jirnal smeinar

1. Pedersen, et. al. J. Biol. Chem. 2021
Departments of Psychiatry & Molecular Pharmacology and Therapeutics,
Colombia University
22/04/19 SB8
1
A novel luminescence-based β-arrestin
recruitment assay for unmodified receptors

2. Introduction
• GPCR: G protein-coupled receptors (GPCRs) and interaction with β-arrestin-
mediated signaling have involved signaling in more targeted therapeutics and
biological phenomena, e. g: heart failure (β-adrenergic receptor),
neuropathology (μ-opioid receptor), and behavior (dopamine D2 receptor).
2
heart failure (β-adrenergic receptor)
neuropathology
(μ-opioid
receptor),

3. Introduction
3

4. The most well-established techniques to
investigate β-arrestin recruitment
4
PathHunter
The Tango-GPCR assay

5. Introduction
• GPCR: G protein-coupled receptors (GPCRs) and interaction with β-arrestin-
mediated signaling have involved signaling in more targeted therapeutics and
biological phenomena, e. g: heart failure (β-adrenergic receptor),
neuropathology (μ-opioid receptor), and behavior (dopamine D2 receptor).
5
Why the assay need to bind to membrane not in GPCR receptor? Notably,
all of these β-arrestin assays require that fusion tags be directly attached to the
C terminus of receptors of interest, which could alter GRK, arrestin, or other
protein interactions with the receptors and therefore impact signaling
Purpose: developed the assay which avoids potential artifacts related to C-
terminal receptor modification for measuring β-arrestin recruitment to diverse
GPCR types in heterologous or native cells.

6. Flow this research
• Models: HEK293 cells (ATTC CRL-1573) and CHO-K1 cell lines
• Plasmid construction: using standard techniques in molecular biology,
including overlapping-PCR and Gibson Assembly
• β-Arrestin complementation assay:
• For initial testing: transfected cells were measured in 96-well black-white
using PHERAstar FS (BMG labtech) plate reader
• For stable cell line testing: in 384 well plate and luminescence was read on a
Flexstation III.
6

7. Validation of a unique NanoLuc split for
complementation assays (1)
Namkung, et. al. Nat. Comm. 2016 7
Preliminary data, They used NanoBiT to
detect membrane recruitment of β-arrestin.
No luminescence detected upon agonist
stimulation

8. Validation of a unique NanoLuc split for
complementation assays (2)
8
They selected a site in a loop region that
divides NanoLuc into two almost equally
sized fragments (N: 1–102; C: 103–172),
without disrupting any secondary structural
element
Cotransfection of both fragments increased
~30-fold luminescence signal by the
addition of rapamycin

9. 9
Validation of a unique NanoLuc split for
complementation assays (3)
the N-terminal Nanoluc fragment was
attached to the C-terminal tail of D2R
and the C-terminal fragment was fused
to the N terminus of β- arrestin2
In this configuration, the endogenous
agonist dopamine produced a time- and
dose-dependent increase in
luminescence

10. Detection of β-arrestin plasma membrane
recruitment by class A receptors (1)
10
The N-terminal fragment of NanoLuc is
anchored to the plasma membrane
through a double palmitoylated domain
of GAP43 (MeN)
the C-terminal fragment is fused to the N
terminus of β-arrestin1 or β-arrestin2
(ArC)
Increase in luminescence upon agonist
treatment (QP/quinpirole) and decay in
luminescence after antagonist stimulation
(SP/sulpiride)

11. Detection of β-arrestin plasma membrane
recruitment by class A receptors (2)
11
To assess the MeNArC assay’s dynamic
range, different level of efficacy agonist
were tested.
MeNArC assay is also capable of
measuring different degrees of partial
agonism of unmodified receptors
Quinpirole (High-efficacy)
Bromocriptine and Terguride (Moderate)
PPP (Low)

12. Detection of β-arrestin plasma membrane and early
endosomal recruitment by a class B receptor (1)
12
Unlike class A receptors, AT1R and
other class B receptors bind β-arrestin
with higher affinity and form longer-lived
complexes that persist after endocytosis
The ArC component described is
coexpressed with the N-terminal
NanoLuc fragment attached to the
FYVE domain of endofin (EeN)
(EeNArC), which was shown previously
to selectively bind endosomes and used
in a direct receptor recruitment assay

13. Detection of β-arrestin plasma membrane and early
endosomal recruitment by a class B receptor (2)
13
As expected, the agonist effect was
dramatically blunted by sucrose for the
EeNArC assay but not for the MeNArC
assay.

14. Development of a polycistronic MeNArC expression
in stably transfected CHO-K1 cells
14
Schematic of the polycistronic MeNArC
Dose–response curves of the MeNArC assay for the
MOR and KOR cells showing increase in luminescence
(au) with increasing concentration of agonist
β-arrestin2 recruitment tested with the MOR
partial agonists morphine and buprenorphine
normalized to the full agonist DAMGO

15. In Conclusion
• MeNArC and EeNArC is β-arrestin membrane and endosomal recruitment
assays using complementation of novel NanoLuc fragments with the potential of
serving as a generic and readily adapted screening method for detecting β-
arrestin recruitment to unmodified receptors.
15
• Note for reader:
• This assays cannot rule out action of a tested agonist on endogenous
receptors or possible off-target receptors when tested in native tissue unless
knockout models are available.
• Need to test the utility of MeNArC and EeNArC in primary cell cultures for
measuring β-arrestin recruitment to endogenous receptors.
• This assay reports on β-arrestin recruitment, like all β-arrestin recruitment
assays, it does not measure β-arrestin “activity” and therefore does not
directly report on downstream signaling, which can involve several
different pathways such as ERK and Src

16. Note
• Imaging data
• Part fragment not lebih berat dari b arrestin
• Check endosomal activity
16