Palmitoylation of the TPβ isoform of the human thromboxane A2 receptor.

Palmitoylation is a prevalent feature amongst G protein-coupled receptors. In this study we sought to establish whether the TPα and TPβ isoforms of the human prostanoid thromboxane (TX) A2 receptor (TP) are palmitoylated and to assess the functional consequences thereof. Consistent with the presence of three cysteines within its unique carboxyl-terminal domain, metabolic labelling and site-directed mutagenesis confirmed that TPβ is palmitoylated at Cys347 and, to a lesser extent, at Cys373,377 whereas TPα is not palmitoylated. Impairment of palmitoylation did not affect TPβ expression or its ligand affinity. Conversely, agonist-induced [Ca2+]i mobilization by TPβC347S and the non-palmitoylated TPβC347,373,377S, but not by TPβC373S or TPβC373,377S, was significantly reduced relative to the wild type TPβ suggesting that palmitoylation at Cys347 is specifically required for efficient Gq/phospholipase Cβ effector coupling. Furthermore, palmitoylation at Cys373,377 is critical for TPβ internalization with TPβC373S, TPβC373,377S and TPβC347,373,377S failing to undergo either agonist-induced or temperature-dependent tonic internalization. On the other hand, whilst TPβC347S underwent reduced agonist-induced internalization, it underwent tonic internalization to a similar extent as TPβ. The deficiency in agonist-induced internalization by TPβC347S, but not by TPβC373,377 nor TPβC347,373,377S, was overcome by over-expression of either β-arrestin1 or β-arrestin2. Taken together, data herein suggest that whilst palmitoylation of TPβ at Cys373,377 is critical for both agonist- and tonic-induced internalization, palmitoylation at Cys347 has a role in determining which pathway is followed, be it by the β-arrestin-dependent agonist-induced pathway or by the β-arrestin-independent tonic internalization pathway.