FUNCTIONAL COUPLING OF THE HUMAN DOPAMINE-D-3 RECEPTOR IN A TRANSFECTED NG-108-15 NEUROBLASTOMA-GLIOMA HYBRID CELL-LINE

Transfection of a human dopamine D-3 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 [I-125]iodosulpiride binding sites. Dopamine and several agonists distinguished two receptor-affinity states in membranes. In the case of dopamine, the high-affinity state (K-i = 0.9 nM, 30% of total binding) was completely converted into a low-affinity state (K-i = 57 nM) in the presence of 10 mu M 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 D-3 receptor mediated two responses: c-fos activation, as measured by the appearance of Fos-like immunoreactivity, and increased mitogenesis, as measured by incorporation of [H-3]thymidine. The Fos-like immunoreactivity appeared within 30 min, lasted 2 h and was blocked by the partially selective dopamine D-3 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 D-2 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 (K-i = 9 nM). Talipexole (B-HT 920, 5-allyl-2-amino5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin) was identified as a partial agonist at the dopamine D-3 receptor. Dopamine D-3 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 D-3 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.