Functional coupling of the human dopamine D3 receptor in a transfected NG 108-15 neuroblastoma-glioma hybrid cell line.
Transfection of a human dopamine D3 receptor cDNA in a neuroblastoma-glioma hybrid cell line (NG 108-15) provided clonal cell lines stably expressing up to 600 fmol per mg protein of [125I]iodosulpiride binding sites. Dopamine and several agonists distinguished two receptor-affinity states in membranes. In the case of dopamine, the high-affinity state (Ki = 0.9 nM, 30% of total binding) was completely converted into a low-affinity state (Ki = 57 nM) in the presence of 10 microM guanosine-5'-O-(3-thiotriphosphate). In addition to these two sites, a site with a very low affinity for dopamine was evidenced in whole cells. The dopamine D3 receptor mediated two responses: c-fos activation, as measured by the appearance of Fos-like immunoreactivity, and increased mitogenesis, as measured by incorporation of [3H]thymidine. The Fos-like immunoreactivity appeared within 30 min, lasted 2 h and was blocked by the partially selective dopamine D3 receptor compound (+)-UH 232 (cis-(+)-5-methoxy-1-methyl-2-(di-n-propylamino)tetralin). The mitogenic effect, which occurred after a lag time (over 2 h stimulation), was produced with subnanomolar potency and full intrinsic activity by several compounds previously identified as dopamine D2 receptor agonists, e.g. quinpirole, (+)-7-OH-DPAT ((+)-7-hydroxy-2-(di-n-propylamino)tetralin) and RU 24926 (N-n-propyl-di-beta(3-hydroxyphenyl)-ethylamine), and was reversibly blocked by (+)-UH 232 (Ki = 9 nM). Talipexole (B-HT 920, 5-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin) was identified as a partial agonist at the dopamine D3 receptor. Dopamine D3 receptor-mediated mitogenesis was potentiated by a phorbol ester and was abolished by pretreatment with pertussis toxin. A mitogenic effect of same amplitude was elicited by bradykinin or carbachol, both acting through constitutive receptors. Bradykinin markedly activated inositol phosphate turnover, and had no effect on forskolin-stimulated cyclic AMP accumulation. Carbachol inhibited forskolin-stimulated cyclic AMP accumulation and had no effect on inositol-phosphate turnover. Quinpirole had no effect on any of these second messenger pathways. Thus, in transfected NG 108-15 cells, the dopamine D3 receptor is coupled to a pertussis toxin-sensitive G protein and mediates two possibly unrelated biological effects, through initial biochemical events that remain to be identified.
Pilon
C
C
Unité de Neurobiologie et Pharmacologie (U. 109), INSERM, Centre Paul Broca, Paris, France.
Lévesque
D
D
Dimitriadou
V
V
Griffon
N
N
Martres
M P
MP
Schwartz
J C
JC
Sokoloff
P
P
D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D004873:Ergolines / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D016762:Genes, fos; D005910:Glioma / Q000473:pathology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D006822:Hybrid Cells; D051379:Mice; D009447:Neuroblastoma / Q000473:pathology; D019257:Quinpirole; D051381:Rats; D011954:Receptors, Dopamine / Q000502:physiology*; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; D013469:Sulpiride / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
C493593:DRD3 protein, human; C493594:Drd3 protein, mouse; C493595:Drd3 protein, rat; D004873:Ergolines; D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; D019257:Quinpirole; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013469:Sulpiride; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D013936:Thymidine