tailieunhanh - Báo cáo khoa học: Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9 ) and in HEK293 cells Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer

Human D2Long(D2L)andD2Short(D2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed inSpodop-tera frugiperda 9(Sf9)cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). | Eur. J. Biochem. 270 3928-3938 2003 FEBS 2003 doi Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 Sf9 and in HEK293 cells Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer Lucien Gazi1 t Juan F. López-Giménez1 t Martin P. Rudiger2 and Philip G. Strange1 1 School of Animal and Microbial Sciences University of Reading Reading UK 2GlaxoSmithKline New Frontiers Science Park Harlow UK Human D2Long D2L and D2Short D2S dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus HIV or a FLAG epitope tag. The receptors were then expressed in Spodop-tera frugiperda 9 Sf9 cells using the baculovirus system and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer FRET . 3H Spiperone labelled D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors with a pKd value of w 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D2L and D2S receptors in Sf9 cells. When the FLAG-tagged D2S and HIV-tagged D2L receptors were co-expressed co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D2 receptors. In both cases constitutive homo-oligomers were revealed for D2L and D2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D2S receptors. The D2 receptor ligands dopamine R- - propylnorapomorphine and raclopride did not affect oligomerization of D2L and D2S in Sf9 and HEK293 cells. Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can