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

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• 作者：Laura E. Kilpatrick ; Stephen J. Briddon ; Nicholas D. Holliday ; ^{nicholas.holliday@nottingham.ac.uk}
• 关键词：BiFC ; bimolecular fluorescence complementation ; BIBO3304 ; (R)-N2-(diphenylacetyl)-N-[(4-(aminocarbonylaminomethyl-)phenyl)methyl]-argininamide ; BSA ; bovine serum albumin ; DMEM ; Dulbecco's modified Eagle's medium ; FCS ; fluorescence correlation spectroscopy
• 刊名：Biochimica et Biophysica Acta (BBA)/Molecular Cell Research
• 出版年：2012
• 期刊代码：20_01674889
• 类别：bio
• 出版时间：June, 2012
• 卷：1823
• 期：6
• 页码：1068-1081
• 文件大小：2046 K

摘要

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 尾-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-尾-arrestin complexes. Diffusion co-efficients (D) for Y1 and Y2-GFP receptors in HEK293 cell plasma membranes were 2.22 and 2.15 脳 10^鈭?#xA0;9 cm² s^鈭?#xA0;1 respectively. At a concentration which promoted only Y1 receptor endocytosis, NPY treatment reduced Y1-GFP motility (D 1.48 脳 10^鈭?#xA0;9 cm² s^鈭?#xA0;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 尾-arrestin recruitment and internalisation; conversely they became apparent in a Y2 receptor mutant with increased 尾-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 尾-arrestin recruitment for these effects was illustrated by reduced lateral mobility (D 1.20-1.33 脳 10^鈭?#xA0;9 cm² s^鈭?#xA0;1) of Y1 receptor-尾-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-尾-arrestin signalling complex.