Palmitoylation is a post-translational modification that consists in the addition of a 16 carbons fatty acid, palmitate, to a cysteine residue through the creation of a thioester link. Unlike other lipid modifications such as myristoylation and prenylation, which are irreversible, palmitoylation is a dynamic modification involving palmitoylation/depalmitoylation cycles suggesting a regulatory role. Several years ago, we and others demonstrated that many G protein-coupled receptors GPCR are palmitoylated on cysteine residues located in their carboxyl tail leading to the formation of a fourth cytoplasmic loop as a result of the insertion of the palmitic acid in the plasma membrane (see Fig 1). To date, no strict palmitoylation consensus site has been identified and the enzymatic reaction involved remain unknown. However, studies on the in vitro autocatalytic palmitoylation of synthetic peptides allowed us to suggest that the presence of basic and hydrophobic residues close to a cysteine facilitates its palmitoylation while the presence of acidic residues inhibits the modification (Belanger et al. 2001).

Fig 1
	Schematic representation of the topological arrangement of the human β2-adrenergic receptor (β2AR) illustrating the positions of the palmitoylation (green) and phosphorylation (red) sites. The residues in yellow surrounding the palmitoylated cysteine are believed to favour the palmitoylation reaction while the residue in orange has a negative influence.

When considering the dynamic nature of the palmitoylation reaction, we found that GPCR palmitoylation is affected by the activity state of the receptor (Mouillac et al. 1992), agonist stimulation leading to an increased turnover of the palmitate on the β2AR (Loisel et al. 1996 and 1999). Heterologous regulation of the receptor palmitoylation state by nitric oxide was also found (Adam et al. 1999). Changes in the palmitoylation state of the receptor were proposed as regulating the formation of the 4th cytoplamic loop and hence modulating the accessibility of regulatory phosphorylation sites located down-stream of the palmitoylated cysteine (Moffett et al. 1993, 1996 and 2001). These studies suggested that palmitoylation could be involved in the regulation of signalling efficacy of the β2AR by PKA and βARK. Consistent with a general role of palmitoylation in the regulation of GPCR signalling, we recently found that the palmitoylation state of the V2-vasopressin receptor (V2R) regulates the recruitment of barrestin to the receptor and as a result modulates the vasopressin-promoted receptor endocytosis and mitogen-activated protein kinase (MAPK) activation (Charest et al. 2003).

Fig 2
	Schematic representation of the topological arrangement of the human V2-vasopressin receptor (V2R) illustrating the positions of the two palmitoylation sites.

Fig 3
	Site directed mutagenesis study confirming that Cysteines 341 and 342 represent the major palmitoylation sites. The palmitoylation state of the receptor was monitored by immunoaffinity purification of the receptor following metabolic labelling of cells with [3H]palmitate. The incorporation of palmitate was detected by autoradiography while the receptor was

revealed by Western blot analysis taking advantage of the presence of a myc-epitope at the N-terminus of the receptor. The four immunoreactive species correspond to the monomeric and dimeric forms of the immature (core-glycosylated) and mature (fully glycosylated) state of the receptor.

Fig 4
	Altered recruitment of βarrestin by the palmitoylation-less mutant C341,342AV2R mutant. The real-time recruitment of βarrestin to the receptor was monitorred by BRET using βarrestin-Luc and V2R-GFP constructs.

Among the projects currently underway, we are trying to determine if the rules established in vitro for the autocatalytic palmitoylation reaction, namely the positive influence of basic and hydrophobic residues and the negative influence of acidic residues, also apply to the GPCR palmitoylation reaction in living cells. Also, the influence of phosphorylation on the palmitoylation of GPCR is under investigation. We are also planning to study the nature of the reaction(s) controling the palmitoylation state of GPCR and G protein receptor kinases (GRK). One of the difficulty impeding the study of palmitoylation is the lack of tools to quantitatively assess the palmitoylation state and turnover rate of the protein-bound palmitate. We are currently developing new tools to accurately determine these parameters. These tolls should allow us to better understand the molecular mechanisms controlling palmitoylation and the general role of this modification for GPCR fnctions.