derived binding characteristics of unmodified ligands for all β-ARs.

o Figure S11. Correlation of kinetic derived pKD with equilibrium derived pKi, log(kon) and koff for

all β-ARs.

o Figure S12. Influence of allosteric modulation on the competitive binding of agonists to β2-AR.

o Figure S13. Influence of allosteric modulation on the competitive binding of partial agonists and

antagonist to β2-AR.

o Figure S14. Influence of allosteric modulation on the competitive binding of agonists to β1-AR.

o Table S4. Concentrations of 50% internalization (Int50) in an endpoint assay at tested setups.

o Figure S15. Influence of antagonists and partial agonists on constitutive internalization of β2-AR.

o Figure S16. Examples of processing and normalizing raw internalization kinetic reads.

o Table S5. Agonist-driven internalization half-lives for various compound concentrations in

different setups.

o Figure S17. Representative inhibition of agonist-induced internalization kinetics for β2-AR.

o Figure S18. pH influence on NanoLuc and HiBiT/LgBiT luminescence signal.

• References

3

SUPPORTING METHODS

Sanger sequence analysis of endogenously-tagged PC3 clones

To assess the quality of a representative sample of clonal populations generated by CRISPR/Cas9 genome

editing, PCR amplifications were performed across the N-terminus of the target gene using 50 ng of PC3

genomic DNA and 10 µM primers. Primers hybridized outside the repair template sequence and contained a

homologous sequence to pF5 CMV-neo Flexi Vector (Promega). Amplified DNAs were purified and

subcloned into pF5A by Gibson Assembly, using HiFi 1-step Gibson Kit (SGI-DNA). The resulting DNA

from each clonal population was transformed into E. coli and plated for growth. A minimum of 24 resulting

colonies representing individual plasmids were selected. Their plasmid DNAs were purified using PureYield

Plasmid Miniprep System (Promega) and subjected to Sanger sequencing analysis. Resulting reads were

compared to unedited PC3 sequences to determine the presence of intact HiBiT sequence, and any potential

changes to the genomic integrity of the clones.

Droplet digital PCR (ddPCR) for detection of HiBiT incorporation

Genomic DNA from PC3 cells was purified using Maxwell RSC Cultured Cells DNA Kit (Promega).

Amplification primers and detection probes were designed using PrimerQuest online tool (IDT). Primer sets

were designed to amplify across repair template sequence, resulting in amplificons ranging from ~150–400

bp. Two probes were designed for each target, one specific to HiBiT sequence, and the other specific to the

gene of interest (GOI), containing either FAM or HEX fluorophores. Probes were multiplexed, linking the

total amplified targets with those containing HiBiT. 30ng of total genomic DNA was pre-digested with XbaI

restriction endonuclease (Promega), then added to ddPCR Supermix (no dUTP; BioRad) containing 250 nM

final concentration of primers and probes. 20 µL reaction mix were added to the cartridge along with 70 µL

of Droplet Counting Oil (BioRad) and emulsified using QX200 Droplet Generator (BioRad). Resulting

emulsified PCR reactions were transferred to PCR plates and sealed using PX1 Plate Sealer (BioRad).

Emulsifications were deemed successful when over 10,000 droplets were reported. Templates were amplified

(95 °C 1 min, [94 °C 30 sec, 60 °C 1 min, 40x], 98 °C 10 min) and resulting droplets were counted using

Absolute Quantitation (ABS) with FAM and HEX channels on QX200 Droplet Reader (BioRad). Ratios of

GOI probe to HiBiT probe reported by Quantasoft v1.7 software (BioRad) were used to determine percentage

of total HiBiT sequence in a clonal population.

