Comparison of the ligand binding and signaling properties of human dopamine D(2) and D(3) receptors in Chinese hamster ovary cells.
Human dopamine D(2) (hD(2)) and D(3) (hD(3)) receptors were expressed at similar, high expression levels in Chinese hamster ovary (CHO) cells, and their coupling to G proteins and further signal transduction pathways were compared. In competition radioligand-binding experiments, guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) treatment of hD(2S)- or hD(3)-CHO cell membranes induced a rightward shift and steeping of the dopamine inhibition curve. This effect was pronounced for hD(2) receptors and small for hD(3) receptors. Activation of G proteins was investigated in [(35)S]GTPgammaS-binding assays. Dopamine stimulated [(35)S]GTPgammaS binding 330 and 70% over basal levels on hD(2)-CHO and hD(3)-CHO cell membranes, respectively. (+)-7-(Dipropylamino)-5, 6,7,8-tetrahydro-2-naphthalenol and PD128907 were partial agonists for both receptors. Haloperidol, risperidone, raclopride, and nemonapride inhibited dopamine-stimulated [(35)S]GTPgammaS binding with potencies comparable to their binding affinities for hD(2) and hD(3) receptors in CHO cell membranes; inverse agonism could not be detected with this assay. Receptor stimulation by dopamine inhibited forskolin-induced cyclic AMP formation in hD(2)-CHO and hD(3)-CHO cells by 70%. Furthermore, the extracellular acidification rate increased when hD(2)-CHO and hD(3)-CHO cells were stimulated by dopamine; this effect was abolished by pertussis toxin pretreatment. In this study, we could demonstrate clear functional effects at different levels of the signaling cascade of hD(2) and hD(3) receptors in CHO cells when expressed at high levels. High-affinity agonist binding to hD(2) and hD(3) receptors was still present, but effects of receptor-G protein uncoupling at hD(3) receptors were small, indicating that hD(3) receptors maintain relatively high-affinity agonist binding in the absence of G proteins.
Vanhauwe
J F
JF
Department of Biochemical Pharmacology, Janssen Research Foundation, Beerse, Belgium.
Fraeyman
N
N
Francken
B J
BJ
Luyten
W H
WH
Leysen
J E
JE
D000818:Animals; D001578:Benzopyrans / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003470:Culture Media; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism / Q000494:pharmacology; D018491:Dopamine Agonists / Q000378:metabolism; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D010078:Oxazines / Q000378:metabolism; D011869:Radioligand Assay; D017448:Receptors, Dopamine D2 / Q000096:biosynthesis / Q000187:drug effects* / Q000378:metabolism; D050637:Receptors, Dopamine D3; D015398:Signal Transduction / Q000187:drug effects*; D013764:Tetrahydronaphthalenes / Q000378:metabolism
D001578:Benzopyrans; D003470:Culture Media; C493593:DRD3 protein, human; D018491:Dopamine Agonists; D008024:Ligands; D010078:Oxazines; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; D013764:Tetrahydronaphthalenes; C062458:3,4,4a,10b-tetrahydro-4-propyl-2H,5H-(1)benzopyrano(4,3-b)-1,4-oxazin-9-ol; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D019204:GTP-Binding Proteins; C031349:7-hydroxy-2-N,N-dipropylaminotetralin; D004298:Dopamine