Functional selectivity of dopamine D1 receptor agonists in regulating the fate of internalized receptors
Introduction
The dopamine receptors are a superfamily of heptahelical G protein-coupled receptors (GPCRs) that have historically been partitioned into “D1-like” and “D2-like” subfamilies (Kebabian and Calne, 1979, Garau et al., 1978). The dopamine D1 receptor is a member of the “D1-like” subfamily, and couples to adenylate cyclase through stimulatory G proteins Gs and Golf (Herve et al., 1993). The early steps in the regulation of the D1 receptor following the binding of dopamine have been addressed in model cell lines. After the binding of dopamine to the D1 receptor, receptor phosphorylation is complete within minutes (Gardner et al., 2001). This can be mediated by GRKs (Gardner et al., 2001, Tiberi et al., 1996) and/or protein kinase A (PKA) (Mason et al., 2002). Both types of kinases may facilitate D1 desensitization, and the contribution of each to the overall extent of receptor phosphorylation and desensitization is probably highly dependent on the cell line being studied. Receptor phosphorylation allows arrestin to bind to the third intracellular loop of the receptor (Kim et al., 2004) leading to D1 receptor internalization. Arrestin is not trafficked into the cell with the receptor, thus the D1 receptor is considered a “Class A” GPCR (Oakley et al., 2000). Following dopamine-induced internalization, the D1 receptor is rapidly recycled back to the cell surface (Vickery and von Zastrow, 1999, Vargas and von Zastrow, 2004). Recent studies indicate that a signal sequence within the proximal C-terminal region of the receptor mediates this process (Vargas and von Zastrow, 2004).
The effects of D1 agonists other than dopamine itself on regulatory events downstream of receptor activation are not well characterized. Besides heuristic interest in these questions, several of the D1 agonists that have been tested as antiparkinson agents in human and non-human primates caused a very rapid tolerance evidenced as an almost complete loss of response within a day or so (Asin and Wirtshafter, 1993, Kebabian et al., 1992, Lin et al., 1996, DeNinno et al., 1991a, Johnson et al., 1992). Thus, such molecular events may be important in understanding the cellular mechanisms that contribute to the development of this therapeutic tolerance. Previously, we have observed that desensitization of adenylate cyclase responsivity and receptor down-regulation are highly dependent upon the agonist used, but largely independent of adenylate cyclase activity and agonist affinity in a stably transfected C6 glioma cell line (Lewis et al., 1998). Recently, we explored the relationship between agonist structure, receptor affinity, and efficacy of receptor internalization and adenylate cyclase activation in greater depth by constructing an HEK cell line stably transfected with a hemaglutinin-tagged human D1 receptor and comparing these endpoints in 13 agonists from three different structural families. We found that D1 agonists exhibit functional selectivity at these early endpoints following receptor activation that are apparently independent of agonist structure or binding affinity (Ryman-Rasmussen et al., 2005).
These results suggested the major hypothesis tested herein, that D1 agonists are functionally selective in regulating receptor function at the endpoint of intracellular trafficking of the D1 receptor, an endpoint that temporally lies downstream of adenylate cyclase activation and internalization, events more immediate of receptor activation. We selected two agonists of therapeutic interest, A-77636 and dinapsoline (DNS), for comparison with dopamine at this endpoint. Both of these synthetic ligands have efficacies of internalization and adenylate cyclase activation comparable to that of dopamine in the HA-hD1 HEK cell line (Ryman-Rasmussen et al., 2005). The isochroman A-77636 elicits profound and rapid in vivo tolerance occurring within approximately 24 h, preventing its use in Parkinson's disease therapy (Lin et al., 1996). Conversely, DNS does not cause such tolerance in a rat model of Parkinson's disease (Gulwadi et al., 2001). The mechanisms of tolerance are unknown, but presumably result from cellular adaptations that lie temporally downstream of receptor internalization and adenylate cyclase activation. The current data demonstrate that although these agonists cause functional changes identical to dopamine immediately following receptor binding, with time they modify D1 receptor trafficking, and thus show a novel pattern of functional selectivity.
Section snippets
Materials
Dopamine and A-77636 [(−)-(1R,3S)-3-adamantyl-1-(aminomethyl)-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyran hydrochloride)] were purchased from RBI/Sigma-Aldrich (St. Louis, MO). Dinapsoline (8,9-dihydroxy-2,3,7, 11b-tetrahydro-1H-naph[1,2,3-de]isoquinoline) and [3H]SCH23390 were synthesized according to published procedures (Ghosh et al., 1996, Wyrick et al., 1986). All other reagents and materials were from Sigma Chemical Company (St. Louis, MO), unless otherwise stated.
HA-hD1 HEK model cell line
The HA-hD1 HEK cell line
Radioreceptor assays
Saturation binding with the D1-selective antagonist, [3H]SCH23390, in membrane homogenates indicated that the assay expression level of HA-hD1 in this cell line is approximately 4 ± 1 pmol/mg membrane protein with a KD of 2.4 ± 0.8 nM (Fig. 1, panel A and Table 1). Competition assays of dopamine, A-77636, and DNS versus [3H]SCH23390 were performed to determine the affinities of these compounds for the HA-hD1 receptor (Fig. 1, panel B and Table 1). Dopamine and DNS best fit a two-site binding model,
Discussion
Utilizing an HA-hD1 HEK cell line (Ryman-Rasmussen et al., 2005), we studied the intracellular trafficking of the D1 receptor following binding of dopamine and two structurally dissimilar agonists, A-77636 and DNS. These agonists were similarly efficacious to dopamine in activating adenylate cyclase, and caused a similar time course of D1 receptor internalization. Conversely, the synthetic agonists had quite different effects on events that were not temporally proximal to initial receptor
Acknowledgments
The confocal data were obtained at the Michael Hooker Microscopy Facility at UNC-Chapel Hill. This work was supported by NIH research grants NS039036 (RM), MH040537 (RM), and MH073910 (WCG,RM), and training grants ES007126 and NS007431.
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Richard B. Mailman and the University of North Carolina at Chapel Hill have a financial interest in Biovalve Technologies, Inc. that holds license rights to dinapsoline. All opinions are those of the authors, and do not represent those of that company, the University of North Carolina at Chapel Hill, or the California Institute of Technology.