Agonist-independent and -dependent oligomerization of doparnine D2 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 \ H-3 \ nemonapride binding and agonist-mediated Stimulation Of \ S-35 \ GTP gammaS binding upon co-expression with a G(alpha0)Cys(351) Ile protein, were not different from the respective wild-type D2S and D2L 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 D2L 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 D2S or D2L receptor subtype, but not with a distinct beta (2)-adrenoceptor. Incubation with the agonist (-)-norpropylapomorphine dose-dependently (EC50: 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 D2 receptor oligomers in intact Cos-7 cells. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.