CRE reporter gene assay

PC3 cells and edited PC3 cells expressing β2-AR that is endogenously tagged with VS-HiBiT or IL6-VS-

HiBiT were transiently transfected with a plasmid encoding the reporter gene CRE-Luc2P. Briefly,

transfected cells were used to monitor the capacity of propranolol and propranolol-NB590 to inhibit

isoproterenol stimulated CRE induced transcription. Cells were transfected using ViaFect (Promega) at a 1:4

ratio of DNA to transfection reagent, seeded in white plates at a density of 2 x 104 cell/well and grown

overnight at 37 °C/5% CO2. Following 24 hours post transfection, cells were stimulated for 6 hours with 400

nM isoproterenol in the presence of increasing concentrations of propranolol or propranolol-NB590.

Expression of the CRE-luc2P reporter gene was measured using ONE-Glo luciferase reagent (Promega)

according to manufacturer recommendations.

Assessing the relative fraction of total NanoLuc donor signal that is intracellular

To restrict luminescence measurements to NanoLuc-tagged GPCRs accumulated in intracellular

compartments, cells were treated with Extracellular NanoLuc Inhibitor (Promega), an impermeable inhibitor

that suppresses extracellular NanoLuc signal and ensures measured luminescence originated from inside

intact cells(1). Total and intracellular filtered luminescence were measured in the absence or presence of the

Extracellular NanoLuc Inhibitor, respectively. The inhibitor was included at a 1:150 dilution in a 10x

detection solution comprising 1:10 dilution of furimazine Live Cell Substrate (Promega) in Opti-MEM. Plates

were mixed, incubated and read as previously described for standard BRET donor luminescence

4

measurements. The fraction (%) of the total signal originating from intracellular compartments was calculated

by dividing the intracellular filtered luminescence by the total filtered luminescence x 100.

Biochemical analysis of pH influence on NanoLuc and HiBiT/LgBiT luminescence signal

Influence of pH on the activity of purified NanoLuc and HiBiT/LgBiT was measured in a universal buffer

comprising 25mM sodium citrate, 25 mM MES, 25 mM PIPES, 25 mM HEPES and 25 mM TAPS that was

supplemented with Tergitol at a final concentration of 0.5% (v/v) and adjusted with either 5M NaOH or 1M

HCl to a wide range of pH’s (4.5-8.5). Briefly, purified NanoLuc (Promega) and LgBiT (Promega) were

diluted to a final concentration of 0.2 nM in TBS supplemented with 0.01% BSA and 0.1% Tergitol.

Following addition of synthetic HiBiT peptide to a LgBiT solution at a final concentration of either 400 nM

or 4 nM, the HiBiT/LgBiT solutions were incubated at room temperature for 20 minutes to allow for

complementation. Equal volumes of enzyme solutions (NanoLuc or HiBiT/LgBiT) and universal buffer

solutions (pH 4.5-8.5) supplemented with 1:100 dilution of furimazine substrate (Promega) were mixed and

incubated for 12 minutes prior to luminescence measurements on a plate luminometer.

Propranolol-NB590 synthesis and characterization

To a solution of (S)-2-((naphthalen-1-yloxy)methyl)oxirane(2) (330 mg, 1.7 mmol) in 9:1 DMF-H2O

(10 mL) was added tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (1.0 g, 4.1 mmol). The resulting

solution was heated at 85 ºC for 4 hours, at which point HPLC analysis indicated complete consumption of

the starting material. Solvents were removed under reduced pressure and the crude residue was purified by

silica gel chromatography (0→30% MeOH/DCM) to provide 535 mg (72% yield) of alcohol S1 as a yellow

oil. 1H NMR (400 MHz, DMSO-d6) δ 8.23 (dd, J = 7.7, 1.9 Hz, 1H), 8.04 – 7.75 (m, 1H), 7.64 – 7.48 (m,

2H), 7.46 (d, J = 8.3 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 6.95 (dd, J = 7.6, 1.1 Hz, 1H), 6.76 (s, 1H), 5.13 (d, J

= 4.5 Hz, 1H), 4.24 – 3.89 (m, 3H), 3.55 – 3.42 (m, 6H), 3.36 (t, J = 6.1 Hz, 2H), 3.05 (q, J = 6.0 Hz, 2H),

2.88 – 2.67 (m, 4H), 1.36 (s, 9H); MS (SI) Calc’d for C24H37N2O6 [M+H]+ 449.3, found 449.6.

