Despite extensive study, the G protein coupling of dopamine D3 receptors is poorly understood.In this study, the authors used guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]-GTPγS) binding to investigate the activation of G proteins coupled to human (h) D3 receptors stably expressed in Chinese hamster ovary (CHO) cells.Although the receptor expression level was high (15 pmol/mg), dopamine only stimulated G protein activation by 1.6-fold.This was despite the presence of marked receptor reserve for dopamine, as revealed by Furchgott anal. after irreversible hD3 receptor inactivation with the alkylating agent, EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline).Thus, half-maximal stimulation of [35S]-GTPγS binding required only 11.8% receptor occupation of hD3 sites.In contrast, although the hD2(short) receptor expression level in another CHO cell line was 11-fold lower, stimulation by dopamine was higher (2.5-fold).G protein activation was increased at hD3 and, less potently, at hD2 receptors by the preferential D3 agonists, PD 128,907 [(+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H- [1]benzopyrano[4,3-b]-1,4-oxazin-9-ol] and (+)-7-OH-DPAT (7-hydroxy-2-(di-n-propylamino)tetralin).Furthermore, the selective D3 antagonists, S 14297 ((+)-[7-(N, N-dipropylamino)-5,6,7,8- tetrahydro-naphtho(2,3b)dihydro-2,3-furane]) and GR 218,231 (2(R,S)-(dipropylamino)-6-(4-methoxyphenylsulfonylmethyl)-1,2,3,4- tetrahydronaphthalene), blocked dopamine-stimulated [35S]GTPγS binding more potently at hD3 than at hD2 sites.Antibodies against Gαi/αo reduced dopamine-induced G protein activation at both CHO-hD3 and -hD2 membranes, whereas GαS antibodies had no effect at either site.In contrast, incubation with anti-Gαq/α11 antibodies, which did not affect dopamine-induced G protein activation at hD2 receptors, attenuated hD3-induced G protein activation.These data suggest that hD3 receptors may couple to Gαq/α11 and would be consistent with the observation that pertussis toxin pretreatment, which inactivates only Gi/o proteins, only submaximally (80%) blocked dopamine-stimulated [35S]GTPγS binding in CHO-hD3 cells.Taken together, the present data indicate that (1) hD3 receptors functionally couple to G protein activation in CHO cells, (2) hD3 receptors activate G proteins less effectively than hD2 receptors, and (3) hD3 receptors may couple to different G protein subtypes than hD2 receptors, including non-pertussis sensitive Gq/11 proteins.
