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) 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 Spodoptera 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 approximately 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 be detected by different approaches, and D2 oligomerization in these cells is not regulated by ligands.
D000818:Animals; D001058:Apomorphine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D018491:Dopamine Agonists / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000494:pharmacology; D000939:Epitopes / Q000235:genetics; D005063:Europium / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics / Q000276:immunology; D008024:Ligands; D008969:Molecular Sequence Data; D011233:Precipitin Tests / Q000379:methods*; D020891:Raclopride / Q000494:pharmacology; D017448:Receptors, Dopamine D2 / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics; D013997:Time Factors
D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D000939:Epitopes; D007136:Immunoglobulins; D008024:Ligands; D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins; C113776:dopamine D2L receptor; D020891:Raclopride; D005063:Europium; C009074:N-n-propylnorapomorphine; D001058:Apomorphine