Ricks Tiffany K, Trejo JoAnn
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7365, USA.
J Biol Chem. 2009 Dec 4;284(49):34444-57. doi: 10.1074/jbc.M109.048942. Epub 2009 Oct 8.
Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor irreversibly activated by extracellular proteases. Activated PAR2 couples to multiple heterotrimeric G-protein subtypes including G alpha(q), G alpha(i), and G alpha(12/13). Most activated G protein-coupled receptors are rapidly desensitized and internalized following phosphorylation and beta-arrestin binding. However, the role of phosphorylation in regulation of PAR2 signaling and trafficking is not known. To investigate the function of phosphorylation, we generated a PAR2 mutant in which all serines and threonines in the C-tail were converted to alanines and designated it PAR2 0P. In mammalian cells, the addition of agonist induced a rapid and robust increase in phosphorylation of wild-type PAR2 but not the 0P mutant, suggesting that the major sites of phosphorylation occur within the C-tail domain. Moreover, desensitization of PAR2 0P signaling was markedly impaired compared with the wild-type receptor. Wild-type phosphorylated PAR2 internalized through a canonical dynamin, clathrin- and beta-arrestin-dependent pathway. Strikingly, PAR2 0P mutant internalization proceeded through a dynamin-dependent but clathrin- and beta-arrestin-independent pathway in both a constitutive and agonist-dependent manner. Collectively, our studies show that PAR2 phosphorylation is essential for beta-arrestin binding and uncoupling from heterotrimeric G-protein signaling and that the presence of serine and threonine residues in the PAR2 C-tail hinder constitutive internalization through a non-canonical pathway. Thus, our studies reveal a novel function for phosphorylation that differentially regulates PAR2 desensitization and endocytic trafficking.
蛋白酶激活受体2（PAR2）是一种G蛋白偶联受体，可被细胞外蛋白酶不可逆地激活。激活的PAR2与多种异源三聚体G蛋白亚型偶联，包括Gα(q)、Gα(i)和Gα(12/13)。大多数激活的G蛋白偶联受体在磷酸化和β-抑制蛋白结合后会迅速脱敏并内化。然而，磷酸化在PAR2信号传导和转运调节中的作用尚不清楚。为了研究磷酸化的功能，我们构建了一种PAR2突变体，其中C末端的所有丝氨酸和苏氨酸都被转化为丙氨酸，并将其命名为PAR2 0P。在哺乳动物细胞中，添加激动剂可导致野生型PAR2的磷酸化迅速而强烈地增加，但0P突变体则不会，这表明磷酸化的主要位点位于C末端结构域内。此外，与野生型受体相比，PAR2 0P信号的脱敏明显受损。野生型磷酸化的PAR2通过经典的发动蛋白、网格蛋白和β-抑制蛋白依赖性途径内化。引人注目的是，PAR2 0P突变体的内化以组成型和激动剂依赖性方式通过发动蛋白依赖性但网格蛋白和β-抑制蛋白非依赖性途径进行。总体而言，我们的研究表明，PAR2磷酸化对于β-抑制蛋白结合以及与异源三聚体G蛋白信号解偶联至关重要，并且PAR2 C末端的丝氨酸和苏氨酸残基的存在阻碍了通过非经典途径的组成型内化。因此，我们的研究揭示了磷酸化的一种新功能，该功能差异调节PAR2脱敏和内吞运输。
