Phosphorylation-independent β-arrestin translocation and internalization of leukotriene B4 receptors

Abstract

Leukotriene B4 (LTB4) activates the G-protein-coupled receptor leukotriene B4 receptor 1 (BLT1) to mediate a diverse array of cellular responses in leukocytes including chemotaxis, calcium mobilization, degranulation, and gene expression. To determine the role of phosphorylation in BLT1 regulation, we generated mutants of BLT1 in which all of the serine/threonine residues in the C-tail are converted to alanine or to aspartate/glutamate. These mutants expressed in rat basophilic leukemia RBL-2H3 cells bound LTB4 with similar affinity and activated all of the known functional activities of BLT1, albeit at different levels. The conversion of phosphorylation sites to alanine resulted in enhanced G-protein-mediated activities, whereas conversion to aspartate/glutamate resulted in reduced responses and a right shift in dose response, indicating that receptor phosphorylation is a critical regulator of G-protein-mediated pathways. Surprisingly, translocation of β-arrestin and receptor internalization was completely independent of BLT1 phosphorylation. Real-time analysis of β-arrestin translocation and receptor internalization using digital fluorescence video microscopy in cells expressing a red fluorescent protein labeled BLT1 and a green fluorescent protein-tagged β-arrestin confirmed phosphorylation-inclependent β-arrestin translocation and internalization of BLT1. In β-arrestin-deficient mouse embryo fibroblasts, the BLT1 receptors failed to display endosomal localization upon stimulation. In these cells, co-expression of β-arrestin-green fluorescent protein with BLT1-red fluorescent protein resulted in co-localization of BLT1 and β-arrestin upon activation. Thus, receptor phosphorylation-dependent mechanisms regulate G-protein-mediated pathways; however, phosphorylation-independent mechanisms regulate β-arrestin association and internalization of BLT1.