Agonist-independent and -dependent oligomerization of dopamine D(2) receptors by fusion to fluorescent proteins.
Oligomerization of the short (D(2S)) and long (D(2L)) isoforms of the dopamine D(2) receptor was explored in transfected Cos-7 cells by their C-terminal fusion to either an enhanced cyan or enhanced yellow fluorescent protein (ECFP or EYFP) and the fluorescent fusion protein interaction was monitored by a fluorescence resonance energy transfer (FRET) assay. The pharmacological properties of the fluorescent fusion proteins, as measured by both displacement of [(3)H]nemonapride binding and agonist-mediated stimulation of [(35)S]GTPgammaS binding upon co-expression with a G(alphao)Cys(351)Ile protein, were not different from the respective wild-type D(2S) and D(2L) receptors. Co-expression of D2S:ECFP+D2S:EYFP in a 1:1 ratio and D2L:ECFP+D2L:EYFP in a 27:1 ratio resulted, respectively, in an increase of 26% and 16% in the EYFP-specific fluorescent signal. These data are consistent with a close proximity of both D(2S) and D(2L) receptor pairs of fluorescent fusion proteins in the absence of ligand. The agonist-independent D(2S) receptor oligomerization could be attenuated by co-expression with either a wild-type, non-fluorescent D(2S) or D(2L) receptor subtype, but not with a distinct beta(2)-adrenoceptor. Incubation with the agonist (-)-norpropylapomorphine dose-dependently (EC(50): 0.23+/-0.06 nM) increased the FRET signal for the co-expression of D2S:ECFP and D2S:EYFP, in support of agonist-dependent D(2S) receptor oligomerization. In conclusion, our data strongly suggest the occurrence of dopamine D(2) receptor oligomers in intact Cos-7 cells.
Wurch
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Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 17 avenue Jean Moulin, 81106 Cedex, Castres, France. thierry.wurch@pierre-fabre.com
Matsumoto
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A
Pauwels
P J
PJ
D000818:Animals; D001058:Apomorphine / Q000031:analogs & derivatives* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001549:Benzamides / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D018491:Dopamine Agonists / Q000378:metabolism; D018492:Dopamine Antagonists / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017448:Receptors, Dopamine D2 / Q000819:agonists* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D001426:Bacterial Proteins; D001549:Benzamides; D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D008164:Luminescent Proteins; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C009074:N-n-propylnorapomorphine; D001058:Apomorphine; C030265:nemonapride