G protein activation by human dopamine D3 receptors in high-expressing Chinese hamster ovary cells: A guanosine-5'-O-(3-[35S]thio)- triphosphate binding and antibody study.
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]-GTPgammaS) 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]-GTPgammaS 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]GTPgammaS binding more potently at hD3 than at hD2 sites. Antibodies against Galphai/alphao reduced dopamine-induced G protein activation at both CHO-hD3 and -hD2 membranes, whereas GalphaS antibodies had no effect at either site. In contrast, incubation with anti-Galphaq/alpha11 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 Galphaq/alpha11 and would be consistent with the observation that pertussis toxin pretreatment, which inactivates only Gi/o proteins, only submaximally (80%) blocked dopamine-stimulated [35S]GTPgammaS 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.
Newman-Tancredi
A
A
Department of Psychopharmacology, Institut de Recherches Servier, Croissy-sur-Seine (Paris), France. newman_tancredi@hotmail.com
Cussac
D
D
Audinot
V
V
Pasteau
V
V
Gavaudan
S
S
Millan
M J
MJ
D000478:Alkylation; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D037342:Pertussis Toxin; D011804:Quinolines / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000187:drug effects / Q000378:metabolism*; D050637:Receptors, Dopamine D3; D013462:Sulfur Radioisotopes / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000906:Antibodies; C493593:DRD3 protein, human; D011804:Quinolines; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; D013462:Sulfur Radioisotopes; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C000470:EEDQ; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins