Phosphorylation-independent beta-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 beta-arrestin and receptor internalization was completely independent of BLT1 phosphorylation. Real-time analysis of beta-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 beta-arrestin confirmed phosphorylation-independent beta-arrestin translocation and internalization of BLT1. In beta-arrestin-deficient mouse embryo fibroblasts, the BLT1 receptors failed to display endosomal localization upon stimulation. In these cells, co-expression of beta-arrestin-green fluorescent protein with BLT1-red fluorescent protein resulted in co-localization of BLT1 and beta-arrestin upon activation. Thus, receptor phosphorylation-dependent mechanisms regulate G-protein-mediated pathways; however, phosphorylation-independent mechanisms regulate beta-arrestin association and internalization of BLT1.

• Authors: Venkatakrishna R Jala, Wen-Hai Shao, Bodduluri Haribabu
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 280 Issue 6 Pg. 4880-7 (Feb 11 2005) ISSN: 0021-9258 [Print] United States
• PMID: 15561704 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Arrestins
• Epitopes
• Ligands
• Receptors, Leukotriene B4
• beta-Arrestins
• Green Fluorescent Proteins
• beta-N-Acetylhexosaminidases
• Alanine
• Calcium

Topics

• Alanine (chemistry)
• Amino Acid Sequence
• Animals
• Arrestins (metabolism)
• Binding Sites
• Biological Transport
• Calcium (metabolism)
• Cell Line, Tumor
• Cells, Cultured
• Chemotaxis
• Dose-Response Relationship, Drug
• Epitopes (chemistry)
• Fibroblasts (metabolism)
• Green Fluorescent Proteins (metabolism)
• Ligands
• Mice
• Microscopy, Fluorescence
• Microscopy, Video
• Molecular Sequence Data
• Mutation
• Phosphorylation
• Protein Transport
• Rats
• Receptors, Leukotriene B4 (metabolism)
• Time Factors
• Transfection
• beta-Arrestins
• beta-N-Acetylhexosaminidases (metabolism)