To a solution of S1 (313 mg, 698 μmol) in DCM (16 mL) was added TiPS (0.2 mL) and TFA (4 mL).

The resulting solution was stirred at 22 ºC for 90 minutes, at which point LCMS analysis indicated complete

consumption of the starting material. Solvent was removed under reduced pressure and the crude residue was

dissolved in MeOH (10 mL). Solvent was removed under reduced pressure and the crude residue S2 was used

in the next step without further purification.

To a solution of S2 (4.1 mg, 7.0 μmol) in DMF (8 mL) was added DIPEA (9 μL, 50 μmol) followed

by NanoBRET 590 SE (3.0 mg, 7.0 μmol, Promega). The resulting solution was allowed to react at 22 ºC for

2 hours at which point HPLC analysis indicated full consumption of the starting material. Solvent was

5

removed under vacuum and the crude residue was purified by preparative RP HPLC (5→95% MeCN/H2O

buffered with 0.5% TFA) to provide 4 mg (74% yield) of propranolol-NB590 as a purple film. HPLC: 98%

purity at 254 nm; 1H NMR (400 MHz, Methanol-d4) δ 8.25 (dd, J = 8.2, 1.5 Hz, 1H), 7.81 (dd, J = 7.8, 1.5

Hz, 1H), 7.58 – 7.41 (m, 3H), 7.37 (t, J = 7.9 Hz, 1H), 7.24 – 7.16 (m, 3H), 7.14 (d, J = 4.5 Hz, 1H), 6.98 (d,

J = 4.6 Hz, 1H), 6.88 (dd, J = 5.7, 4.2 Hz, 2H), 6.41 – 6.21 (m, 2H), 4.40 (dtd, J = 10.1, 5.2, 3.1 Hz, 1H), 4.17

(dd, J = 9.9, 4.9 Hz, 1H), 4.11 (dd, J = 9.9, 5.4 Hz, 1H), 3.75 (t, J = 5.0 Hz, 2H), 3.69 – 3.57 (m, 4H), 3.53 (t,

J = 5.5 Hz, 2H), 3.44 – 3.34 (m, 3H), 3.29 – 3.18 (m, 4H), 2.64 (t, J = 7.7 Hz, 2H); HRMS (SI) Calc’d for

C35H41BF2N5O5 [M+H]+ 660.3163, found 660.3158.

6

SUPPORTING FIGURES AND TABLES

Figure S1. Characterizing relationships between DNA titration used for transfection, resulting donor signal,

and BRET saturation ligand binding assay. (A) Filtered donor luminescence for endogenously-tagged PC3

clones expressing HiBiT-tagged β2-AR at native levels and for HEK293 and PC3 cells transiently transfected

with varying concentrations of plasmid DNA encoding HiBiT-β2-AR. (B) Correlation between filtered donor

luminescence from transiently transfected cells and corresponding equilibrium dissociation constants (KD)

calculated from BRET-based saturation binding assays (Fig S2). Box indicates transfection conditions used

for further binding studies. Error bars indicate SEM of three independent experiments.

7

Figure S2. Influence of transient expression level on saturation binding of propranolol-NB590 to β2-AR.

Analysis performed in HEK293 and PC3 cells transiently transfected with varying concentrations of plasmid

DNA. Error bars represent SEM of three independent experiments. Calculated equilibrium dissociation

constants (KD) were correlated with filtered donor luminescence from corresponding transfection conditions

(Fig S1).

8

Figure S3. Inhibition of CRE signaling in PC3 cells by propranolol derivatives. Error bars show SD in an

experimental quadruplicate. IC50 error is shown as a 95% confidence interval.

9

Table S1 Literature pKi values of unmodified ligands for β2-AR.

