5-hydroxytryptamine 4(a) receptor expressed in Sf9 cells is palmitoylated in an agonist-dependent manner

The mouse 5-hydroxytryptamine 4(a) receptor [5-HT4(a)] was expressed with a baculovirus system in insect cells and analysed for acylation. [H-3]Palmit ic acid was effectively incorporated into 5-HT4(a) and label was sensitive to the treatment with reducing agents indicating a thioester-type bond. Ana lysis of protein-bound fatty acids revealed that 5-HT4(a) contains predomin antly palmitic acid. Treatment of infected Sf9 (Spodoptera frugiperda) cell s with BIMU8 {(endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dehydro-2-o xo-3-(prop-2-yl)- 1H-benzimid-azole-1-carboxamide), a 5-HT4 receptor-select ive agonist, generated a dose-dependent increase in [H-3]palmitate incorpor ation into 5HT(4(a)) with an EC50 of approx. 10 nM. The change in receptor labelling after stimulation with agonist was receptor-specific and did not result from general metabolic effects.. We also used both pulse labelling a nd pulse-chase labelling to address the dynamics of 5-HT4(a) palmitoylation . Incorporation studies revealed that the rate of palmitate incorporation w as increased approx. 3-fold after stimulation with agonist. Results of puls e-chase experiments show that activation with BIMU8 promoted the release of radiolabel from 5-HT4(a), thereby reducing the levels of receptor-bound pa lmitate to approximately one-half. Taken together. our results demonstrate that palmitoylation of 5-HT4(a) is a reversible process and that stimulatio n of 5-HT4(a) with agonist increases the turnover rate for receptor-bound p almitate. This provides evidence for a regulated cycling of receptor-bound palmitate and suggests a functional role for palmitoylation/depalmitoylatio n in 5-hydroxytryptamine-mediated signalling.