Investigating Phosphorylation Differences in FFA4 Short and Long Isoforms

Abstract

The G protein‐coupled receptor Free Fatty Acid Receptor 4 (FFA4) is an attractive drug target due to its profound influence on a variety of endocrine, inflammatory, and metabolic processes. Humans contain a unique long splice variant of FFA4, which contains an additional 16 amino acid insert within the 3rd intracellular loop, and has not been found to be expressed in any other mammals. Although the shorter FFA4 isoform (FFA4‐S) is ubiquitously expressed, human FFA4‐L has only been found in colorectal tissue. More interestingly, although both isoforms are agonized by the same ligands, upon agonism, FFA4‐L has been demonstrated to possess an intrinsic bias towards β‐arrestin signaling rather than coupling to G proteins. Since it is known that receptor phosphorylation is required for interaction with β‐arrestin, the purpose of the current study was to determine whether any differences exist between the long and short isoform of FFA4 in terms of basal, homologous, or heterologous phosphorylation.MethodsWhole cell phosphorylation assays using 32P‐labeled orthophosphate were performed in HEK293 cells transiently expressing FFA4 isoforms or mutants. Inquiries into G‐protein receptor kinase (GRK) activity were performed using small interfering RNA (siRNA) followed by whole cell phosphorylation assay. Inquiries into protein kinase A (PKA) or protein kinase C (PKC) activity utilized the adenylyl cyclase activator forskolin to induce PKA activity, PKA inhibitor H‐89, PKC activator phorbol‐myristol acetate (PMA), and PKC inhibitor BIMII. Arrestin recruitment assays were conducted using yellow fluorescence protein (YFP) tagged arrestin‐3 and visualized via confocal microscopy. Truncated mutants were generated by sequential deletions via overlap primers and PCR mutagenesis.ResultsSimilar to our previously reported results with FFA4‐S, homologous phosphorylation of FFA4‐L is mediated by GRK6, whereas heterologous phosphorylation is mediated by PKC. FFA4‐L has lower basal phosphorylation compared to FFA4‐S. Truncation of FFA4‐L at S356 fully inhibits receptor phosphorylation and abrogates arrestin recruitment, matching reported observations for FFA4‐S at corresponding S340.ConclusionThe same kinases are responsible for homologous and heterologous phosphorylation of FFA4‐S and FFA4‐L, while basal phosphorylation of FFA4‐L remains lower compared to FFA4‐S. The arrestin sensor for both isoforms is located on the C‐terminal tail of the receptor following S340 (FFA4‐S) or S356 (FFA4‐L).Support or Funding InformationNIH/NIDDK DK098730 to NHM and Diabetes Action Research and Education Foundation grants to NHMThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.