Literature pKi Reference

timolol 9.7 (3)

salmeterol 8.8 (3)

pindolol 9.4 (4)

propranolol 9.1 (3)

alprenolol 9.0 (3)

carvedilol 9.4 (3)

formoterol 8.6 (5)

isoproterenol 6.4 (6)

xamoterol 6.1 (3)

salbutamol 6.1 (3)

10

Figure S4. Filtered luminescence for HEK293 cells transiently transfected with varying concentrations of

NanoLuc and HiBiT constructs. Error bars indicate SEM of three independent experiments.

11

105.5 106 106.5 107 107.5 108

0.001

0.01

0.1

RLUs

KD

(

M) NanoLuc-1-AR

HiBiT-1-AR

1-AR

105 106 107 108

0.0001

0.001

0.01

RLUs

KD

(

M) NanoLuc-2-AR

HiBiT-2-AR

2-AR

105.5 106 106.5 107

0.01

0.1

1

RLUs

KD

(

M) NanoLuc-3-AR

HiBiT-3-AR

3-AR

Figure S5. Correlation between transient donor signals and corresponding equilibrium dissociation constants

(KD) of propranolol-NB590 for NanoLuc and HiBiT-β-AR fusions. KD values were calculated from BRET-

based saturation binding assays. Transfection conditions and corresponding donor signals are shown in Fig

S4. Error bars indicate SEM of three independent experiments.

12

Figure S6. Correlation between transient donor signals for NanoLuc-β-ARs and the fraction of those signals

that is originating from intracellular compartments. The fractions of total signals originating from intracellular

compartments were determined as described in the Supporting Methods. Briefly, total and intracellular signals

were measured in the absence or presence of an impermeable NanoLuc inhibitor that suppresses extracellular

NanoLuc signal and ensures measured luminescence originated from inside intact cells. The fraction (%) of

the total signal originating from intracellular compartments was calculated by dividing the intracellular

filtered luminescence by the total filtered luminescence x 100. Error bars indicate SEM of three independent

experiments.

13

Figure S7. Competitive displacement of propranolol-NB590 by increasing concentrations of unmodified

ligands for β1-AR and β3-AR. Error bars indicate SEM of ≥3 independent experiments.

Table S2. Unmodified ligand affinities (pKi) for β1-AR and β3-AR. Data represents the mean ± SEM of N

independent experiments.

β1-AR β3-AR

pKi N Literature

pKi Ref. pKi N

Literature

pKi Ref.

formoterol 6.2 ± 0.2 5 6.5 (7) – 4 5.1 (8)

isoproterenol 6.5 ± 0.2 5 6.6 (6) – 5 5.2 (6)

propranolol 8.8 ± 0.2 5 8.2 (3) 7.4 ± 0.2 5 6.9 (3)

salbutamol 5.7 ± 0.7 5 4.7 (3) – 4 4.3 (3)

salmeterol 6.0 ± 0.1 5 5.4 (3) – 4 5.7 (3)

timolol 9.2 ± 0.2 5 8.3 (3) 7.1 ± 0.1 5 6.8 (3)

alprenolol 8.40 ± 0.09 5 7.8 (3) 7.2 ± 0.2 5 6.9 (3)

carvedilol 8.8 ± 0.2 5 9.5 (9) 8.3 ± 0.2 5 8.3 (3)

pindolol 9.0 ± 0.1 5 9.3 (4) 7.3 ± 0.1 5 7.4 (8)

xamoterol 8.2 ± 0.1 5 7.0 (10) – 3 4.5 (3)

14

0 5 10 15 200

1

2

3

4timolol

Time (min)

BR

ET

(m

BU

)

15

7.5

3.75

0nM

0 5 10 15 200

1

2

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4

5alprenolol

Time (min)

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ET

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)15

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5pindolol

Time (min)

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ET

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1.875

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0 5 10 15 200

1

2

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Time (min)

BR

ET

(m

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)

