The D2 dopamine receptor (D2R) is a clinically important G-protein coupled receptor (GPCR). It is a major target of drugs used to alleviate symptoms of Parkinson's disease and depression (Missale et al. 1998; Neve et al. 2004) and a common property of all currently available anti-psychotic drugs is that they block D2R at therapeutic concentrations (Remington 2003). There is evidence that the cellular regulation of D2R plays an important role in the physiological responses to long-term treatment with these drugs as well as in the underlying disease pathophysiology. For example, schizophrenia patients have increased levels of a ‘high affinity’ form of striatal D2R (Seeman et al. 2006), and D2R desensitization or down-regulation could explain the loss of efficacy of receptor-targeted anti-Parkinsonian after prolonged use (Rascol and Fabre 2001).
The classical model for agonist-dependent regulation of GPCRs begins with phosphorylation of the agonist-bound receptor by a G-protein coupled receptor kinase (GRK) followed by the binding of an arrestin molecule to the receptor. Arrestin-binding can terminate receptor-mediated activation of G-protein signals and link the receptor to the cellular endocytotic machinery to facilitate receptor endocytosis (DeWire et al. 2007; Gurevich et al. 2008). Interestingly, the formation of the arrestin-GPCR complex can also initiate cellular signals that are distinct from those produced by the receptor-mediated activation of G proteins (Gurevich et al. 2008).
GRK-phosphorylated receptor residues help to trigger the conformational change in arrestin, which enables it to recognize and bind the activated receptor (Gurevich and Gurevich 2004, 2006). However, for some GPCRs, negatively charged amino acids or residues phosphorylated by other kinases can substitute for GRK phosphorylation and promote arrestin binding to the agonist-activated receptor (Xiang et al. 2001; Mukherjee et al. 2002; Galliera et al. 2004).
For D2R, the molecular events which trigger arrestin activation and binding are not clear. Serine and threonine residues within D2R, which are phosphorylated in an agonist-dependent manner, have been previously mapped. However, alanine substitution of these residues in D2R did not block D2R internalization in mammalian cells and these agonist-dependent D2R phosphorylation sites were not required for agonist-dependent recruitment of arrestin to D2R (Namkung et al. 2009a). Even more, recent study examining the role of receptor-attached phosphates in binding of arrestins to GPCRs also suggests that the arrestin interactions with D2R are largely independent of GRK phosphorylation (Gimenez et al. 2012).
It has been demonstrated that D2R that is expressed in HEK293 cells is basally phosphorylated at residues that are distinct from those that are phosphorylated after agonist treatment. Furthermore, the level of phosphorylation at these sites is enhanced after treatment with protein kinase C (PKC) activators such as phorbol esters and phorbol ester treatment can induce D2R internalization in the absence of agonist-treatment (Namkung and Sibley 2004; Thibault et al. 2011). Finally, the mutation of sites that are basally phosphorylated or phosphorylated after phorbal ester-treatment significantly decreased agonist dependent internalization of D2R (Cho et al. 2010). These studies suggest that it is phosphorylation of D2R by PKC, and not GRK, that serves as the trigger for arrestin activation and the formation of the D2R/arrestin complex.
It has been previously demonstrated that the mutation of one set of serine and threonine residues in the mu opioid receptor or the cannabinoid receptor could block internalization of the GPCRs while allowing for arrestin-mediated uncoupling of the GPCRs from coupled G proteins. Conversely, mutation of another set of serine and threonine residues blocked arrestin-mediated uncoupling while allowing for agonist-mediated internalization of the GPCR to occur (Jin et al. 1999; Celver et al. 2004). A related finding—that arrestins can form distinct complexes with the same receptor depending on the position of phosphorylation sites on the receptor was also established for N-formyl peptide receptor (Key et al. 2003). Thus, phosphorylation at distinct sites may determine whether arrestin binding uncouples the receptor from G protein or promotes internalization of the receptor instead. Desensitization of D2R-elicited cellular responses could result from either GPCR-G protein uncoupling and/or from receptor internalization. As these processes occur concurrently in mammalian cells it has not been established if the formation of the arrestin-D2R complex can uncouple D2R from associated G proteins in addition to allowing for receptor internalization.
GPCRs expressed in mammalian cells can simultaneously undergo uncoupling, internalization, and recycling that could either be dependent on, or independent of endogenously expressed GRKs and arrestins (Pak et al. 1999; Shapira et al. 2001; Qiu et al. 2003; Rasmussen et al. 2004). Thus, a multitude of processes acts concurrently in mammalian cells to regulate GPCRs. Furthermore, the endogenous expression, in mammalian cells, of numerous regulatory molecules including arrestins and kinases such as GRKs make it difficult to clearly define and study the separate cellular processes that are involved in regulating D2R.
