Palmitoylation of the TPβ isoform of the human thromboxane A(2) receptor.: Modulation of G protein: Effector coupling and modes of receptor internalization

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) A(2) 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 Cys(347) and, to a lesser extent, at Cys(373,377) whereas TPα is not palmitoylated. Impairment of palmitoylation did not affect TPβ expression or its ligand affinity. Conversely, agonist-induced [Ca(2+)](i) mobilization by TPβ(C347S) and the non-palmitoylated TPβ(C347,373,377S), but not by TPβ(C373S) or TPβ(C373,377S), was significantly reduced relative to the wild type TPβ suggesting that palmitoylation at Cys(347) is specifically required for efficient Gq/phospholipase Cβ effector coupling. Furthermore, palmitoylation at Cys(373,377) is critical for TPβ internalization with TPβ(C373S), TPβ(C373,377S) and TPβ(C347,373,377S) failing to undergo either agonist-induced or temperature-dependent tonic internalization. On the other hand, whilst TPβ(C347S) underwent reduced agonist-induced internalization, it underwent tonic internalization to a similar extent as TPβ. The deficiency in agonist-induced internalization by TPβ(C347S), but not by TPβ(C373,377) nor TPβ(C347,373,377S), was overcome by over-expression of either β-arrestin1 or β-arrestin2. Taken together, data herein suggest that whilst palmitoylation of TPβ at Cys(373,377) is critical for both agonist- and tonic-induced internalization, palmitoylation at Cys(347) 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.