Role of palmitoylation in the serotonin receptor functioning

We analyzed role of 5-HT1A and 5-HT4(a) receptor palmitoylation in the receptor functions. In case of 5-HT4(a) receptor we demonstrated that receptor palmitoylation affects phosphorylation. We showed that upon 5-HT stimulation, the 5-HT4(a) receptor undergoes rapid (τ1⁄2=2 min) and dose-dependent (EC50=180 nM) phosphorylation on serine residues by a staurosporine-insensitive receptor kinase. The acylation deficient (Cys328/29-Ser) mutant, which is constitutive active in the absence of ligand, exhibited enhanced receptor phosphorylation under both basal and agonist-stimulated conditions and was more effective desensitized and internalized via GRK2/β-arrestin pathway compared with the wild-type 5-HT4(a) receptor. G-protein activation, phosphorylation, desensitization and internalization of the other acylaiton deficient receptor mutants were affected differently. Analysis of dynamic interaction between receptor and s-arrestin! 2 BRET2 assay demonstrated that wild-type and all palmitoylation deficient mutants show accelerated interaction with β-arrestin2 upon agonist stimulation. Acylation deficient mutants demonstrated a higher basal level of interaction with β-arrestin2 than the wild-type, but a slower interaction kinetic (τ1⁄2=12 sec. for Cys328/29-S vs. 10 seconds for the wild-type) after stimulation with agonist. Co-expression of wild-type form of the GRK2 promotes receptor β-arrestin2 interaction, while dominant negative mutant slightly reduce this affect, suggesting that the positive effect of GRK on receptor β-arrestin2 interaction is mediated both by enzymatic activity as well as by direct interaction of GRK2 with the receptor. These findings suggest that palmitoylation plays an important role in modulating 5-HT4(a) receptor functions and that G-protein activation, phosphorylation, desensitization, and internalization of the recep! tor are regulated by this dynamic modification. In the second part of the study we addressed the question on the molecular mechanisms by which receptor palmitoylation may regulate communication between receptors and Gαi-proteins. In contrast to 5-HT4(a) receptor, which undergoes dynamic pamitoylation, palmitoylation of 5-HT1A is an irreversible modification. Our data demonstrate that activation of the 5-HT1A receptor caused an activation of Gαi3 protein. In contrast,acylation-defi-n contrast, acylation-deficient 5-HT1A mutant failed to reproduce Gαi3 activation upon agonist stimulation. By using gradient centrifugation and co-patching assays, we also demonstrated that a significant fraction of the 5-HT1A receptor resides in lipid rafts, while the yield of the palmitoylation-de-the yield of the palmitoylation-deficient receptor in these membrane microdomains is considerably reduced. These data sugest that receptor palmitoylation serves as a targeting signal responsible for the retention of the 5-HT1A recep! tor in lipid rafts, and the raft localization of the 5-HT1A receptor appears to be involved in receptor-mediated signaling.