Agonism with the omega-3 fatty acids α-linolenic acid and docosahexaenoic acid mediates phosphorylation of both the short and long isoforms of the human GPR120 receptor

doi:10.1016/j.bbrc.2010.05.057

Biochemical and Biophysical Research Communications

Volume 396, Issue 4, 11 June 2010, Pages 1030-1035

https://doi.org/10.1016/j.bbrc.2010.05.057 Get rights and content

Abstract

The newly discovered G protein-coupled receptor GPR120 has recently been shown to stimulate secretion of the gut hormones glucagon-like peptide-1 and cholecystokinin upon binding of free fatty acids, thrusting it to the forefront of drug discovery efforts for treatment of type 2 diabetes as well as satiety and obesity. Although sequences for two alternative splice variants of the human GPR120 receptor have been reported, there have been no studies which directly compare the signaling of these isoforms. We have identified an additional 16 amino acid gap containing four phospho-labile serine/threonine residues which is localized to the third intracellular loop of the GPR120-long (GPR120-L) isoform. Based on this finding, we hypothesized that the agonist-stimulated phosphorylation profiles of this isoform would be distinct from that of the short isoform (GPR120-S). Using a clonal HEK293 cell model, we examined agonist-mediated phosphorylation of GPR120-S and GPR120-L with the omega-3 fatty acids α-linolenic acid (ALA) and docosahexaenoic acid (DHA). Our results show rapid phosphorylation of both isoforms following agonism by either ALA or DHA. Moreover, we show no significant difference in the degree or rate of phosphorylation of both isoforms upon agonism with either ALA or DHA, suggesting that the additional gap in the longer variant is not phosphorylated. Importantly, our results demonstrate that the shorter variant exhibits significantly more pronounced basal phosphorylation in the absence of agonist, suggesting that the additional gap in the long variant may contribute to masking of constitutive phosphorylation sites. These are the first results which demonstrate specific phosphorylation of GPR120 isoforms upon agonism by free fatty acids and the first which distinguish the phosphorylation profiles of the two GPR120 isoforms.

Introduction

GPR120 is a recently discovered G protein-coupled receptor (GPCR) that was identified as a member of the rhodopsin-like GPCRs based on database mining and phylogenetic analysis [1]. Since its discovery, GPR120 has become an attractive target for treatment of type 2 diabetes due to its ability to stimulate the release of the insulin secretagogue glucagon-like peptide-1 from secretory L-cells of the intestinal lumen [2], [3]. Stimulation of GPR120 has also been shown to elicit secretion of cholecystokinin in vivo as well as in mouse intestinal enteroendocrine cells [4], suggesting that GPR120 may have a broad role in regulating secretion of intestinal peptides.

While it has been demonstrated that GPR120 is agonized by long chained free fatty acids (FFA), which include the omega-3 fatty acids (Ω3FA), the molecular aspects involved in GPR120 signaling remain elusive. Interestingly, while the rat and mouse GPR120 genes are comprised of a single 1086 nucleotide sequence which encodes a protein of 361 amino acids, the human GPR120 gene can be alternatively spliced yielding a 361 amino acid protein (GPR120 Short, GPR120-S) and a distinct longer isoform of 377 amino acids which contains an extra exon (GPR120 Long, GPR120-L) [2], [3]. Surprisingly, a recent study using a non-human primate (Cynomolgus monkey) model revealed presence of only the shorter 361 amino acid protein which was 97.5% homologous to human GPR120-S, suggesting that GPR120-L is specific to humans [5].

In this study, we examined the sequence differences between the two human GPR120 isoforms and note an additional 16 amino acid gap encoded for in GPR120-L. Importantly, we identify this addition to be localized to the third intracellular loop, a receptor domain that is critical to both G protein-coupling as well as agonist-induced receptor phosphorylation and subsequent desensitization. Moreover, this additional gap consists of four phospho-labile serine/threonine residues, suggesting that GPR120-S and GPR120-L could have distinct agonist-stimulated phosphorylation profiles. Herein, we utilize a human embryonic kidney clonal cell model to assess whether agonism of cloned human GPR120-S and GPR120-L with the Ω3FA α-linolenic acid (ALA) and docosahexaenoic acid (DHA) would facilitate receptor phosphorylation. We also examined the phosphorylation profiles of both human GPR120 isoforms in response to each Ω3FA to determine if the additional gap in GPR120-L contributes to receptor phosphorylation.

