Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 278, Issue 12, Pages 10562-10571Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M205747200
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Up to now neuropeptide Y (NPY) receptors, which belong to the large family of G-protein-coupled receptors and are involved in a broad range of physiological processes, are believed to act as monomers. Studies with the Y-1-receptor antagonist and Y-1-receptor agonist GR231118, which binds with a 250-fold higher affinity than its monomer, led to the first speculation that NPY receptors can form homodimers. In the present work we used the fluorescence resonance energy transfer (FRET) to study homodimerization of the hY(1)-, hY(2)-, and hY(5)-receptors in living cells. For this purpose, we generated fusion proteins of NPY receptors and green fluorescent protein or spectral variants of green fluorescent protein (cyan, yellow, and red fluorescent protein), which can be used as FRET pairs. Two different FRET techniques, fluorescence microscopy and fluorescence spectroscopy, were applied. Both techniques clearly showed that the hY(1)-, hY(2)-, and hY(5)-NPY receptor subtypes are able to form homodimers. By using transiently transfected cells, as well as a stable cell line expressing the hY(2)-GFP fusion protein, we could demonstrate that the Y-GFP fusion proteins are still functional and that dimerization varies from 26 to 44% dependent on the receptor. However, homodimerization is influenced neither by NPY nor by Got protein binding.
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