Electrophysiological recordings of GPCR-mediated activation of co-expressed G-protein gated inwardly rectifying potassium channels (Kir3) reconstituted in the Xenopus oocyte expression system have been used extensively to specifically examine the effects of GRKs and arrestins on agonist-dependent GPCR-G protein uncoupling (Kovoor et al. 1997; Appleyard et al. 1999; Jin et al. 1999; Celver et al. 2004). A convenient property of the Xenopus oocyte expression system is that it lacks functional expression of endogenous GRKs (see Figure S2) and arrestins, and furthermore, exogenous expression of GRKs and arrestins can specifically reconstitute agonist-mediated GPCR uncoupling without producing significant receptor internalization (Kovoor et al. 1997; Appleyard et al. 1999; Jin et al. 1999; Celver et al. 2004)(see Figure S1). In this study, we have utilized the Xenopus oocyte expression system to examine the effects of GRK and arrestin co-expression on the agonist-mediated uncoupling of D2R and the delta opioid receptor (DOR) with the goals of (i) more clearly identifying the molecular triggers required for functional interaction between D2R and arrestin, and (ii) asking if the determinants implicated in the arrestin-mediated internalization of D2R in mammalian cells are the same as those required for D2R uncoupling.
- Top of page
- Materials and methods
- Supporting Information
In this study, we utilized the Xenopus oocytes expression system to identify the molecular triggers which enable arrestin to interact with D2R in an agonist-dependent manner and produce D2R uncoupling. We used cell surface biotinylation to confirm that the desensitization of the receptor-activated Kir3 response observed for D2R and DOR occurred without detectable receptor internalization (see Figure S1) and most likely occurred as a result of receptor uncoupling from associated G proteins. The novel findings of the study are as follows: we found that arrestin could produce dopamine-dependent uncoupling of D2R from G-protein pathways even in the absence of functional GRK expression. Unlike what was observed for DOR in the same cells GRK co-expression was not required for and did not enhance arrestin-mediated uncoupling of D2R. The novel agonist-induced ARRB2-dependent but GRK3 independent uncoupling observed with D2R could be conferred to DOR by substituting the third cytoplasmic loop of DOR with the third cytoplasmic loop of D2R. Arrestin-dependent GRK3-independent uncoupling of D2R was blocked by staurosporine treatment and by alanine substitution of putative PKC phosphorylation sites in the third cytoplasmic loop of D2R. Finally, phosphorylation by GRK could not substitute for PKC phosphorylation to produce arrestin-mediated uncoupling of D2R.
Arrestins can bind and uncouple GPCRs from G proteins, serve as adapters that link the GPCR to the cellular internalization machinery and function as molecular scaffolds to support the activation of alternate signaling pathways by the GPCR (Gurevich et al. 2008). Importantly, the complex of a GPCR with an arrestin molecule does not exist as a singular entity or in a single fixed conformation. For example, some GPCRs have distinct phosphorylation requirements that determine whether arrestin-binding will mediate uncoupling or internalization of the GPCR indicating the existence of distinct conformations of the GPCR-arrestin produced by phosphorylation of the GPCR at different sites (Jin et al. 1999; Key et al. 2003; Celver et al. 2004). Furthermore, biased ligands identified for some GPCRs preferentially activate distinct arrestin-dependent signal transduction mechanisms revealing further potential for variability in the nature of the ligand/GPCR/arrestin complex (Reiter et al. 2012).
The molecular trigger utilized by D2R for ‘activating’ arrestin is unclear. However, specific residues in the D2R third cytoplasmic loop (212–215) have previously been identified as being required for the high-affinity interaction between D2R and arrestin, and for agonist-dependent internalization of D2R in mammalian cells (Lan et al. 2009). In the Xenopus oocyte expression system, we showed that the mutation of these residues (i.e. the D2R212-215(IYIV)A mutant) blocked the arrestin-mediated uncoupling of D2R. Thus, it appears that the arrestin-D2R complex that forms in Xenopus oocytes, to uncouple D2R, shares binding properties of the D2R-arrestin complex that forms in mammalian cells to mediate agonist-dependent internalization. Thus, our data suggest that an important structural interface of the D2R-arrestin that exists in mammalian cells is also established in the Xenopus oocyte system and supports the use of the oocyte expression system as a physiologically valid system for further investigations into the D2R-arrestin complex.
The stark difference in the requirement for GRK co-expression to produce agonist-dependent uncoupling between DOR and D2R (Fig. 1) support the suggestion that arrestin-mediated regulation of D2R diverges from classical models of arrestin action and does not require phosphorylation of D2R by GRKs. For example, these data suggest that another kinase phosphorylates D2R or a proximate substrate and such phosphorylation triggers arrestin activation and allows for binding to the agonist-activated D2R. Alternatively, arrestin can uncouple D2R in a manner that does not require D2R phosphorylation. Our observation that staurosporine can inhibit the arrestin-depended uncoupling of D2R suggests the former explanation is the correct one and indicates that phosphorylation of D2R by an endogenously expressed serine/threonine kinase can trigger arrestin activation. Furthermore, the finding that arrestin-dependent uncoupling of D2R was blocked by mutation of serine and threonine residues (i.e. the D2RT225,S228,S229A mutant) suggests that it is phosphorylated at these D2R sites that trigger arrestin activation. Such phosphorylation then allows arrestin to bind to the agonist-bound receptor in a manner that uncouples D2R from associated G proteins.
