Cloning, expression and elucidation of the phosphorylation profiles of the short and long isoforms of the Omega-3 Fatty Acid Receptor 1.

摘要

The orphan G protein-coupled receptor, Omega-3 Fatty Acid Receptor-1 (0O3FAR-1), has been thrust to the forefront of diabetes research due to its ability to stimulate release of the insulin secretagogue glucagon-like peptide-1 upon stimulation by omega-3, as well as other long chained fatty acids. Interestingly, the receptor has both a long and short splice variant with O3FAR1-Long containing additional amino acids located in the third intracellular loop (ICL3) domain. ICL3 is an integral portion of GPCRs responsible for G-protein coupling and desensitization, a process which renders the receptor functionally incompetent. Furthermore, several putative phosphorylation sites are located in the added amino acids which could facilitate varied desensitization between the two splice variants as phosphorylation can initiate desensitization. Therefore, we aimed to elucidate differences in phosphorylation of O3FAR1-Long and -Short with respect to agonist-induced phosphorylation (homologous) and agonist-independent phosphorylation (heterologous). We cloned and expressed both splice variants of the receptor in vitro and developed an immunoprecipitation protocol allowing the assessment of phosphorylation using radioactive phosphate through a metabolic labeling assay. Comparing agonist-induced phosphorylation of O3FAR1-Long and -Short revealed no significant differences in phosphorylation following agonism by omega-3 fatty acids DHA and ALA. However, it was noted that basal (heterologous) phosphorylation was significantly higher for O3FAR1-Short when compared to O3FAR1-Long evoking the question of whether O3FAR1-Short and -Long utilized different kinases. To determine this, we used small interference RNA against isoforms 2, 3, 5 and 6 of the G-protein receptor kinases (GRK), which are associated with agonist-induced phosphorylation, and pharmacologic inducers and inhibitors along with co-transfection experiments to assess involvement of protein kinase A and C (PKA and PKC), which are involved in heterologous phosphorylation. Results from these experiments indicated involvement of GRK 6 and PKC with PKC having a stronger effect on O3FAR1-Long phosphorylation. We then used a computational approach to predict potential amino acid phosphorylation sites specific for GRK and/or PKC. Following mutation of identified amino acids on each receptor variant, phosphorylation was tested to locate GRK and PKC phosphorylation sites on both isoforms. Future studies will determine the functional effect of eliminating PKC or GRK 6 specific phosphorylation.