| Autor: |
Coleman JLJ; Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Australia.; St Vincent's Clinical School, UNSW Sydney, Darlinghurst, Australia., Ngo T; Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Australia.; St Vincent's Clinical School, UNSW Sydney, Darlinghurst, Australia., Smythe RE; Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Australia.; Department of Pharmacology, School of Medical Sciences, UNSW Sydney, Kensington, Australia., Cleave AJ; Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Australia.; Department of Pharmacology, School of Medical Sciences, UNSW Sydney, Kensington, Australia., Jones NM; Department of Pharmacology, School of Medical Sciences, UNSW Sydney, Kensington, Australia., Graham RM; Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Australia.; St Vincent's Clinical School, UNSW Sydney, Darlinghurst, Australia., Smith NJ; Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Australia. nicola.smith@unsw.edu.au.; St Vincent's Clinical School, UNSW Sydney, Darlinghurst, Australia. nicola.smith@unsw.edu.au.; Department of Pharmacology, School of Medical Sciences, UNSW Sydney, Kensington, Australia. nicola.smith@unsw.edu.au.; Orphan Receptor Laboratory, School of Medical Sciences, UNSW Sydney, Kensington, Australia. nicola.smith@unsw.edu.au. |
| Abstrakt: |
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. |
