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

Sci Rep. 2020 Nov 17;10(1):19995. doi: 10.1038/s41598-020-76384-9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cerebellum / metabolism
  • Female
  • GTP-Binding Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Male
  • Matrix Metalloproteinases / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Nerve Growth Factors / metabolism
  • Peptide Hydrolases / metabolism
  • Proteolysis
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, G-Protein-Coupled / metabolism*
  • Signal Transduction / physiology

Substances

  • GPR37L1 protein, human
  • Nerve Growth Factors
  • Receptors, G-Protein-Coupled
  • prosaptide
  • Peptide Hydrolases
  • Matrix Metalloproteinases
  • GTP-Binding Proteins