Abstract
Sphingosine 1-phosphate (S1P) is a pleiotropic lipid mediator involved in the regulation of immune cell trafficking and vascular permeability acting mainly through G-protein-coupled S1P receptors (S1PRs). However, mechanism underlying how S1PRs are coupled with G-proteins remains unknown. Here we have uncovered that palmitoylation of a prototypical subtype S1P1R is prerequisite for subsequent inhibitory G-protein (Gi) coupling. We have identified DHHC5 as an enzyme for palmitoylation of S1P1R. Under basal conditions, S1P1R was functionally associated with DHHC5 in the plasma membranes (PM) and was fully palmitoylated, enabling Gi coupling. Upon stimulation, the receptor underwent internalisation leaving DHHC5 in PM, resulting in depalmitoylation of S1P1R. We also revealed that while physiological agonist S1P-induced endocytosed S1P1R readily recycled back to PM, pharmacological FTY720-P-induced endocytosed S1P1R-positive vesicles became associated with DHHC5 in the later phase, persistently transmitting Gi signals there. This indicates that FTY720-P switches off the S1P signal in PM, while switching on its signal continuously inside the cells. We propose that DHHC5-mediated palmitoylation of S1P1R determines Gi coupling and its signalling in a spatio/temporal manner.
Highlights
Protein palmitoylation is the attachment of the 16-carbon saturated fatty acid palmitate to cysteine residues on proteins through thioester linkages, and plays a role in the regulation of localisation, trafficking and function of a variety of proteins including GPCRs2
Since S1P1R is localised predominantly at plasma membranes (PM) in SH-SY5Y cells as previously reported[21] we reasoned that DHHC5, DHHC20 and DHHC21, which are known to show plasma membrane localisation[22], may be potential candidates for the enzymes for S1P1R
Knockdown of DHHC20 or DHHC21 had little effect on the palmitoylation of the receptor (Fig. 1a, DHHC20-siRNA, DHHC21-siRNA). These results suggest that DHHC5 is a potential palmitoyl transferase for S1P1R
Summary
Protein palmitoylation is the attachment of the 16-carbon saturated fatty acid palmitate to cysteine residues on proteins through thioester linkages, and plays a role in the regulation of localisation, trafficking and function of a variety of proteins including GPCRs2. Akr[1] was shown to be an enzyme for S-palmitoylation of the yeast casein kinase Yck[211]. These initial studies in yeast have lead to the discovery of a family of 23 mammalian DHHC (Asp-His-His-Cys) proteins as S-acyltransferases[12]. It has previously been shown that S1P1R is palmitoylated at three Cys residues in the cytoplasmic tail of the receptor[20]. In the present studies we have identified DHHC5 as an enzyme for palmitoylation of S1P1R. Physiological relevance of the association of DHHC5 with the S1P1R during vesicular trafficking is discussed
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