Abstract

Despite extensive study, the G protein coupling of dopamine D₃ receptors is poorly understood. In this study, we 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 analysis 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 hD_2(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- tetrahydronaphtalene), blocked dopamine-stimulated [³⁵S]GTPγS binding more potently at hD₃than at hD₂ sites. Antibodies against Gα_i/α_o reduced dopamine-induced G protein activation at both CHO-hD₃ and -hD₂ membranes, whereas Gα_S antibodies had no effect at either site. In contrast, incubation with anti-Gα_q/α₁₁antibodies, 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 Gα_q/α₁₁ and would be consistent with the observation that pertussis toxin pretreatment, which inactivates only G_i/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 nonpertussis sensitive G_q/11 proteins.