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 bindi ng sites. Dopamine and several agonists distinguished two receptor-aff inity states in membranes. In the case of dopamine, the high-affinity state (K-i = 0.9 nM, 30% of total binding) was completely converted in to a low-affinity state (K-i = 57 nM) in the presence of 10 mu M guano sine-5'-O-(3-thiotriphosphate). In addition to these two sites, a site with a very low affinity for dopamine was evidenced in whole cells. T he 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 block ed by the partially selective dopamine D-3 receptor compound(+)-UH 232 -5-methoxy-1-methyl-2-(di-n-propylamino)tetralin). The mitogenic effe ct, which occurred after a lag time (over 2 h stimulation), was produc ed with subnanomolar potency and full intrinsic activity by several co mpounds 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-propyl-di-beta(3-hydroxyphenyl)-ethylamine),and was re versibly blocked by (+)-UH 232 (K-i = 9 nM). Talipexole (B-HT 920, -am ino5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin) was identified as a pa rtial agonist at the dopamine D-3 receptor. Dopamine D-3 receptor-medi ated mitogenesis was potentiated by a phorbol ester and was abolished by pretreatment with pertussis toxin. A mitogenic effect of same ampli tude was elicited by bradykinin or carbachol, both acting through cons titutive receptors. Bradykinin markedly activated inositol phosphate t urnover, and had no effect on forskolin-stimulated cyclic AMP accumula tion. Carbachol inhibited forskolin-stimulated cyclic AMP accumulation and had no effect on inositol-phosphate turnover. Quinpirole had no e ffect on any of these second messenger pathways. Thus, in transfected NG 108-15 cells, the dopamine D-3 receptor is coupled to a pertussis t oxin-sensitive G protein and mediates two possibly unrelated biologica l effects, through initial biochemical events that remain to be identi fied.