GPR37 and GPR37L1 are receptors for the neuroprotective and glioprotective factors prosaptide and prosaposin

Abstract

GPR37 (also known as Pael-R) and GPR37L1 are orphan G protein-coupled receptors that are almost exclusively expressed in the nervous system. We screened these receptors for potential activation by various orphan neuropeptides, and these screens yielded a single positive hit: prosaptide, which promoted the endocytosis of GPR37 and GPR37L1, bound to both receptors and activated signaling in a GPR37- and GPR37L1-dependent manner. Prosaptide stimulation of cells transfected with GPR37 or GPR37L1 induced the phosphorylation of ERK in a pertussis toxin-sensitive manner, stimulated (35)S-GTPγS binding, and promoted the inhibition of forskolin-stimulated cAMP production. Because prosaptide is the active fragment of the secreted neuroprotective and glioprotective factor prosaposin (also known as sulfated glycoprotein-1), we purified full-length prosaposin and found that it also stimulated GPR37 and GPR37L1 signaling. Moreover, both prosaptide and prosaposin were found to protect primary astrocytes against oxidative stress, with these protective effects being attenuated by siRNA-mediated knockdown of endogenous astrocytic GPR37 or GPR37L1. These data reveal that GPR37 and GPR37L1 are receptors for the neuroprotective and glioprotective factors prosaptide and prosaposin.

Keywords: GPCR; MAPK; deorphanization; neurodegeneration; peptide.

Fig. 1.

Prosaptide binds to and induces endocytosis of GPR37 and GPR37L1. (A and B) Five different peptides were examined for their abilities to induce internalization of Flag-tagged GPR37 (A) and GPR37L1 (B) in HEK-293T cells. Each peptide was added to the cells at 1 µM for 30 min. Only treatment with prosaptide induced significant receptor internalization (n = 3–5 for each peptide; all points done in triplicate, **P < 0.01, ***P < 0.001). (C and D) The ability of prosaptide to induce internalization of GPR37L1 in transfected COS-7 cells was visualized using confocal microscopy. Cell surface GPR37L1 is labeled in red, with green labeling indicating internalized GPR37L1. Representative images of GPR37L1 localization in vehicle-treated cells (C) and cells treated with 1 µM prosaptide (D) are shown. (E) Quantification of surface vs. internalized GPR37L1. COS-7 cells were analyzed for ratio of internalization by comparing the quantity of internalized receptor (green) to the total receptor found on the cell surface (red). In this analysis, 20 cells each from vehicle- vs. prosaptide-treated GPR37L1-transfected COS-7 cells were randomly selected in a blinded manner and analyzed to assess the extent of GPR37L1 internalization (***P < 0.001). (F) The ability of prosaptide to bind to different GPCRs was assessed using biotinylated prosaptide attached to streptavidin beads (+) or streptavidin beads alone (−). Flag-tagged GPR37 and GPR37L1, but not other Flag-tagged (α1A- and β1-adrenergic) or HA-tagged receptors (D1 dopamine and endothelin ET_B), were pulled down with the biotinylated prosaptide adsorbed to the beads, indicating specific binding of the peptide to GPR37 and GPR37L1.

Fig. 2.

Prosaptide stimulates G protein-mediated signaling via GPR37 and GPR37L1. (A) HEK-293T cells transiently transfected with either empty vector (Mock), GPR37/syntenin-1 or GPR37L1 were treated for 10 min with 100 nM prosaptide and ERK phosphorylation was assessed. pERK, phosphorylated ERK; tERK, total ERK. (B) Quantification of changes in pERK levels in response to prosaptide treatment. All experiments determined prosaptide-induced ERK phosphorylation over vehicle treatment (n = 3; all points done in duplicate, ***P < 0.001). (C and D) Dose–response curves for prosaptide stimulation of HEK-293T cells transfected with either GPR37/syntenin-1 (C) or GPR37L1 (D) (n = 6). (E) Effects of prosaptide treatment (30 min, 100 nM) on cAMP levels in HEK-293T cells transfected with empty vector (Mock), GPR37/syntenin-1, or GPR37L1. All experiments determined prosaptide-induced cAMP inhibition compared with vehicle treatment (n = 5; all points done in triplicate, **P < 0.01). (F) Effects of prosaptide treatment on ³⁵S-GTPγS binding to membranes derived from HEK-293T cells transfected with Gα_i1 alone (Mock), GPR37/syntenin-1/Gα_i1, or GPR37L1/Gα_i1. All experiments determined prosaptide-induced ³⁵S-GTPγS binding over vehicle treatment (n = 3; all points done in duplicate, **P < 0.01, *P < 0.05). Total counts for vehicle-treated samples averaged ∼6,000 cpm and did not vary significantly between transfection conditions.

Fig. 3.

Prosaptide actions on primary astrocytes are mediated by GPR37 and GPR37L1. (A) The relative expression levels of GPR37 and GPR37L1 in primary cortical astrocyte lysates vs. whole-brain lysates were assessed via Western blot. (B) Prosaptide-induced ERK phosphorylation in primary cortical astrocytes was assessed under several conditions: no siRNA treatment, treatment with a scrambled siRNA, or treatment with siRNAs directed against GPR37 and/or GPR37L1. KD, knockdown. (C) Quantification of the studies illustrated in B (n = 3; all points done in duplicate, ***P < 0.001). (D) Prosaptide-mediated protection of cortical astrocytes from a hydrogen peroxide insult was assessed by measuring lactate dehydrogenase release. Labels are the same as in C (n = 3; all points done in triplicate, ***P < 0.001).

Fig. 4.

Full-length prosaposin protects primary astrocytes via activation of GPR37 and GPR37L1. (A) Treatment of HEK-293T cells transiently transfected with empty vector (Mock), GPR37/syntenin-1, or GPR37L1 with prosaposin (100 nM, 10 min) resulted in enhanced ERK phosphorylation in only GPR37- or GPR37L1-transfected cells (n = 3; all points done in duplicate, ***P < 0.001). (B) Endocytosis of Flag-GPR37 and Flag-GPR37L1 was assessed following treatment with 100 nM prosaposin for 30 min (n = 3; all points done in triplicate, *P < 0.05). (C) Primary cortical astrocytes were treated with either no siRNA, a scrambled siRNA, or siRNAs directed against GPR37 and/or GPR37L1. These astrocytes were then stimulated with prosaposin (100 nM, 10 min) and activation of ERK was assessed (n = 3; all points done in duplicate, ***P < 0.001). (D) Quantification of the experiments illustrated in C. (E) Primary cortical astrocytes were exposed to H₂O₂ and cell death was assessed via measuring the release of lactate dehydrogenase into the media. Prosaposin pretreatment (100 nM) protected the astrocytes from the oxidative stress, but the effects of prosaposin were attenuated when GPR37 and/or GPR37L1 was knocked down via siRNA (n = 3; all points done in triplicate, **P < 0.01).