The N-terminus of GPR37L1 is proteolytically processed by matrix metalloproteases

Abstract

GPR37L1 is an orphan G protein-coupled receptor expressed exclusively in the brain and linked to seizures, neuroprotection and cardiovascular disease. Based upon the observation that fragments of the GPR37L1 N-terminus are found in human cerebrospinal fluid, we hypothesized that GPR37L1 was subject to post-translational modification. Heterologous expression of GPR37L1-eYFP in either HEK293 or U87 glioblastoma cells yielded two cell surface species of approximately equivalent abundance, the larger of which is N-glycosylated at Asn105. The smaller species is produced by matrix metalloprotease/ADAM-mediated proteolysis (shown by the use of pharmacological inhibitors) and has a molecular weight identical to that of a mutant lacking the entire N-terminus, Δ122 GPR37L1. Serial truncation of the N-terminus prevented GPR37L1 expression except when the entire N-terminus was removed, narrowing the predicted site of N-terminal proteolysis to residues 105–122. Using yeast expressing different G protein chimeras, we found that wild type GPR37L1, but not Δ122 GPR37L1, coupled constitutively to Gpa1/Gαs and Gpa1/Gα16 chimeras, in contrast to previous studies. We tested the peptides identified in cerebrospinal fluid as well as their putative newly-generated N-terminal ‘tethered’ counterparts in both wild type and Δ122 GPR37L1 Gpa1/Gαs strains but saw no effect, suggesting that GPR37L1 does not signal in a manner akin to the protease-activated receptor family. We also saw no evidence of receptor activation or regulation by the reported GPR37L1 ligand, prosaptide/TX14A. Finally, the proteolytically processed species predominated both in vivo and ex vivo in organotypic cerebellar slice preparations, suggesting that GPR37L1 is rapidly processed to a signaling-inactive form. Our data indicate that the function of GPR37L1 in vivo is tightly regulated by metalloprotease-dependent N-terminal cleavage.

Cytosolic GPR37, but not GPR37L1, multimerization and its reversal by Parkin: A live cell imaging study

“…Although they have different length of their N‐terminal ectodomains, both are subject to metalloprotease‐dependent proteolytic N‐terminus processing. Both GPR37 and GPR37L1 N‐terminal peptides have been found in cerebrospinal fluid (CSF) 32–34 . Interestingly, peptides from the ectodomain of GPR37 are increased in CSF from PD patients 33 .…”

Emerging Roles for the Orphan GPCRs, GPR37 and GPR37 L1, in Stroke Pathophysiology

“…A recent study highlighting the role of GPR37 in mediating glial cell responses following stroke examined prosaposin levels within the peri-infarct region of wild-type and GPR37 knock-out ( Gpr37 -/- ) mice and did not observe detectable differences in prosaposin expression [ 23 ]. Similar to studies proposing HA as the endogenous ligand for GPR37, there have been a number of studies which have failed to recapitulate specific prosaposin/prosaptide-mediated effects on GPR37/GPR37 L1 signaling [ 32 , 38 , 39 ]. It has been posited that the conflicting findings being presented by various groups could specifically relate to the cellular context in which the receptor was studied [ 40 ].…”

“…Both GPR37 and GPR37 L1 undergo constitutive a metalloproteinase cleavage of their N-terminus [ 38 , 46 , 47 ]. This phenomenon of ectodomain shedding has been extensively described for the following GPCRs: protease-activated receptors (PARs) [ 48 ] and adhesion receptors [ 49 ].…”

“…This phenomenon of ectodomain shedding has been extensively described for the following GPCRs: protease-activated receptors (PARs) [ 48 ] and adhesion receptors [ 49 ]. For GPR37 L1, the cleavage of the N-terminus was linked to a loss of constitutive (ligand independent) receptor signaling through Gαs [ 38 ]. These data were assessed within heterologous cells overexpressing a tagged receptor construct, therefore the physiological relevance of this effect remains unclear.…”

“…It has, however, been established that its ectodomain cleavage is specifically modulated by the metalloproteinase’s ADAM10 and furin [ 47 ]. Interestingly, the proteolytically processed species of both GPR37 and GPR37L1 seem to dominate in vivo [ 38 , 47 ]. This suggests that under physiological conditions, both receptors are being rapidly processed into their proteolytically cleaved counterparts.…”