3000

1500

750

0nM

0 5 10 15 200.0

0.5

1.0

1.5carvedilol

Time (min)

BR

ET

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7.5

3.75

0nM

0 5 10 15 200

1

2

3fomoterol

Time (min)

BR

ET

(m

BU

)

1000

500

250

0nM

0 5 10 15 200

2

4

6isoproterenol

Time (min)

BR

ET

(m

BU

)

1000

500

250

0nM

0 5 10 15 200

1

2

3

4xamoterol

Time (min)

BR

ET

(m

BU

)

25

12.5

6.125

0nM

0 5 10 15 200

1

2

3

4salbutamol

Time (min)

BR

ET

(m

BU

)

4000

2000

1000

0nM

Figure S8. Binding kinetics of unmodified ligands to β1-AR. Kinetic analyses competing varying

concentrations of unmodified ligands with 25 nM propranolol-NB590. Data presented as specific BRET ratios.

Error bars indicate SD in an experimental triplicate.

15

0 10 20 300

2

4

6timolol

Time (min)

BR

ET

(m

BU

)

2.5

1.25

0.625

0nM

0 10 20 300

2

4

6

8alprenolol

Time (min)

BR

ET

(m

BU

)

2.5

1.25

0.625

0nM

0 10 20 300

2

4

6pindolol

Time (min)

BR

ET

(m

BU

)

2.5

1.25

0.625

0nM

0 10 20 300.0

0.5

1.0

1.5

2.0

2.5salmeterol

Time (min)

BR

ET

(m

BU

)

2.5

1.25

0.625

0nM

0 5 10 15 200

2

4

6

8carvedilol

Time (min)

BR

ET

(m

BU

)

5

2.5

1.25

0nM

0 10 20 300

2

4

6

8

10formoterol

Time (min)

BR

ET

(m

BU

)12.5

6.125

3.06

0nM

0 10 20 300

2

4

6isoproterenol

Time (min)

BR

ET

(m

BU

)

750

375

187.5

0nM

0 10 20 300

2

4

6

8xamoterol

Time (min)

BR

ET

(m

BU

)

500

250

125

0nM

0 10 20 300

2

4

6

8

10

12salbutamol

Time (min)

BR

ET

(m

BU

)

2000

1000

500

0nM

Figure S9. Binding kinetics of unmodified ligands to β2-AR. Kinetic analyses competing varying

concentrations of unmodified ligands with 2 nM propranolol-NB590. Data presented as specific BRET ratios.

Error bars indicate SD in an experimental triplicate.

16

0 5 10 150.0

0.5

1.0

1.5

2.0

2.5timolol

Time (min)

BR

ET

(m

BU

)

600

300

150

0nM

0 5 10 150.0

0.5

1.0

1.5

2.0

2.5alprenolol

Time (min)

BR

ET

(m

BU

)

600

300

150

0nM

0 5 10 150.0

0.5

1.0

1.5

2.0pindolol

Time (min)

BR

ET

(m

BU

)

600

300

150

0nM

0 5 10 150.00

0.25

0.50

0.75

1.00

1.25carvedilol

Time (min)

BR

ET

(m

BU

)

200

100

50

0nM

Figure S10. Binding kinetics of unmodified ligands to β3-AR. Kinetic analyses competing varying

concentrations of unmodified ligands with 80 nM propranolol-NB590. Data presented as specific BRET ratios.

Error bars indicate SD in an experimental triplicate.

17

Table S3. Kinetic derived binding characteristics of unmodified ligands for all β-ARs. Data represents the

mean ± SD in an experimental triplicate.

