Phosphorylation and Signaling of the Long Splice Isoform of Free-fatty Acid Receptor 4 (FFA4)
PHOSPHORYLATION AND SIGNALING OF THE LONG SPLICE ISOFORM OF FREE FATTY-ACID RECEPTOR 4 (FFA4) Under the direction of Dr. Nader H. Moniri ABSTRACT The G protein-coupled receptor Free-fatty Acid Receptor 4 (FFA4) is expressed ubiquitously throughout the human body where it plays role in modulation of endocrine, inflammatory, and metabolic processes. FFA4 has become a very attractive drug target because it has shown great promise in alleviating systemic inflammation as well as managing malfunctions in diseases resulting from inflammation such as obesity and type 2 diabetes. Humans contain a unique splice variant of FFA4 which has not been found in any other mammals investigated. This variant contains an additional 16 amino acid insert and thereby divides the receptor into FFA4-Short (FFA4-S) and FFA4-Long (FFA4-L) isoforms. Although FFA4-S is found throughout the entire body, FFA4-L has only been found in colorectal tissue, a site where FFA4 expression and signaling has been strongly linked to tumorigenesis. More interestingly, although FFA4-L reacts to the same ligands as FFA4-S, FFA4-L has been demonstrated to possess an intrinsic bias towards β-arrestin signaling rather than coupling to G proteins. Due to the fact that there are currently many efforts to develop therapeutic agonists for FFA4 systemically, it is important to decipher signaling differences between the two receptors to help shed light on what outcomes may result from systemic agonist of both isoforms. Given that β-arrestin signaling is dependent on receptor phosphorylation, the first objective of this study was to determine whether a difference existed in the kinases which recognize and phosphorylate FFA4-S and FFA4-L, or whether their individual residues composing the “barcode” of phosphorylation were different. Using siRNA-mediated knockdown, PCR-driven mutagenesis, pharmacological inducers, inhibitors, and autoradiography, we demonstrate that GRK6 mediates homologous phosphorylation of both isoforms, while PKC mediates heterologous phosphorylation of both isoforms. However, their individual sites of phosphorylation proved to be different, although all sites were localized to the same region of their C-terminal tails. The second objective was to see whether the additional 16 amino acid insert differentiating FFA4-L from FFA4-S could rescue β-arrestin signaling in the absence of the C-terminal phosphosensor. Using Bioluminescence Resonance Energy Transfer (BRET) measuring direct β-arrestin recruitment to the receptors in real time, in live cells, we demonstrate that the loss of the C-terminal phosphosensor abolishes β-arrestin signaling in both FFA4-S and FFA4-L The final objective was to determine whether downstream signaling differences could be detected as a result of differential phosphosensors between FFA4-S and FFA4-L. We demonstrate via time-response immunoblotting that ERK1/2 activation via FFA4-L is exclusively mediated through β-arrestin and is sustained over 60 minutes, while ERK1/2 activation through FFA4-S is mediated through both Gαq/11 and β-arrestin resulting in a transient signal, confirming a major functional signaling difference between the isoforms.
Senatorov, Ilya S