Background
Leukotriene D(4) (LTD(4)) belongs to the bioactive lipid group known as eicosanoids and has implications in pathological processes such as inflammation and cancer. Leukotriene D(4) exerts its effects mainly through two different G-protein-coupled receptors, CysLT(1) and CysLT(2). The high affinity LTD(4) receptor CysLT(1)R exhibits tumor-promoting properties by triggering cell proliferation, survival, and migration in intestinal epithelial cells. In addition, increased expression and nuclear localization of CysLT(1)R correlates with a poorer prognosis for patients with colon cancer. Methodology/principal findings: Using a proximity ligation assay and immunoprecipitation, this study showed that endogenous CysLT(1)R formed heterodimers with its counter-receptor CysLT(2)R under basal conditions and that LTD(4) triggers reduced dimerization of CysLTRs in intestinal epithelial cells. This effect was dependent upon a parallel LTD(4)-induced increase in CysLT(1)R tyrosine phosphorylation. Leukotriene D(4) also led to elevated internalization of CysLT(1)Rs from the plasma membrane and a simultaneous increase at the nucleus. Using sucrose, a clathrin endocytic inhibitor, dominant-negative constructs, and siRNA against arrestin-3, we suggest that a clathrin-, arrestin-3, and Rab-5-dependent process mediated the internalization of CysLT(1)R. Altering the CysLT(1)R internalization process at either the clathrin or the arrestin-3 stage led to disruption of LTD(4)-induced Erk1/2 activation and up-regulation of COX-2 mRNA levels. Conclusions/significance: Our data suggests that upon ligand activation, CysLT(1)R is tyrosine-phosphorylated and released from heterodimers with CysLT(2)R and, subsequently, internalizes from the plasma membrane to the nuclear membrane in a clathrin-, arrestin-3-, and Rab-5-dependent manner, thus, enabling Erk1/2 signaling and downstream transcription of the COX-2 gene.
Significance
Our data suggests that upon ligand activation, CysLT(1)R is tyrosine-phosphorylated and released from heterodimers with CysLT(2)R and, subsequently, internalizes from the plasma membrane to the nuclear membrane in a clathrin-, arrestin-3-, and Rab-5-dependent manner, thus, enabling Erk1/2 signaling and downstream transcription of the COX-2 gene.