| Summary: | 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.
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