Metalloprotease cleavage of the N terminus of the orphan G protein–coupled receptor GPR37L1 reduces its constitutive activity

Abstract

Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gα(s) when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3',5'-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gα(s) or Gα(i) signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue.

GPR37L1 modulates seizure susceptibility: Evidence from mouse studies and analyses of a human GPR37L1 variant

“…However, neither prosaptide nor HA stimulation significantly increased ERK phosphorylation further (Figure 3A & B). Similarly, transfection with either Flag-GPR37L1 WT or the Flag-K349N mutant resulted in comparable increases in constitutive signaling to CRE luciferase (Figure 3C), another readout that has been reported to be downstream of GPR37L1 46 . Again, neither prosaptide nor HA increased GPR37L1 signaling to CRE luciferase.…”

“…The natural ligand for GPR37L1 remains uncertain, and in the studies described here, we assessed two peptides that have been proposed as ligands for GPR37L1 and/or GPR37: prosaptide 25; 42; 43 and head activator (HA) 44; 45 However, no significant increases in receptor signaling activity were observed with either peptide, and therefore the previously-reported agonistic actions of these two peptides could not be confirmed here. It is possible that the regulation of GPR37/GPR37L1 signaling activity by these peptides is dependent on cellular context and/or other variables, although another recent study also reported a lack of activation of GPR37L1 signaling by prosaptide 46 . Thus, while the in vitro data described here cannot confirm the pathogenicity of the K349N variant, it is plausible that functional differences between GPR37L1 WT and the K349N mutant may be observed only with agonist-induced signaling.…”

RETRACTED ARTICLE: Orphan receptor ligand discovery by pickpocketing pharmacological neighbors

“…We purchased eight OX receptor antagonists with varying selectivity for OX 1 and OX 2 receptors, one non-selective BB 1 /BB 2 receptor antagonist, PD176252 (peptide agonists for the BB receptors did not bind to GPR37L1 31,32 ) and one NPS receptor antagonist SHA-68. Our previous studies established that GPR37L1 is a constitutively active receptor coupled to the Gα s –cAMP pathway 34 , so we tested each ligand for either agonism or antagonism using a CRE-luciferase assay 35 . Several OX receptor antagonists, namely ACT 335827, TCS 1102, JNJ 10397049 and SB 674042, and the NPS receptor antagonist SHA-68, showed promise as GPR37L1 inverse agonists, reducing GPR37L1-mediated CRE-luciferase levels (Fig.…”

“…We have previously described the capacity of GPR37L1 to constitutively signal downstream of Gα s using cAMP-Response Element (CRE) luciferase reporter gene assays 34 ; this allows the detection of both agonism and inverse agonism. The pcDNA3-GPR37L1 construct was generated as described previously and verified by DNA sequencing 34 .…”

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage

“…TM, transmembrane domain. 42 , and again, the glycopeptide was less efficiently cleaved. C, schematic overview of the total observed cleavage sites by ADAM10, ADAM17, and MMPs, and their relation to the observed glycosylation sites.…”