Fluorescence correlation spectroscopy, combined with bimolecular fluorescence complementation, reveals the effects of ß-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors.
Biochim Biophys Acta
; 1823(6): 1068-81, 2012 Jun.
Article
em En
| MEDLINE
| ID: mdl-22487268
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 ß-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-ß-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) respectively. 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 ß-arrestin recruitment and internalisation; conversely they became apparent in a Y2 receptor mutant with increased ß-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 ß-arrestin recruitment for these effects was illustrated by reduced lateral mobility (D 1.20-1.33 × 10(-9)cm(2)s(-1)) of Y1 receptor-ß-arrestin BiFC complexes. Thus NPY-induced changes in Y receptor motility 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-ß-arrestin signalling complex.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Fluorescência
/
Membrana Celular
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Receptores de Neuropeptídeo Y
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Arrestinas
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Complexos Multiproteicos
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Endocitose
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Biochim Biophys Acta
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Reino Unido