β1-AR KD (nM) kon (M-1 min-1) koff (min-1) τ (min)

timolol 4.2 ± 0.4 1.10 ± 0.07 x108 0.46 ± 0.03 2.2 ± 0.1

alprenolol 5.3 ± 0.6 1.5 ± 0.1 x108 0.80 ± 0.07 1.3 ± 0.1

pindolol 1.9 ± 0.2 4.5 ± 0.4 x108 0.85 ± 0.07 1.2 ± 0.1

salmeterol 1700 ± 400 6.6 ± 0.9 x105 1.1 ± 0.2 0.9 ± 0.1

carvedilol 4.6 ± 0.4 4.6 ± 0.3 x107 0.21 ± 0.01 4.7 ± 0.3

formoterol 600 ± 200 3.4 ± 0.9 x106 1.9 ± 0.5 0.5 ± 0.1

isoproterenol 220 ± 20 3.3 ± 0.2 x106 0.71 ± 0.05 1.4 ± 0.1

xamoterol 15 ± 8 3 ± 1 x108 4 ± 1 0.3 ± 0.1

salbutamol 2000 ± 300 1.9 ± 0.2 x105 0.42 ± 0.04 2.4 ± 0.2

β2-AR KD (nM) kon (M-1 min-1) koff (min-1) τ (min)

timolol 0.56 ± 0.04 2.5 ± 0.1 x108 0.139 ± 0.008 7.2 ± 0.4

alprenolol 0.6 ± 0.2 2.7 ± 0.7 x109 1.7 ± 0.4 0.6 ± 0.2

pindolol 0.9 ± 0.4 1.9 ± 0.6 x109 1.8 ± 0.5 0.6 ± 0.2

salmeterol 1.0 ± 0.1 2.6 ± 0.2 x108 0.26 ± 0.02 3.8 ± 0.3

carvedilol 4.4 ± 0.8 9 ± 1 x107 0.39 ± 0.05 2.6 ± 0.3

formoterol 5.4 ± 0.8 1.0 ± 0.1 x108 0.54 ± 0.06 1.9 ± 0.2

isoproterenol 170 ± 30 7.0 ± 0.8 x106 1.2 ± 0.1 0.8 ± 0.1

xamoterol 400 ± 100 6 ± 1 x106 2.0 ± 0.5 0.5 ± 0.1

salbutamol 400 ± 300 8 ± 3 x106 3 ± 1 0.3 ± 0.1

β3-AR KD (nM) kon (M-1 min-1) koff (min-1) τ (min)

timolol 300 ± 50 5.0 ± 0.6 x106 1.5 ± 0.2 0.66 ± 0.08

alprenolol 270 ± 40 4.2 ± 0.4 x106 1.2 ± 0.1 0.87 ± 0.09

pindolol 140 ± 40 1.9 ± 0.4 x107 2.7 ± 0.6 0.37 ± 0.08

salmeterol – – – –

carvedilol 37 ± 7 5.2 ± 0.7 x107 1.9 ± 0.3 0.52 ± 0.07

formoterol – – – –

isoproterenol – – – –

xamoterol – – – –

salbutamol – – – –

18

Figure S11. Correlation of kinetic derived pKD with (A) equilibrium derived pKi, (B) log(kon) and (C) koff for

all β-ARs.

19

Figure S12. Influence of allosteric modulation on the competitive binding of agonists to β2-AR. Competitive

displacements of a fixed concentration of propranolol-NB590 by increasing concentrations of agonists in the

presence of increasing concentration of a PAM modulator. Distinct 95% confidence intervals (CIs) without

overlap at 0 µM and 30 µM PAM indicate significant shifts (p ≤ 0.05) in IC50 values. Error bars indicate the

SD of an experimental quadruplicate.

20

Figure S13. Influence of allosteric modulation on the competitive binding of partial agonists and antagonist

to β2-AR. Competitive displacements of a fixed concentration of propranolol-NB590 by increasing

concentrations of partial agonists or antagonist in the presence of increasing concentration of a PAM modulator.

Error bars indicate the SD of the mean of an experimental quadruplicate.

