Biochemical and Biophysical Research Communications
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
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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