Agonist-dependent phosphorylation, presumably by GRKs, at a different set of D2R serine and threonine residues has previously been reported. However, the co-expression of both GRK and arrestin was unable to produce dopamine-dependent uncoupling of the D2RT225,S228,S229A mutant, suggesting that GRK phosphorylation of D2R cannot substitute for the actions of the endogenous kinase; a subtle but important distinction which is difficult to discern in other systems. Our results support the conclusions from a recent study which examined the role of receptor-attached phosphates in binding of arrestins to GPCRs and suggested that arrestin interactions with D2R are largely independent of GRK phosphorylation (Gimenez et al. 2012).
From this study it is not clear whether the D2R is basally phosphorylated, thus priming arrestin for binding to D2R once the receptor is activated by dopamine, or whether the phosphorylation events occur in an agonist-dependent manner through an endogenously expressed GRK. However, Xenopus oocytes do not endogenously express detectable levels of GRK (see Figure S2) and phosphorylation studies in mammalian cells suggest the former: phosphorylation of the residues mutated in D2RT225,S228,S229A may be blocked by PKC inhibitors and enhanced by PKC activation but is not affected by agonist activation of D2R (Cho et al. 2010). Thus, our data, taken together with previous studies (Namkung et al. 2009a,b; Cho et al. 2010; Thibault et al. 2011), suggest a scenario where alterations in PKC activity in D2R-expressing cells can have marked effects on the rates of both dopamine-mediating uncoupling and internalization of D2R. In this study, while we have not identified the kinase responsible for phosphorylating D2R in Xenopus oocytes, we believe we have more specifically linked the putative PKC phosphorylation sites, Thr225, T228, and S229, to the formation of an arrestin/D2R complex which can uncouple D2R from G protein in an agonist-dependent manner.
Mechanisms for the regulation of D2R remain poorly understood. For example, agonist-induced phosphorylation has recently been implicated in the recycling of D2R; that is, mutation of agonist-dependent phosphorylation sites has been reported as causing the D2R mutant to recycle at a slower rate to the plasma membrane. Consequently, by increasing the rate of reinsertion of internalized D2R back to the plasma membrane, GRK phosphorylation should decrease the net loss of D2R from the cell surface after agonist treatment. Instead, it has been found that GRK over-expression enhances the amount of D2R removed from the cell surface following agonist activation of D2R in mammalian cells (Ito et al. 1999; Celver et al. 2010; Cho et al. 2010). In another example, Cho and colleagues reported that a D2R-elicited cellular response, thought to be mediated via the second messenger, cAMP, was not desensitized after 40 min of dopamine pre-treatment even though such pre-treatment caused more than 40% of the receptor to be internalized. These apparently paradoxical observations were made in mammalian cell systems where GPCR uncoupling, internalization, recycling, and arrestin-mediated signaling can all occur simultaneously and in parallel with multiple other regulatory processes (Pak et al. 1999; Shapira et al. 2001). Mammalian cells also support GPCR internalization that occurs through mechanisms that do not involve GRK and arrestin (Shapira et al. 2001; Qiu et al. 2003; Namkung and Sibley 2004; Rasmussen et al. 2004). The concurrent occurrence of many of these processes in mammalian cells poses an impediment to untangling the multiple molecular determinants that may regulate D2Rs. The goal of fully untangling these processes may be supported by reconstitution experiments in multiple expression systems, each of which expresses a different set of regulatory molecules or which lack the full complement of regulatory elements found in vivo.
We believe this study is one such example. Agonist-induced internalization is not observed in Xenopus oocytes (Appleyard et al. 1999; Jin et al. 1999; Celver et al. 2004) and Xenopus oocytes do not express functional GRKs (see Figure S2) or arrestins (Kovoor et al. 1997; Appleyard et al. 1999; Jin et al. 1999; Celver et al. 2004). Here, in the absence of receptor internalization (see Figure S1) the effects of phosphorylation at specific D2R residues and the action of arrestin were uniquely associated with the functional uncoupling of D2R.
- Top of page
- Materials and methods
- Supporting Information
The authors have no potential conflict of interest with the publication of this manuscript. The project described was supported by grants from the National Institute of Mental Health (R15MH091639-01 to AK and MS), from the National Center for Research Resources (5P20RR016457-11) and the National Institute for General Medical Science (8 P20 GM103430-11), components of the National Institutes of Health (NIH), and its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIMH, NCRR, NIGMS or the NIH. Additional support includes work conducted using the Rhode Island Genomics and Sequencing Center which is supported in part by the National Science Foundation under EPSCoR Grants Nos. 0554548 & EPS-1004057.
The authors declare that they have reviewed the ARRIVE guidelines and these guidelines have been followed. Furthermore, the authors have no conflict of interest to declare.