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Fluorescence correlation spectroscopy, combined with bimolecular fluorescence complementation, reveals the effects of ?-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors

Kilpatrick, Laura E.; Briddon, Stephen J.; Holliday, Nicholas D.

Authors

Stephen J. Briddon

Nicholas D. Holliday



Abstract

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 investigated 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 fluorescence 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 cm2 s−1 respectively. At a concentrationwhich promoted only Y1 receptor endocytosis, NPY treatment reduced Y1-GFPmotility
(D 1.48×10−9 cm2 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 cm2 s−1) of Y1 receptor-β-arrestin BiFC complexes.
Thus NPY-induced changes in Y receptormotility and brightness reflect early events surrounding arrestin dependent endocytosis at the plasma membrane, results supported by a novel combined BiFC/FCS approach to detect
the underlying receptor-β-arrestin signalling complex.

Citation

Kilpatrick, L. E., Briddon, S. J., & Holliday, N. D. (2012). Fluorescence correlation spectroscopy, combined with bimolecular fluorescence complementation, reveals the effects of ?-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1823(6),

Journal Article Type Article
Publication Date Jun 1, 2012
Deposit Date Apr 30, 2014
Publicly Available Date Mar 28, 2024
Journal BBA Molecular Cell Research
Electronic ISSN 0167-4889
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 1823
Issue 6
Keywords G protein coupled receptor; Neuropeptide Y; Arrestin; Fluorescence correlation spectroscopy; Bimolecular fluorescence complementation; Endocytosis;
Public URL https://nottingham-repository.worktribe.com/output/1007370
Publisher URL http://www.sciencedirect.com/science?_ob=ArticleListURL&_method=list&_ArticleListID=-568470760&_sort=r&_st=13&view=c&_acct=C000009959&_version=1&_urlVersion=0&_userid=5939061&md5=79c1a1c97d9265365983216cf7c2eadf&searchtype=a

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