RT Journal Article
SR Electronic
T1 G Protein Activation by Human Dopamine D<sub>3</sub> Receptors in High-Expressing Chinese Hamster Ovary Cells: A  Guanosine-5′-<em>O</em>-(3-[<sup>35</sup>S]thio)-Triphosphate Binding and Antibody Study
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 564
OP 574
VO 55
IS 3
A1 Adrian Newman-Tancredi
A1 Didier Cussac
A1 Valérie Audinot
A1 Valérie Pasteau
A1 Samantha Gavaudan
A1 Mark J. Millan
YR 1999
UL http://molpharm.aspetjournals.org/content/55/3/564.abstract
AB 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 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- tetrahydronaphtalene), blocked dopamine-stimulated [35S]GTPγS binding more potently at hD3than 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/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 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 nonpertussis sensitive Gq/11 proteins. The American Society for Pharmacology and Experimental Therapeutics