Section snippets

Cloning and FLAG-epitope tagging of human GPR120

Cloning of human GPR120-S began with reverse transcription of human colon total RNA (Applied Biosystems, Austin, TX) to complimentary DNA (cDNA) using Powerscript reverse transcriptase (Takara, Mountain View, CA) and random primers. Following their synthesis, cDNA templates were amplified for 30 cycles by polymerase chain reaction (PCR) with primers corresponding to the 5′- and 3′- ends of the published human GPR120-S sequence (NCBI ID: BC101175). The A-overhang containing amplified band

GPR120 isoform topology

Two gene sequences for the human GPR120 receptor have been identified [2], [4], [5] and are distinguished by an additional 48 nucleotides in the longer sequence, which encodes for an added 16 amino acids. We examined the structural topology of the two GPR120 isoform proteins using TMpred, which predicts membrane-spanning regions and their orientations [6]. Results of this analysis demonstrated that the strongly preferred model for both sequences corresponded to the typical topology of a GPCR,

Discussion

A common dogma of GPCR function is that receptor signaling is rapidly abrogated via phosphorylation and subsequent functional uncoupling or internalization of the receptor [12]. In this study, we wished to first examine if the two GPR120 isoforms would be phosphorylated upon agonism by FFA, and our results shown here are the first to report that agonist stimulation facilitates GPR120 phosphorylation in a rapid and transient manner. In our analysis of the differences between the two human GPR120

Acknowledgments

This study was funded by a Grant (to N.H.M.) from the Diabetes Action Research and Education Foundation. Mrs. Burns is supported by a predoctoral fellowship from the American Foundation for Pharmaceutical Education. We thank Mrs. Vivienne Oder for her excellent secretarial assistance.

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Citation Excerpt :
The primary structural distinction between GPR120S and GPR120L is that GPR120L has 16 additional amino acid gaps containing four phospho-labile serine/threonine residues in the third intracellular loop [20]. This could partly explain the reported signaling differences, such as the phosphorylation/β-arrestin interactions between the two FFAR4 isoforms [21]. Early studies showed that Agonism of FFAR4-S in HEK-293 cells could signal via both Gαq/11 and β-arrestin-2 cascades, while that of FFAR4-L was incapable of promoting Ca2+−dependent signals via Gαq/11, yet could signal fully via β-arrestin [22].
Fatty acid metabolism contributes to energy supply and plays an important role in regulating immunity. Free fatty acids (FFAs) bind to free fatty acid receptors (FFARs) on the cell surface and mediate effects through the intra-cellular FFAR signaling pathways. FFAR4, also known as G-protein coupled receptor 120 (GPR120), has been identified as the primary receptor of omega-3 polyunsaturated fatty acids (ω-3 PUFAs). FFAR4 is a promising target for treating metabolic and inflammatory disorders due to its immune regulatory functions and the discovery of highly selective and efficient agonists. This review summarizes the reported immune regulatory functions of ω-3 PUFAs and FFAR4 in immune cells and immune-related diseases. We also speculate possible involvements of ω-3 PUFAs and FFAR4 in other types of inflammatory disorders.
Structure based prediction of a novel GPR120 antagonist based on pharmacophore screening and molecular dynamics simulations
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The G-protein coupled receptor, GPR120, has ubiquitous expression and multifaceted roles in modulating metabolic and anti-inflammatory processes. Recent implications of its role in cancer progression have presented GPR120 as an attractive oncogenic drug target. GPR120 gene knockdown in breast cancer studies revealed a role of GPR120-induced chemoresistance in epirubicin and cisplatin-induced DNA damage in tumour cells. Higher expression and activation levels of GPR120 is also reported to promote tumour angiogenesis and cell migration in colorectal cancer. Some agonists targeting GPR120 have been reported, such as TUG891 and Compound39, but to date development of small-molecule inhibitors of GPR120 is limited.
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Agonism with the omega-3 fatty acids α-linolenic acid and docosahexaenoic acid mediates phosphorylation of both the short and long isoforms of the human GPR120 receptor

Abstract

Introduction

Section snippets

Cloning and FLAG-epitope tagging of human GPR120

GPR120 isoform topology

Discussion

Acknowledgments

FEBS Lett.

Comp. Biochem. Physiol. B, Biochem. Mol. Biol.

Biochem. Pharmacol.

Trends Pharmacol. Sci.

J. Biol. Chem.

J. Biol. Chem.

Trends Endocrinol. Metab.

Prog. Neurobiol.

Eur. J. Pharmacol.