FFAs concentration response curves for increasing intracellu

FFAs concentration–response curves for increasing intracellular calcium and receptor phosphorylation showed a difference of ≈1 order of magnitude indicating a relatively large receptor reserve, which is expected for a transfected model, and that high receptor occupancy is necessary to induce receptor phosphorylation. Current ideas indicate that receptor phosphorylation is a very initial step in the GPCR desensitization of G protein-dependent signaling and the initiation of β-arrestin-dependent events (García-Sáinz et al., 2011, Vázquez-Prado et al., 2003, Violin and Lefkowitz, 2007). G protein-coupled kinases (GRKs), second messenger-activated kinases, and some receptor tyrosine kinases appear to be key players in these events (García-Sáinz et al., 2011, Vázquez-Prado et al., 2003, Violin and Lefkowitz, 2007). PKC phosphorylates many GPCRs including, among others, α1-adrenergic receptors (García-Sáinz et al., 2001, Vázquez-Prado et al., 1997, Vázquez-Prado et al., 2000), AT1 angiotensin II receptors (García-Caballero et al., 2001) and LPA1 lysophosphatidic Albendazole receptors (Avendaño-Vázquez et al., 2005). In the majority of cases, receptor phosphorylation is associated with clear attenuation of function. However, the α1A-adrenergic receptor, such as GPR120, is phosphorylated in response to phorbol esters but is only marginally affected in its function (Vázquez-Prado et al., 2000). Our present data indicate that despite the fact that GPR120 is a PKC substrate; the phosphorylation catalyzed by this kinase, did not attenuate receptor function. The absence of effect of PKC inhibitors on FFA-induced receptor phosphorylation suggests that other kinases, such as GRK2 or GRK3 (Penn et al., 2000), could be the major modulators. Consequently, it appears likely that the GPR120 phosphorylation patterns induced by FFAs and PKC activation might differ. This possibility is supported by the fact that the phosphorylation state of the receptor was greater when DHA and PMA were present than when any one of these stimulated the cells alone; a likely possibility is that among the phosphorylated residues, some might be shared but others might not be. Agonist-induced GPR120 internalization has been previously observed (Hirasawa et al., 2005, Hudson et al., 2013) and was confirmed here using confocal and TIRF microscopy added to the observation that PMA is also able to do so. The fact that DHA- and PMA-induced internalizations show partial additivity is of interest, particularly considering that a similar pattern was observed in the receptor phosphorylation experiments. To what extent these two processes are related remains to be determined; but it is possible that the phosphorylation patterns might participate in defining the receptor's internalization pathways. PKC is a heterogeneous subfamily of protein kinases classified into three groups based on their structure and cofactor regulation: conventional, novel and atypical (Newton, 2010). To obtain some insight into the PKC isozymes involved in the actions of PMA we employed the non isoform-selective inhibitor, bis-indolyl-maleimide I and the following isoform-selective PKC inhibitors: PKC α-selective, Gö 6976 (Martiny-Baron et al., 1993), isoform β-selective, hispidin (Gonindard et al., 1997), and for isoform δ, rottlerin (Gschwendt et al., 1994). Although precise definition of the PKC isoform(s) involved in GRP120 phosphorylation will require the use of a variety of molecular biological tools, the very clear effect of Gö 6976 led us to suggest that conventional PKC isoforms participate in PMA-induced GPR120 receptor phosphorylation.
Acknowledgments This research was partially supported by a Grant from CONACyT [153278]. O. B. Sánchez-Reyes and J. A. Castillo-Badillo are students of the Programa de Maestría y Doctorado en Ciencias Bioquímicas-UNAM and recipients of fellowships from CONACyT; this work constitutes a major part of O. B. Sánchez-Reyes thesis. We thank Dr. Luis Vaca for allowing us to use some of his equipment, the members of the Microscopy Unit of our Institute, Jonathan Pacheco and Aurelio Hernández-Méndez for their help and advice, and Dr. Rocío Alcántara-Hernández, Aurey Galván, Manuel Ortínez and Juan Barbosa for technical help. We also express our gratitude to Mrs. Margaret Brunner, MA, for style corrections. J.A. García-Sáinz dedicates this work to his former advisors, Drs. Victoria Chagoya and John N. Fain.