Fluorescence correlation spectroscopy, combined with bimolecular fluorescence
complementation, reveals the effects of β-arrestin complexes and endocytic targeting
on the membrane mobility of neuropeptide Y receptors
Laura E. Kilpatrick, Stephen J. Briddon, Nicholas D. Holliday
⁎
Cell Signalling Research Group, School of Biomedical Sciences, University of Nottingham, The Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, UK
abstractarticle info
Article history:
Received 7 September 2011
Received in revised form 29 February 2012
Accepted 1 March 2012
Available online 8 March 2012
Keywords:
G protein coupled receptor
Neuropeptide Y
Arrestin
Fluorescence correlation spectroscopy
Bimolecular fluorescence complementation
Endocytosis
Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis are powerful
ways to study mobility and stoichiometry of G protein coupled receptor complexes, within microdomains
of single living cells. However, relating these properties to molecular mechanisms can be challenging. We in-
vestigated the influence of β-arrestin adaptors and endocytosis mechanisms on plasma membrane diffusion
and particle brightness of GFP-tagged neuropeptide Y (NPY) receptors. A novel GFP-based bimolecular fluores-
cence complementation (BiFC) system also identified Y1 receptor-β-arrestin complexes. Diffusion co-efficients
(D) for Y1 and Y2-GFP receptors in HEK293 cell plasma membranes were 2.22 and 2.15×10
−9
cm
2
s
−1
respec-
tively. At a concentration which promoted only Y1 receptor endocytosis, NPY treatment reduced Y1-GFP motility
(D 1.48×10
−9
cm
2
s
−1
), but did not alter diffusion characteristics of the Y2-GFP receptor. Agonist induced
changes in Y1 receptor motility were inhibited by mutations (6A) which prevented β-arrestin recruitment and
internalisation; conversely they became apparent in a Y2 receptor mutant with increased β-arrestin affinity.
NPY treatment also increased Y1 receptor-GFP particle brightness, changes which indicated receptor clustering,
and which were abolished by the 6A mutation. The importance of β-arrestin recruitment for these effects was
illustrated by reduced lateral mobility (D 1.20–1.33 × 10
−9
cm
2
s
−1
)ofY1receptor-β-arrestin BiFC complexes.
Thus NPY-induced changes in Y receptor motility and brightness reflect early events surrounding arrestin depen-
dent endocytosis at the plasma membrane, results supported by a novel combined BiFC/FCS approach to detect
the underlying receptor-β-arrestin signalling complex.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
G protein coupled receptors (GPCRs) constitute a large and diverse
array of cell surface receptors, which respond to signalling molecules
ranging from metal ions to large polypeptide hormones. They are
now known to activate a wide variety of signalling and regulatory
pathways, beyond those mediated by classical heterotrimeric G pro-
teins. Other effector proteins, most prominently the two non-visual
isoforms of the β-arrestin family, can interact with the agonist
bound GPCR to form alternative complexes [1,2]. β-Arrestins were
originally described as simple terminators of G protein signalling,
but their roles have rapidly expanded to those of multifunctional
adaptors. Their association with clathrin, AP-2 and phosphotidylinositides
drives internalisation of many GPCRs, and the stability of the interna-
lised GPCR-β-arrestin complexes can dictate subsequent receptor traf-
ficking to recycling or degradative pathways [2–8]. Moreover they
recruit a range of other enzymes to the GPCR which can both regulate
G protein dependent events [9,10] and initiate G protein independent
signalling, for example through scaffolding of mitogen activated protein
kinase cascades [1,2,11]. Structurally, several adaptor binding domains
overlap on the β-arrestin surface, suggesting that some β-arrestin-
based signalling complexes must form to the exclusion of others [2].Un-
derstanding the spatiotemporal formation of different GPCR-β-arrestin
scaffolds is thus an important element in defining how subsequent sig-
nalling is orchestrated. Potentially, this also influences the ability of
some GPCR ligands to direct these events in a pathway specific manner
[1]. Studying this organisation in part requires techniques to investigate
such complexes within microdomains of single cells, rather than the
overall population response.
Fluorescence correlation spectroscopy (FCS) is an imaging tech-
nique that is in theory capable of this resolution in living cells [12–14].
As fluorescent molecules pass in and out of a confocal detection
volume fixed in position, they generate time-dependent fluctuations
in intensity. Analysis of the fluctuations then provides information
Biochimica et Biophysica Acta 1823 (2012) 1068–1081
Abbreviations: BiFC, bimolecular fluorescence complementation; BIBO3304, (R)-N
2
-
(diphenylacetyl)-N-[(4-(aminocarbonylaminomethyl-)phenyl)methyl]-argininamide; BSA,
bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FCS, fluorescence cor-
relation spectroscopy; FRAP, fluorescence recovery after photobleaching; Gc, sfGFP fragment
155–238; Gn, sfGFP fragment 2–173; GPCR, G protein coupled receptor; HBSS, HEPES buff-
ered saline solution; NPY, neuropeptide Y; PCH, photon counting histogram; (sf)GFP, (super-
folder) green fluorescent protein; YFP, yellow fluorescent protein
⁎ Corresponding author. Tel.: +44 115 82 30084; fax: +44 115 82 30081.
E-mail address: nicholas.holliday@nottingham.ac.uk (N.D. Holliday).
0167-4889/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2012.03.002
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