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

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

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 β-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: Apr 30, 2014
• 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