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
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 challe...
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| Format: | Article |
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Elsevier
2012
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| Online Access: | https://eprints.nottingham.ac.uk/3067/ |
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| author | Kilpatrick, Laura E. Briddon, Stephen J. Holliday, Nicholas D. |
| author_facet | Kilpatrick, Laura E. Briddon, Stephen J. Holliday, Nicholas D. |
| author_sort | Kilpatrick, Laura E. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T18:20:40Z |
| format | Article |
| id | nottingham-3067 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T18:20:40Z |
| publishDate | 2012 |
| publisher | Elsevier |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-30672020-05-04T20:21:37Z https://eprints.nottingham.ac.uk/3067/ 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. 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. Elsevier 2012-06 Article PeerReviewed Kilpatrick, Laura E., Briddon, Stephen J. and Holliday, Nicholas 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. BBA Molecular Cell Research, 1823 (6). pp. 1068-1081. ISSN 0167-4889 G protein coupled receptor; Neuropeptide Y; Arrestin; Fluorescence correlation spectroscopy; Bimolecular fluorescence complementation; Endocytosis; 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 |
| spellingShingle | G protein coupled receptor; Neuropeptide Y; Arrestin; Fluorescence correlation spectroscopy; Bimolecular fluorescence complementation; Endocytosis; Kilpatrick, Laura E. Briddon, Stephen J. Holliday, Nicholas D. 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 |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | G protein coupled receptor; Neuropeptide Y; Arrestin; Fluorescence correlation spectroscopy; Bimolecular fluorescence complementation; Endocytosis; |
| url | https://eprints.nottingham.ac.uk/3067/ https://eprints.nottingham.ac.uk/3067/ |