21

Figure S14. Influence of allosteric modulation on the competitive binding of agonists to β1-AR. Competitive

displacements of a fixed concentration of propranolol-NB590 by increasing concentrations of agonists in the

presence of increasing concentration of a PAM modulator. Distinct 95% confidence intervals (CIs) without

overlap at 0 µM and 30 µM PAM indicate significant shifts (p ≤ 0.05) in IC50 values. Error bars indicate the

SD of the mean of an experimental quadruplicate.

22

Table S4. Concentrations of 50% maximal internalization (Int50) in an endpoint assay at tested setups as shown

in Figure 8.

Int50 (nM)

HEK293

(transient)

PC3

VS-HiBiT-β2-AR

formoterol 0.23 0.82

isoproterenol 70 190

salbutamol 170 120

salmeterol 0.31 0.12

23

Figure S15. Influence of antagonists and partial agonists on constitutive internalization of β2-AR. Endpoint

internalization analyses for β2-AR transiently expressed in HEK293 cell following treatment with increasing

concentrations of antagonists and partial agonists. Increased signal likely signifies inhibition of basal,

constitutive β2-AR internalization. Error bars indicate SD of four replicates from two independent experiments.

24

-20 0 20 40 60 80 1000

50000

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Time (min)

RL

Us

0 nM

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Us

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100 nM

1000 nM

-20 0 20 40 60 80 10050

60

70

80

90

100

110

Time (min)

No

rma

lize

d R

es

po

nse 0 nM

10 nM

100 nM

1000 nM

10000 nM

Figure S16. Examples of processing and normalizing raw internalization kinetic reads. Kinetic

internalization of endogenously expressed HiBiT-β2-AR that is induced by various concentrations of the

agonists (A) formoterol and (B) isoproterenol. Raw luminescence reads (A.1 and B.1) were normalized to

the initial luminescence reads of each well (A.2 and B.2), then baseline corrected to the no compound control

reads at each time point (A.3 and B.3).

A.1

A.3

A.2

B.1

B.3

B.2

25

Table S5. Agonist-driven internalization half-lives for various compound concentrations in different setups as

shown in Figure 9. Half-lives are recorded in minutes.

formoterol isoproterenol

[compound]

nM

HEK293

(transient)

PC3

VS-HiBiT-β2-AR

HEK293

(transient)

PC3

VS-HiBiT-β2-AR

1 22 33 – –

10 11 9.4 – >1000

100 10 7.2 15 12

1000 9.2 6.6 9.1 5.3

10000 – – 9.6 4.5

26

Figure S17. Representative inhibition of agonist-induced internalization kinetics for β2-AR. Inhibition of

formoterol-induced internalization of HiBiT-β2-AR in (A) transiently transfected HEK293 and (B) endogenously-

tagged PC3 clone by either propranolol (antagonist) or two dynamin inhibitors, OcTMAB and MiTMAB. Cells

were pretreated for one hour with LgBiT and extended release Nano-Glo Vivazine Live Cell substrate to allow

substrate deprotection by cellular and serum esterases. Following an additional one-hour treatment with either

Opti-MEM, 10 µM propranolol, 20 µM OcTMAB or 20 µM MiTMAB, cells were treated with 100 nM formoterol

and luminescence was measured over time. (C) Following 80 minutes kinetics reads, formoterol-induced

internalizations were blocked by subsequent treatment with 10 µM propranolol. The increased signal likely

signifies rapid recycling to the cell-surface. Raw luminescence measurements were normalized as described in

the Experimental Procedures and in Figure S16. Error bars indicate SD of an experimental triplicate.

27

Figure S18. pH influence on NanoLuc and HiBiT/LgBiT luminescence signal. Influence of pH (4.5–8.5) on the

activity of purified NanoLuc and HiBiT/LgBiT was measured as described in the Supporting Methods. Briefly,

equal volumes of enzyme solutions (NanoLuc or HiBiT/LgBiT) and buffers (pH 4.5–8.5) supplemented with

1:100 dilution of furimazine substrate were mixed and incubated for 12 minutes prior to luminescence

measurements. Error bars indicate SEM of three independent experiments.

28

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