br Experimental section br Acknowledgements This study
Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (Grants 81673299 and 81273376).
Introduction Free fatty acids (FFAs) are important metabolic fuels and those that are polyunsaturated are essential components of the human diet. In addition to these metabolic roles, it is now clear that they are bioactive ligands for several types of receptors, including nuclear receptors, such as peroxisome proliferator-activated receptors, and a family of G protein-coupled receptors (GPCRs) (Chawla et al., 2001, Hirasawa et al., 2008, Stoddart et al., 2008). Four GPCRs have been deorphanized/identified as FFA receptors: GPR40, GPR41, GPR43 (Stoddart et al., 2008) and, recently, GPR120 (Hirasawa et al., 2005). GPR40 (FFA receptor 1) and GPR120 (FFA receptor 4) are considered of particular interest because they appear to be involved in physiological and pathophysiological processes related with metabolic disorders such as the so-called “metabolic syndrome” and type 2 diabetes mellitus (Hara et al., 2011). It has been observed that GPR120-deficient mice develop obesity, glucose intolerance, and fatty liver, and that a dysfunctional variant of GPR120 (R270H) is associated with obesity and other metabolic disturbances in humans (Ichimura et al., 2012). Additionally, this receptor mediates insulin-sensitizing and anti-inflammatory effects in scr 11 (adipocytes and macrophages) and in whole organisms (mice) (Oh et al., 2010). Two splice variants of GPR120 exist in humans, a long form of 361 residues and a short one (lacking 16 residues at the third intracellular loop) (Moore et al., 2009); the short receptor (but not the long one), couples to Gαq/11 and activates calcium signaling, whereas both receptors interact with β-arrestin and internalize in response to agonists (Watson et al., 2012). It has also been reported that both variants forms are phosphorylated in response to agonists (Burns and Moniri, 2010). In the present work we show that both, receptor stimulation with agonists, and activation of protein kinase C (PKC) resulted in strong receptor phosphorylation. The magnitude and time-course of these effects were similar, but showed some differences. PKC activation with phorbol esters, did not induce receptor desensitization but increased GPR120 internalization. PKC inhibitors block phorbol ester-induced GPR120 phosphorylation but not that due to agonist action; some of these data were presented in an international meeting (Sánchez-Reyes et al., 2013).
Materials and methods
Discussion Our data show that GPR120 is a phosphoprotein whose phosphorylation state is modulated by agonists: DHA and α-LA, as well as by direct activation of PKC by PMA. The time-course of GPR120 phosphorylation (long and short forms) in response to a single concentration of DHA and α-LA was previously reported by other authors employing FLAG-tagged receptors; they detected bands of ≈42kDa (Burns and Moniri, 2010). In our experiments, a major band of ≈70–75kDa was observed that corresponds to the expected mass of short variant GPR120-Venus construction (i.e., GPR120 ≈42kDa plus Venus ≈27kDa). While this paper was under review, a manuscript was published on-line that studied agonist-mediated GPR120 phosphorylation and high molecular weight bands were also observed, although no major comment was made on the finding (Hudson et al., 2013). In our opinion, the presence of higher molecular weight bands, detected by Western blotting and metabolic labeling, is of interest because it suggests the possibility that this receptor might form complexes with receptors and/or other signaling entities. Homo- and heterodimerization are certainly very attractive options, considering their potential for signaling and cell trafficking (Salahpour et al., 2000). It should be stated that our data only suggest this possibility, but obviously several different approaches would be required to confirm or discard such a suggestion.