Agonist-independent and -dependent oligomerization of doparnine D-2 receptors by fusion to fluorescent proteins

Oligomerization of the short (D-2S) and long (D-2L) isoforms of the dopamin e 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 (E CFP or EYFP) and the fluorescent fusion protein interaction was monitored b y, a fluorescence resonance energy transfer (FRET) assay. The pharmacologic al properties of the fluorescent fusion proteins, as measured by both displ acement 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 rece ptors. Co-expression of D2S:ECFP+D2S:EYFP in a 1:1 ratio and D2L:ECFP+D2L:E YFP in a 27:1 ratio resulted, respectively, in an increase of 26% and 16% i n the EYFP-specific fluorescent signal. These data are consistent with a cl ose proximity of both D-2S and D2L receptor pairs of fluorescent fusion pro teins in the absence of ligand. The agonist-independent D-2S receptor oligo merization could be attenuated by co-expression with either a wild-type, no n-fluorescent D2S or D2L receptor subtype, but not with a distinct beta (2) -adrenoceptor. Incubation with the agonist (-)-norpropylapomorphine dose-de pendently (EC50: 0.23 +/-0.06 nM) increased the FRET signal for the co-expr ession of D2S:ECFP and D2S:EYFP, in support of agonist-dependent D-2S recep tor oligomerization. In conclusion, our data strongly suggest the occurrenc e of dopamine D2 receptor oligomers in intact Cos-7 cells. (C) 2001 Publish ed by Elsevier Science B.V. on behalf of the Federation of European Biochem ical Societies.