Abstract 13463: N-Terminal Processing of an Orphan GPCR: A Novel Mechanistic Target for the Treatment of Cardiovascular Disease?
GPR37L1 is an orphan G protein-coupled receptor found in the heart. Recently, GPR37L1 knock-out mice were reported to develop profoundly elevated blood pressure and concomitant left ventricular hypertrophy. We therefore hypothesised that GPR37L1 is a negative regulator of blood pressure and now report that GPR37L1 levels are significantly down-regulated in a large cohort of human heart failure patients when compared to donor hearts. In addition, GPR37L1 was also down-regulated in the left ventricles of mice subjected to either thoracic-aortic constriction, angiotensin II infusion or transgenic over-expression of a mutant form of myosin heavy chain. Given the potential therapeutic relevance of GPR37L1, we sought to gain insight into the pharmacology of GPR37L1 by generating stable, doxycycline-inducible, FlpIN T-REx HEK293 cells expressing human GPR37L1 tagged with C-terminal enhanced Yellow Fluorescent Protein (GPR37L1-eYFP). Confocal microscopy and cell-surface biotinylation experiments showed wild type (WT) GPR37L1-eYFP reached the cell surface but resided predominantly in intracellular vesicles, suggestive of constitutive activation and internalisation. Interestingly, Western blot analysis revealed that GPR37L1-eYFP is cleaved at the N-terminus in a manner entirely blocked by broad-spectrum matrix metalloprotease/A Disintegrin and Metalloprotease inhibitors. The N-terminally processed fragment of WT GPR37L1-eYFP migrated at the same apparent molecular weight as a truncated version of GPR37L1-eYFP lacking the first 122 amino acids and was also insensitive to enzymatic deglycosylation, allowing us to identify a region of 18 amino acids within which N-terminal cleavage must occur. Using a yeast G protein coupling assay, we discovered the specific signalling pathways activated by GPR37L1 and found that signalling is altered by N-terminal cleavage. Finally, we find no evidence for the recently described ligand prosaptide as an agonist at GPR37L1. Given that GPR37L1 is down-regulated in cardiovascular diseases known to also be associated with elevated matrix metalloprotease levels, these findings have important implications for the development of an entirely new class of anti-hypertensive therapies.
- © 2013 by American Heart Association, Inc.