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GPR37, also known as parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR) that aggregates intracellularly in a juvenile form of Parkinson's disease. However, little is known about the structure or function of this receptor. Here, in order to better understand the functioning of this receptor, we focused on the GPR37 C-terminal tail, in particular on a cystein-enriched region. Thus, we aimed to reveal the role of these residues on receptor plasma membrane expression and function, and also whether the presence of this cysteine-rich domain is linked to the previously described receptor-mediated cytotoxicity. Interestingly, while the deletion of six cysteine residues within this region did not affect receptor internalization it promoted GPR37 plasma membrane expression and signaling. Furthermore, the removal of the C-terminal cysteine-rich domain protected against GPR37-mediated apoptosis and cell death. Overall, we identified a GPR37 domain, namely the C-terminal tail cysteine-rich domain, which played a critical role in receptor cell surface expression, function and GPR37-mediated cytotoxicity. These results might contribute to better comprehend the pathophysiology (i.e. in Parkinson's disease) of this rather unknown member of the GPCR family.
Parkin-associated endothelin-like receptor (Pael-R), also called GPR37, is an orphan G protein-coupled receptor (GPCR) first cloned in 1997 from human brain (Marazziti et al. 1997). Within the brain, this receptor is particularly enriched in cerebellum (i.e. Purkinje cells), corpus callosum, medulla, putamen, caudate nucleus, substantia nigra and hippocampus (i.e. pyramidal and granule cells of the dentate gyrus) (Donohue et al. 1998; Marazziti et al. 1997; Takahashi and Imai 2003; Zeng et al. 1997). Interestingly, GPR37 has been identified as a parkin substrate (Imai et al. 2001; Takahashi and Imai 2003). Parkin is a protein-ubiquitin ligase E3 involved in the ubiquitination and proteasome-mediated protein degradation and in the clearance of aggregated proteins (Dev et al. 2003). Therefore, parkin loss of function, such as in autosomal recessive juvenile Parkinson (Dev et al. 2003), prevents degradation of parkin substrates (i.e. GPR37) which results in their toxic accumulation (Shimura et al. 2000; Sriram et al. 2005; Zhang et al. 2000). Indeed, GPR37 has been described to be up-regulated in the brains of autosomal recessive juvenile Parkinson patients (Imai et al. 2001). In addition, the presence of GPR37 in the core of Lewy bodies in Parkinson disease (PD) patients has been reported (Murakami et al. 2004), thus suggesting a role of GPR37 aggregates in PD pathology. Finally, viral-mediated GPR37 over-expression in vivo (i.e. in the substantia nigra) constitutes a good PD animal model because it results in progressive degeneration of nigral dopaminergic neurons (Dusonchet et al. 2009; Low and Aebischer 2012).
Collectively, these results established a good correlation between the expression of this orphan receptor and PD. However, very little information exists regarding the functional and structural characteristics of this receptor. For instance, GPR37 has a significant sequence homology (40%) with the mammalian peptide activated class A GPCRs (i.e. endothelin-B receptor, bombesin-BB1 and bombesin-BB2 receptors; Marazziti et al. 1997, 1998), but agonists for these receptors (i.e. endothelin and bombesin) failed to activate GPR37 (Leng et al. 1999; Valdenaire et al. 1998; Zeng et al. 1997). However, it has been reported, not without some controversy (Dunham et al. 2009), that the neuropeptide head activator (HA), which is derived from the freshwater coelenterate Hydra, is a high-affinity ligand for GPR37 (Rezgaoui et al. 2006). Interestingly, in this last study it was reported that HA challenge induced GPR37-mediated intracellular Ca2+ accumulation and activation of both Ca2+-dependent calmodulin kinase and phosphoinositide-3-kinase (Rezgaoui et al. 2006). However, although some early publications suggested that HA is present in the human brain (Bodenmuller et al. 1980) and proposed a potential role in some brain tumors (Schaller et al. 1988), no equivalent neuropeptide has been identified in vertebrates, thus casting doubt on the existence of a human version of the HA neuropeptide. Therefore, the existence of HA neuropeptide in humans is still an open question, a fact that may be posed as an additional setback in the description of GPR37 biological functions.
In this study, we aimed to shed some light on GPR37 structure, function and toxicity, trying to elucidate some of the molecular determinants that mediate these processes and that may help to explain the mechanisms driving the toxic accumulation of the receptor. Interestingly, GPR37 possesses an inherent difficulty for folding, a fact that complicates its ectopic expression in living cells and plasma membrane trafficking (Takahashi and Imai 2003). Related to this, several studies have shown that different covalent post-translational modifications of cysteine residues can have distinct effects on protein trafficking (Greaves and Chamberlain 2007) or on receptor coupling to G-proteins and thus in intracellular strength of signaling (Chini and Parenti 2009). Furthermore, it has also been shown that S-nitrosylation and further oxidation of critical cysteine residues can lead to protein misfolding, and that these misfolded proteins can form aggregates in many neurodegenerative diseases (Muchowski and Wacker 2005). Based on these data, we decided to explore the role of a cysteine-rich domain, located at the C-terminal tail of the GPR37, on receptor's trafficking, function and also its relationship with cytotoxicity.
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Neurodegenerative diseases are characterized by the progressive loss of neurons, a phenomenon largely associated to ER and oxidative stress (Forman et al. 2003). Thus, a large body of studies has shown that ER stress-induced apoptosis is implicated in numerous human diseases, including diabetes and neurogenerative diseases (Szegezdi et al. 2006). The build up of unfolded/misfolded proteins activates adaptative responses in cells, the UPR, that protect them from the toxic rise of these proteins (Rao and Bredesen 2004). However, a gradual overtime accumulation of unfolded/misfolded proteins, together with an UPR failure, will finally promote not only cellular stress responses related to the ER but also the induction of specific death pathways (i.e. apoptosis) to remove the stressed cells (Kaufman 2002; Rao and Bredesen 2004). In such way, it has been described that the intracellular accumulation of GPR37 leads to ER stress, which ultimately triggers the activation of apoptotic pathways (e.g. activation of caspase 3) both in neurons and in stable cell lines (Rao and Bredesen 2004). Accordingly, a role of GPR37 aggregates in PD has been suggested(Kitao et al. 2007; Omura et al. 2006). It would then seem likely that the aggregation of GPR37 due to ineffective receptor folding would favor the presence of ubiquitinated protein deposits in the neuronal cytoplasm (i.e. Lewy bodies), thus prompting the loss of dopaminergic neurons from the substantia nigra pars compacta that occurs in PD (Murakami et al. 2004). However, despite this possible role of GPR37 in the pathology – i.e. mediating cell death upon receptor aggregation – very little is known about this orphan receptor and the molecular determinants driving its intracellular accumulation. Hence, in the present study, we focused on a cysteine-rich domain located at the C-terminal tail of the receptor as a possible cornerstone explaining the expression, function, ER-induced stress and cytotoxicity of the receptor.
First of all, we performed several mutations at the C-terminal tail of the receptor by deleting different cysteine residues-containing fragments and started examining whether they affected the cell surface expression of the receptor. As previously shown (Imai et al. 2001; Murakami et al. 2004), a large amount of the wild-type receptor was found in intracellular compartments. However, when deleting the cysteine-rich domain located between the residues 563 and 568, a robust increase in plasma membrane expression was detected (in both GPR37Δ563 and GPR37Δ563-568). These results pointed out to a crucial role of this domain on receptor trafficking, although they diverge from those previously reported where the deletion of the whole GPR37 C-terminal tail prevented cell surface expression (Cookson 2005). Yet, the works differ in several experimental approaches, for instance the number of amino acid residues deleted, the technique used to determine the surface expression and other general methodologies (e.g. transfecting agent, vector where the constructs were subcloned, etc.). Nevertheless, we decided to next assess whether the mutations affected receptor's internalization and functionality, because it would seem likely that a change on signal transduction pathways should be observed due to the effect of the cysteine-rich domain on receptor expression at the cell surface. Interestingly, when the receptor was challenged with the HA neuropeptide it could be observed that the deletion of the GPR37 cysteine-rich domain produced both an increase in Ca2+ mobilization and in inhibition of cAMP accumulation. However, the receptor's cysteine-rich domain did not affect the HA-mediated GPR37 cell surface clustering and internalization. These results are consistent with those proposing that the HA neuropeptide is an agonist of GPR37 that activates and induces GPR37 internalization in a pertussis toxin sensitive manner (Dunham et al. 2009; Imai et al. 2001; Rezgaoui et al. 2006). Overall, the functional experiments were in line with the immunocytochemistry, internalization and cell surface biotinylation assays, indicating that the studied cysteine-rich domain was important to retain the receptor at the cytoplasm, and as a consequence it was also responsible of regulating its membrane G-protein coupled signaling.
Next, based on the relevance of this cysteine-rich domain on receptor's trafficking and function, and because of the relationship of cytoplasmatic GPR37 aggregates with low survival rates in transfected cells (Dunham et al. 2009), we investigated whether this cysteine-rich region could constitute a molecular determinant of GPR37-mediated ER stress and cytotoxicity upon receptor expression. To this end, we first studied GPR37-mediated UPR triggering in cells transfected with the wild-type and the mutated receptor in which the cysteine-rich domain was deleted. Interestingly, the deletion of the GPR37 cysteine-rich domain efficiently reduced the receptor-mediated GRP78 up-regulation and UPR signaling activation, thus pointing out to a prominent role of this GPR37 domain in mediating ER stress. Also, the role of the GPR37 cysteine-rich domain on cell viability and also on caspase 3 pathway activation was analyzed. Interestingly, we effectively found that GPR37 over-expression in living cells induced cell death, as described previously (Rezgaoui et al. 2006). It is important to mention that dying cells expressing GPR37 showed features of both necrosis and apoptosis, which would suggest the concurrence of different pathways leading to the same physiological state. In addition, we also demonstrated that this GPR37-mediated cytotoxicity was significantly reduced when the cysteine-rich domain was removed. This is a particularly important finding because it revealed for the first time the importance of this GPR37 discrete amino acid sequence in the biology of this rather unknown receptor. Thus, the GPR37 cysteine-rich domain not only participated in the control of receptor plasma membrane trafficking and the concomitant receptor-mediated signal transduction, but it also was involved in ER stress and the cytotoxic effects associated to receptor expression. However, it remains to be determined what signals might control the subcellular distribution and function of GPR37 through modification of its cysteine-rich domain.
The ectopic expression of GPR37 may end in the formation of toxic receptor aggregates and ER stress, as mentioned above. In such situation, transfected cells trigger the protective UPR in an attempt to contain the deleterious effects of GPR37 intracellular accumulation. The UPR constitutes a concerted cellular response mediated by three ER transmembrane receptors: protein kinase R-like ER kinase, inositol-requiring enzyme 1, and ATF6. Under normal conditions, all three ER stress receptors are kept inactive through their direct interaction with the ER chaperone, GRP78. However, when an unfolded/misfolded protein accumulates (i.e. GPR37), GRP78 dissociates from the three receptors, leading to their activation and triggering the activation of UPR signaling pathways (Rezgaoui et al. 2006). Interestingly, UPR is a pro-survival response intended to reduce the accumulation of unfolded proteins from the ER (Kitao et al. 2007; Omura et al. 2006). However, under persistent protein aggregation and prolonged ER stress, as it could happen upon GPR37 over-expression, the UPR will activate unique pathways switching the pro-survival response to a pro-apoptotic effect (Szegezdi et al. 2006). These pathways implicate the increased translation of ATF4 (Schroder and Kaufman 2005) and the concomitant induction of the transcription factor C/EBP homologous protein, which play a key role in switching UPR from pro-survival to pro-death signaling (Schroder and Kaufman 2005). Indeed, in our hands the ectopic expression of GPR37 in HEK293 cells induced ATF4 up-regulation, a phenomenon that was abolished when the GPR37 cysteine-rich domain was deleted. As a result, it could be postulated that the cysteine-rich region within the C-terminal tail of the GPR37 constitutes a receptor folding sensor, and thus either the absence or the blockade of this check point would facilitate receptor folding, trafficking and abolish its toxic accumulation. Interestingly, it has been reported that GPR37 is able to interact with other GPCRs, for instance the dopamine D2 receptor (D2R), and that this interaction facilitates GPR37 cell surface expression (Harding et al. 2000). Indeed, GPCR heterodimerization often modulates receptor trafficking (Schroder and Kaufman 2005), thus it might be feasible that the negative effect of GPR37 cysteine-rich domain not only on receptor plasma membrane expression and signaling but also on mediating cytotoxic effects could be precluded by interacting (i.e. heterodimerize) with appropriate receptor partners (i.e. D2R). Overall, GPR37 heteromerization should be also contemplated as a way to attain proper receptor cell surface expression and function, and to diminish GPR37-mediated cytotoxicity.
In conclusion, the results obtained here point out to the following possible mechanistic scenario: while newly synthesized GPR37 is folded in the ER before being targeted to the plasma membrane, the misfolded GPR37 translocates across the ER membrane into the cytoplasm and is degraded through a parkin-dependent ubiquitin-proteasome pathway. However, when a dysregulation of this cellular control system occurs the aggregation of misfolded GPR37 takes place, triggering pro-death pathways, a process in which the cysteine-rich domain located at the C-terminal tail of the receptor might play a pivotal role. Interestingly, our results might be important to understand the pathogenesis associated to the accumulation of this expression-dependent misfolding receptor and ultimately to comprehend its relationship with PD.