Differential Dependence of the D1 and D5Dopamine Receptors on the G Protein γ7 Subunit for Activation of Adenylylcyclase

Abstract

The D 1 dopamine receptor, G protein ␥ 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 . Studies suggest that imbalances between these two opposing classes lead to deficiencies in movement and cognitive performance (2, 3). In particular, alterations in the D 1 -like dopamine receptors are implicated in a variety of neurologic and psychiatric disorders, such as Parkinson's disease, Tourette's syndrome, and schizophrenia. Thus, achieving a better understanding of the D 1 -like dopamine receptors and the signaling pathways they activate may suggest more selective therapeutic targets in these diseases.The D 1 and D 5 dopamine receptors stimulate adenylylcyclase activity through their coupling to heterotrimeric G proteins (1, 4 -6). Because the function of these heterotrimeric G proteins was originally ascribed to the ␣ subunit, most research has focused on determining its identity. Of the several ␣ subunits identified to date, reconstitution studies have shown that coupling of D 1 dopamine receptors to adenylylcyclase can be mediated only by the ␣ s and ␣ olf subunits of the G s subclass (4, 5, 7-9). Although sharing 88% amino acid homology, the ␣s and ␣ olf subunits show very divergent expression patterns, ranging from the ubiquitous expression of the ␣ s subunit to the olfactory and neuron-specific expression of the ␣ olf subunit (10). Immunoprecipitation studies (11) have confirmed the interaction between the ␣ s subunit and the D 1 dopamine receptors in cells, whereas in situ hybridization (12, 13) and gene targeting (14) studies have suggested a possible interaction between the ␣ olf subunit and the D 1 dopamine receptor in striatum.By contrast, little effort has focused on determining the identity of the ␤␥ subunits of the G protein despite mounting evidence for their importance in receptor recognition (15,16). Of particular interest, reconstitution studies (17-21) and reverse genetic approaches (22,23) have shown that the nature of the ␥ subunit is an important determinant of its interaction with receptor. Consistent with such a role, 12 ␥ subunit genes have been identified that show extensive structural diversity (24). Recently, we used a ribozyme approach to begin to elucidate their functions (23,25,26). This approach identified the ␥ 7 subunit as a specific component of the G protein that couples the ␤-adrenergic receptor, but not the prostaglandin E 1 receptor, to stimulation of adenylylcyclase in HEK 293 cells (23). Although expressed in a variety of tissues and cell types (27,28), the ␥ 7 subunit expression is most abundant in medium spiny neurons in striatum (13). This pattern of expression is shared by the D 1 dopamine receptor and adenylylcyclase (4, 12, 29 -31), raising the possibility that all of these components may be involved in the same signaling pathway. In the present study, we used the ribozyme ...

Lipid Rafts Keep NADPH Oxidase in the Inactive State in Human Renal Proximal Tubule Cells

“…4 D 1 -like receptors are endogenously expressed in hRPT cells 38 but not in HEK-293 cells. 39 Similar to the studies in hRPT cells, ␤CD increased Nox activity, which was completely reversed by cholesterol repletion ( Figure 4A and Table S5). Of note was the greater increase in NADPH oxidase activity caused by ␤CD in HEK-293 cells, which do not express either the D 1 or D 5 receptor.…”

“…Of note was the greater increase in NADPH oxidase activity caused by ␤CD in HEK-293 cells, which do not express either the D 1 or D 5 receptor. 39 This could be taken to indicate that NADPH oxidase activity may be negatively regulated by D 1 -like receptors, because HEK-293 cells that do not express D 1 -like receptors have higher Nox activity than cells that express them ( Figure 4A). …”

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells

“…Fen decreased the approximately 100-120 kDa band, which presumably represents the glycosylated AT 1 R plus the EGFP tag, but not the approximately 65-85 kDa AT 1 R band, which presumably represents the nonglycosylated AT 1 R plus the EGFP tag, as early as 20 minutes after treatment (Figure 3A). Because the D 1 R is not expressed in HEK293 cells (20,21), these results indicate that the effect of Fen on AT 1 R protein expression is solely the result of D 5 R activation.…”

“…dopamine receptors (20,21). To investigate the mechanism of the regulation of AT 1 R protein expression by the D 5 R, we transfected human full-length AT 1 R tagged at the C terminus with EGFP into HEK293 cells expressing the D 5 R. Flow cytometry (Supplemental Figure 1A) and immunoblot analyses (Supplemental Figure 1B Figure 1D), indicating that the EGFP tag did not impair AT 1 R function in the transfected cells.…”

The Expanding Roles of Gβγ Subunits in G Protein–Coupled Receptor Signaling and Drug Action

“…Reduction in Ga olf and Gb 2 levels, by .85 and 30% compared with wild-type animals, respectively, was found in the striatum of Gg 7 2/2 mice (Schwindinger et al, 2003(Schwindinger et al, , 2010. A decrease in SKF-81297 (6-chloro-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol)-stimulated adenylyl cyclase activity was measured in HEK293 cells stably expressing the dopamine D 1 receptor but not in cells expressing the dopamine D 5 receptor (Wang et al, 2001). These results implicate Gg 7 in both dopamine D 1 receptor and adenosine A 2A receptor signaling, and provide evidence that all members of the same receptor family do not necessarily couple to the same Gg subunit.…”