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.
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)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 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)cm(2)s(-1)) of Y1 receptor-β-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-β-arrestin signalling complex.
authors.affiliation: Cell Signaling Research Group, School of Biomedical Sciences, University of Nottingham, the Medical School, Queen's Medical Centre, Nottingham, UK.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Kilpatrick
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Briddon
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Holliday
mesh_terms: D019390:Arrestins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004058:Diffusion; D004705:Endocytosis; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D046912:Multiprotein Complexes / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D017785:Photons; D021381:Protein Transport; D017476:Receptors, Neuropeptide Y / Q000819:agonists / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D046912:Multiprotein Complexes; D050505:Mutant Proteins; D017476:Receptors, Neuropeptide Y; D000071557:beta-Arrestins; C097212:neuropeptide Y-Y1 receptor; C095889:neuropeptide Y2 receptor; D049452:Green Fluorescent Proteins