Elsevier

Neuropharmacology

Volume 52, Issue 2, February 2007, Pages 562-575
Neuropharmacology

Functional selectivity of dopamine D1 receptor agonists in regulating the fate of internalized receptors

https://doi.org/10.1016/j.neuropharm.2006.08.028Get rights and content

Abstract

Recently, we demonstrated that D1 agonists can cause functionally selective effects when the endpoints of receptor internalization and adenylate cyclase activation are compared. The present study was designed to probe the phenomenon of functional selectivity at the D1 receptor further by testing the hypothesis that structurally dissimilar agonists with efficacies at these endpoints that equal or exceed those of dopamine would differ in ability to influence receptor fate after internalization, a functional endpoint largely unexplored for the D1 receptor. We selected two novel agonists of therapeutic interest that meet these criteria (the isochroman A-77636, and the isoquinoline dinapsoline), and compared the fates of the D1 receptor after internalization in response to these two compounds with that of dopamine. We found that dopamine caused the receptor to be rapidly recycled to the cell surface within 1 h of removal. Conversely, A-77636 caused the receptor to be retained intracellularly up to 48 h after agonist removal. Most surprisingly, the D1 receptor recovered to the cell surface 48 h after removal of dinapsoline. Taken together, these data indicate that these agonists target the D1 receptor to different intracellular trafficking pathways, demonstrating that the phenomenon of functional selectivity at the D1 receptor is operative for cellular events that are temporally downstream of immediate receptor activation. We hypothesize that these differential effects result from interactions of the synthetic ligands with aspects of the D1 receptor that are distal from the ligand binding domain.

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.

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