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

• Published in: Scientific reports 2020-11, Vol.10 (1), p.19995-19995, Article 19995
• Main Authors: Coleman, James L. J., Ngo, Tony, Smythe, Rhyll E., Cleave, Andrew J., Jones, Nicole M., Graham, Robert M., Smith, Nicola J.
• Format: Article
• Language: English
• Subjects: 631/45/468; 631/45/612/1237; 631/45/612/194; Animals; Binding sites; Cardiovascular diseases; Cell Line, Tumor; Cell surface; Cerebellum; Cerebellum - metabolism; Cerebrospinal fluid; Chimeras; Female; Glioblastoma; Glioblastoma cells; GTP-Binding Proteins - metabolism; HEK293 Cells; Humanities and Social Sciences; Humans; Ligands; Male; Matrix metalloproteinase; Matrix Metalloproteinases - metabolism; Metalloproteinase; Mice; Mice, Inbred C57BL; Molecular weight; multidisciplinary; N-Terminus; Nerve Growth Factors - metabolism; Neuroprotection; Peptide Hydrolases - metabolism; Peptides; Post-translation; Proteins; Proteolysis; Rats; Rats, Sprague-Dawley; Receptor mechanisms; Receptors, G-Protein-Coupled - metabolism; Science; Science (multidisciplinary); Seizures; Signal Transduction - physiology; Species; Yeasts
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-76384-9

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 Asn 105 . 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.