Biosensor Technical Data Sheet 3

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BETA-ARRESTIN MEMBRANE RECRUITMENT BIOSENSORS

BETA-ARRESTIN MEMBRANE RECRUITMENT BIOSENSORS

Summary: β-arrestins are multifunctional adaptor proteins classically associated with the “arrest” of G protein-

coupled receptor (GPCR)-mediated signaling by promoting receptor desensitization and internalization. However,

it is now evident that β-arrestins (mainly β-arrestin 1 and β-arrestin 2) can also scaffold signaling complexes

proximal to a given GPCR and modulate the activity of various signaling networks (including ERK1/2, NF-κB, and

PI3-K). Importantly, β-arrestin-mediated signaling may be spatially and temporally distinct, and result in different

biological outcomes, compared to G protein-mediated signal transduction. Consequently, GPCR-downstream

signaling can occur via G protein-dependent and/or β-arrestin-dependent (G protein-independent) mechanisms

(1-2). β-arrestins are recruited to activated GPCRs following receptor phosphorylation by G protein-coupled

receptor kinases (GRKs) and/or other protein kinases (e.g., PKA, PKC). GPCRs are broadly categorized into two

classes based on the stability of their interaction with β-arrestin proteins. Class A GPCRs (e.g., mu opioid receptor,

dopamine D1A receptor) generally form transient complexes with β-arrestins at the plasma membrane,

internalize into endosomes without β-arrestins and are rapidly recycled to the plasma membrane. Conversely,

Class B receptors (e.g., angiotensin II type 1A receptor, vasopressin V2 receptor) form longer-lived complexes with

β-arrestins, stably internalize into endosomes with their associated β-arrestins and display slower recycling

kinetics (3).

The bioSensAllTM β-arrestin membrane recruitment sensors allow for real-time spatio-temporal monitoring of β-

arrestins following GPCR activation. The multimolecular β-arrestin biosensors consists of a β-arrestin 1 or 2 fused

at their C-terminus to Renilla luciferase (RLuc; R in figure below) that translocates to the plasma membrane

anchored green fluorescent protein (GFP; G in following figure) or early endosome anchored GFP. Specifically,

these sensors were designed to detect the recruitment of β-arrestin proteins to either the plasma membrane or

early endosomes, with localization to each compartment resulting in an increased BRET signal (4).

Biosensor Technical Data Sheet 3

Page 2 of 3 TD SHEET 03

BETA-ARRESTIN MEMBRANE RECRUITMENT BIOSENSORS

-arrestin plasma membrane recruitment biosensors

-arrestin early endosome recruitment biosensors

βarr

R

G G

βarr

R

Activation

Inactivation

Biosensor Technical Data Sheet 3

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BETA-ARRESTIN MEMBRANE RECRUITMENT BIOSENSORS

Results

-1 5 -1 4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

b A r r1 -P M

P T H R 1

[P T H (1 -3 4 )] , lo g (M )

uB

RE

T

E C 5 0 = 4 .3 5 n M

-1 5 -1 4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

b A r r2 -P M

P T H R 1

[P T H (1 -3 4 )] , lo g (M )

uB

RE

T

E C 5 0 = 3 .9 0 n M

-1 5 -1 4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

b A rr2 -P M + G R K 2 W T

M O R

[D A M G O ], lo g (M )

uB

RE

T

E C 5 0 = 2 0 4 n M

-1 5 -1 4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4

0

5 0 0

1 0 0 0

1 5 0 0

b A r r1 -e n d o E

P T H R 1

[P T H (1 -3 4 )] , lo g (M )

uB

RE

T

E C 5 0 = 0 .8 2 n M

-1 5 -1 4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

b A r r2 -e n d o E

P T H R 1

[P T H (1 -3 4 )] , lo g (M )

uB

RE

T

E C 5 0 = 0 .6 3 n M

HEK293 cells were transfected with a receptor coding plasmid (human parathyroid hormone type 1 receptor (PTHR1) or the human mu

opioid receptor (MOR)) as well as plasmids coding for the β-arrestin membrane recruitment biosensor or early endosome recruitment

biosensor. The MOR-mediated plasma membrane recruitment of β-arrestin 2 was assessed in the presence of overexpressed WT GRK2.

On the day of BRET, cells were rinsed with assay buffer, incubated with coelenterazine and increasing amounts of PTH(1-34) or DAMGO

for 10 minutes (β-arrestin-PM sensors) or 30 minutes (β-arrestin-endoE sensors) and BRET subsequently measured.

References

1- Shukla AK, Xiao K, Lefkowitz RJ. Emerging paradigms of β-arrestin-dependent seven transmembrane

receptor signaling. 2011. Trends Biochem Sci. 36(9):457-69. PMID: 21764321.

2- Smith JS, Rajagopal S. The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors. 2016. J

Biol Chem. 291(17):8969-77. PMID: 26984408.

3- Oakley RH, Laporte SA, Holt JA, Caron MG, Barak LS. Differential affinities of visual arrestin, beta arrestin1,

and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. 2000. J Biol

Chem. 275(22):17201-10. PMID: 10748214.

4- Namkung Y, Le Gouill C, Lukashova V, Kobayashi H, Hogue M, Khoury E, Song M, Bouvier M, Laporte SA.

Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander

BRET. 2016. Nat Commun. 7:12178. PMID: 27397672.