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ANALYSIS OF NPY RECEPTOR DIMERIZATION IN LIVING CELLS BY
FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)
Karin Mörl, Ilka Böhme, Michaela Dinger, Robert Rennert and Annette G. Beck-Sickinger
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of
Leipzig, 04103 Leipzig, Germany
Recent growing evidence suggests the existence of G-protein coupled receptors as dimers or
even higher order oligomers. In contrast to indirect biochemical and physiological
experiments, fluorescence resonance energy transfer (FRET) allows the direct and
noninvasive visualization of receptor multimers in living cells. FRET is a nonradiative form
of energy transfer between two fluorescent molecules, with an appropriate spectral overlap of
the donor emission spectrum and the acceptor absorption spectrum, provided that they are
between 10–100 Å apart. Its exquisite distance dependency allows to measure distances, and
thus intermolecular interactions, much smaller than the resolution limit of a light microscope
in living cells.
NPY receptor chimeras with spectral variants of GFP, namely CFP as donor molecule and
YFP as acceptor, were cloned. After transfection of BHK (baby hamster kidney) and CHO
(chinese hamster ovary) cells, the expression of the receptor fusion proteins was analyzed by
microscopy, including confocal laser scanning microscopy, to prove receptor distribution on
the membranes. Functionality of the receptor-fusion protein was assured by competition
binding assays and by inhibition of forskolin stimulated cAMP production.
Homodimerization of of hY1, hY2, hY4 and hY5 receptor subtypes could be shown by two
different FRET techniques, fluorescence microscopy and fluorescence spectroscopy. The
dimerization is independent of the receptor expression level and neither influenced by agonist
stimulation nor GTPγS incubation. [1]
The functional repertoire of proteins might be controlled and enhanced by protein-protein
interactions, which could also contribute to pharmacological diversity. With respect to the
existence of multiple receptor subtypes and the broad range of physiological actions mediated
by NPY, the existence of NPY receptor subtype heterodimers is of great interest and was
analyzed in further experiments. For this purpose hY1-CFP and hY5-YFP (as well as hY5CFP and hY1-YFP) fusion proteins were coexpressed in BHK cells. Interestingly, it turned out
that the expression of both proteins is influenced on the level of translational regulation.
Especially the integrity of the Kozak-Sequence present in the two constructs modulates the
ratio of hY1-CFP / hY5-YFP (or hY5-CFP / hY1-YFP) protein levels. First FRET experiments
show heterodimerization of hY1 and hY5 receptors.
[1] Dinger, M.C.; Bader, J.E.; Kóbor, A.D.; Kretzschmar, A.K. and Beck-Sickinger, A.G.;
J.Biol.Chem. 278, 10562-10571 (2003)