Phosphorylation of the N-Formyl Peptide Receptor Is Required for Receptor Internalization but Not Chemotaxis

Matthew H Hsu,Stephanie C Chiang,Richard D Ye,Eric R Prossnitz

doi:10.1074/jbc.272.47.29426

Abstract

The human N-formyl peptide receptor (FPR) is a member of the family of leukocyte, G protein-coupled, chemoattractant receptors. To determine the role(s) of receptor phosphorylation in FPR processing and formylmethionylleucylphenylalanine (fMLF)-mediated chemotaxis, we utilized U937 cells expressing the recombinant wild type receptor and a mutant form of the FPR. This mutant, which lacks all of the serine and threonine residues in the C terminus of the receptor, DeltaST, has recently been shown to produce a receptor capable of fMLF binding and G protein activation but was demonstrated not to undergo fMLF-dependent phosphorylation or desensitization of the calcium mobilization response upon repeated exposure to agonist (Prossnitz, E. R. (1997) J. Biol. Chem. 272, 15213-15219). In this report, we examined the role of receptor phosphorylation in FPR internalization and leukocyte chemotaxis. Whereas the wild type receptor was rapidly internalized upon stimulation, the phosphorylation-deficient mutant was not, remaining entirely on the cell surface. In addition, contrary to the hypothesis that receptor processing and recycling are required for chemotaxis, we found no defect in the ability of the mutant FPR to migrate up a concentration gradient of fMLF. These results indicate that phosphorylation of the FPR is a necessary step in receptor internalization but that receptor phosphorylation, desensitization, and internalization are not required for chemotaxis.

Highlights

Neutrophils normally exist in a resting state as they circulate though the body
Our results demonstrate that receptor phosphorylation is absolutely essential to receptor desensitization and internalization, neither phosphorylation nor receptor internalization is required for cell chemotaxis
It has been well established that G protein-coupled chemoattractant receptors mediate leukocyte chemotaxis [22]

Summary

Introduction

Neutrophils normally exist in a resting state as they circulate though the body. upon interaction with small molecules known as chemoattractants, they rapidly respond with endothelial adhesion followed by emigration from the vasculature and chemotaxis to the site of inflammation [1]. Receptor Internalization—Receptor internalization was first determined as the loss of FPR from the cell surface as follows. This conclusion was based on a number of experiments, which correlated a block in the re-expression of internalized chemoattractant receptors with a lack of chemotactic ability despite normal ligand binding, cell activation of superoxide generation and degranulation, and normal receptor internalization [15, 28].

Results

Conclusion