Differential dependence of the D1 and D5 dopamine receptors on the G protein gamma 7 subunit for activation of adenylylcyclase.
The D(1) dopamine receptor, G protein gamma(7) subunit, and adenylylcyclase are selectively expressed in the striatum, suggesting their potential interaction in a common signaling pathway. To evaluate this possibility, a ribozyme strategy was used to suppress the expression of the G protein gamma(7) subunit in HEK 293 cells stably expressing the human D(1) dopamine receptor. Prior in vitro analysis revealed that the gamma(7) ribozyme possessed cleavage activity directed exclusively toward the gamma(7) RNA transcript (Wang, Q., Mullah, B., Hansen, C., Asundi, J., and Robishaw, J. D. (1997) J. Biol. Chem. 272, 26040-26048). In vivo analysis of cells transfected with the gamma(7) ribozyme showed a specific reduction in the expression of the gamma(7) protein. Coincident with the loss of the gamma(7) protein, there was a noticeable reduction in the expression of the beta(1) protein, confirming their interaction in these cells. Finally, functional analysis of ribozyme-mediated suppression of the beta(1) and gamma(7) proteins revealed a significant attenuation of SKF81297-stimulated adenylylcyclase activity in D(1) dopamine receptor-expressing cells. By contrast, ribozyme-mediated suppression of the beta(1) and gamma(7) proteins showed no reduction of SKF81297-stimulated adenylylcyclase activity in D(5) dopamine receptor-expressing cells. Taken together, these data indicate that the structurally related D(1) and D(5) dopamine receptor subtypes utilize G proteins composed of distinct betagamma subunits to stimulate adenylylcyclase in HEK 293 cells. Underscoring the physiological relevance of these findings, single cell reverse transcriptase-polymerase chain reaction analysis revealed that the D(1) dopamine receptor and the G protein gamma(7) subunit are coordinately expressed in substance P containing neurons in rat striatum, suggesting that the G protein gamma(7) subunit may be a new target for drugs to selectively alter dopaminergic signaling within the brain.
Wang
Q
Q
Henry Hood Research Program, Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822, USA.
Jolly
J P
JP
Surmeier
J D
JD
Mullah
B M
BM
Lidow
M S
MS
Bergson
C M
CM
Robishaw
J D
JD
D000262:Adenylyl Cyclases / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D016337:RNA, Catalytic / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000378:metabolism*; D050641:Receptors, Dopamine D5; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection
C493605:DRD5 protein, human; D016337:RNA, Catalytic; D017447:Receptors, Dopamine D1; D050641:Receptors, Dopamine D5; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases