GPR37 Surface Expression Enhancement via N-Terminal Truncation or Protein−Protein Interactions

Abstract

GPR37, also known as the parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR) that exhibits poor plasma membrane expression when expressed in most cell types. We sought to find ways to enhance GPR37 trafficking to the cell surface in order to facilitate studies of GPR37 functional activity in heterologous cells. In truncation studies, we found that removing the GPR37 N-terminus (NT) dramatically enhanced the receptor's plasma membrane insertion. Further studies on sequential NT truncations revealed that removal of the first 210 amino acids increased surface expression nearly as much as removal of the entire NT. In studies examining the effects of co-expression of GPR37 with a variety of other GPCRs, we observed significant increases in GPR37 surface expression when the receptor was co-expressed with the adenosine receptor A 2A R or the dopamine receptor D 2 R. Co-immunoprecipitation experiments revealed that full-length GPR37 and, to a greater extent, the truncated GPR37 were capable of robustly associating with D 2 R, resulting in modestly-altered D 2 R affinity for both agonists and antagonists. In studies examining potential interactions of GPR37 with PDZ scaffolds, we observed a specific interaction between GPR37 and syntenin-1, which resulted in a dramatic increase in GPR37 surface expression in HEK-293 cells. These findings reveal three independent approaches -N-terminal truncation, coexpression with other receptors and co-expression with syntenin-1 -by which GPR37 surface trafficking in heterologous cells can be greatly enhanced to facilitate functional studies on this orphan receptor.The orphan G-protein coupled receptor (GPCR) GPR37, also known as the parkin-associated endothelin-like receptor (Pael-R), is highly expressed in the mammalian central nervous system, but its function still remains largely unknown. GPR37 is most closely related to another CNS-enriched orphan receptor known as GPR37-like 1 (GPR37L1), and both orphans share significant sequence homology with the endothelin B receptor and other related peptideactivated GPCRs (1-5). However, none of the mammalian peptides tested so far, including endothelins, bombesin and others, have produced activation of any signaling pathways in heterologous cells or Xenopus oocytes expressing either GPR37 or GPR37L1 (2-5). A peptide called "head activator" (HA), which is derived from the freshwater coelenterate Hydra, has been reported to be capable of activating GPR37 (6), but no peptide equivalent to HA has been definitively identified in vertebrates. Thus, GPR37 and GPR37L1 must still be considered orphan GPCRs at the present time.A major stumbling block impeding progress in understanding the ligand binding and signaling of GPR37 is the receptor's poor trafficking to the plasma membrane in most heterologous cell lines. GPR37 is commonly misfolded and therefore aggregated in the endoplasmic reticulum † This work was supported by the Pharmacological Sciences training grant T32 GM008602, the NIH and the...

The Parkinson's-disease-associated receptor GPR37 undergoes metalloproteinase-mediated N-terminal cleavage and ectodomain shedding

“…This implies that the cleavage of GPR37 is evolutionarily conserved in a similar manner as has been demonstrated for ET B R (Kozuka et al, 1991;Grantcharova et al, 2002) and the β 1 adrenergic receptor (Hakalahti et al, 2013). Interestingly, one of the studies aiming to enhance GPR37 plasma membrane trafficking demonstrated that the receptor cell surface expression could be increased significantly by N-terminal truncations (Dunham et al, 2009). In this study, an N-terminal FLAG epitope tag was used to detect the full-length and truncated receptors.…”

“…GPR37 carries a PSD-95, Discs-large, ZO-1-homology (PDZ)-domain-binding motif at its C-terminus that is known to mediate interaction with a few PDZ-domain-containing proteins (Dunham et al, 2009;Dutta et al, 2014). Such interactions are known to alter receptor function and trafficking (Romero et al, 2011), and thus could modify the susceptibility to cleavage.…”

“…Thus, the intracellular aggregation and impaired ubiquitylation of unfolded GPR37 by parkin have been proposed to be linked with the death of dopaminergic neurons characteristic of Parkinson's disease (Imai et al, 2001;Kitao et al, 2007). Based on these early findings, the research on GPR37 has mainly focused on strategies to improve its plasma membrane trafficking and to reduce the receptor-induced cell toxicity (Dunham et al, 2009;Gandía et al, 2013;Lundius et al, 2013;Dutta et al, 2014). Recently, GPR37, together with its closest homolog GPR37-like 1 (GPR37L1; Valdenaire et al, 1998), has been reported to act as a receptor for prosaposin and a prosaposin-derived peptide prosaptide (Meyer et al, 2013).…”

PDZ Protein Regulation of G Protein–Coupled Receptor Trafficking and Signaling Pathways

“…Although PICK1 regulates mGluR7 phosphorylation, clustering, and membrane expression, it is not yet clear what role syntenin-1 may play in this regulation (Boudin et al, 2000;Dev et al, 2000;Suh et al, 2008). Nonetheless, syntenin-1 has been demonstrated to enhance the membrane expression of G protein-coupled receptor 37 (endothelin receptor type B-like) (Dunham et al, 2009). In regards to signaling, syntenin-1 interacts with frizzled-7 and promotes c-Jun phosphorylation, CDC42 activation, and PKCa recruitment to the membrane (Luyten et al, 2008).…”

Role of the N-Terminal Region in G Protein–Coupled Receptor Functions: Negative Modulation Revealed by 5-HT2BReceptor Polymorphisms

“…In some 7TMRs, such as the cannabinoid CB1 receptor, a1-adrenergic receptor, and GPR37, published data show that truncation of the N-terminal region increases plasma membrane expression of these receptors (Andersson et al, 2003;Hague et al, 2004;Dunham et al, 2009). The extracellular surface of biogenic amine 7TMRs affects ligand recognition, ligand "escorting" into the binding pocket, and ligand binding kinetics (Wittmann et al, 2011).…”