Constitutive Endocytosis of the Chemokine CX3CL1 Prevents Its Degradation by Cell Surface Metalloproteases

Yi-Wei Huang,Paul Su,Guang Ying Liu,Min Rui Crow,Deanna Chaukos,Harry Yan,Lisa A Robinson

doi:10.1074/jbc.m109.045682

Abstract

CX(3)CL1, a chemokine with transmembrane and soluble species, plays a key role in inflammation by acting as both chemoattractant and adhesion molecule. CX(3)CL1 is the only chemokine known to undergo constitutive internalization, raising the possibility that dynamic equilibrium between the endocytic compartment and the plasma membrane critically regulates the availability and processing of CX(3)CL1 at the cell surface. We therefore investigated how transmembrane CX(3)CL1 is internalized. Inhibition of dynamin using a nonfunctional allele or of clathrin using specific small interfering RNA prevented endocytosis of the chemokine in CX(3)CL1-expressing human ECV-304 cells. Perusal of the cytoplasmic domain of CX(3)CL1 revealed two putative adaptor protein-2 (AP-2)-binding motifs. Accordingly, CX(3)CL1 co-localized with AP-2 at the plasma membrane. We generated a mutant allele of CX(3)CL1 lacking the cytoplasmic tail. Deletion of the cytosolic tail precluded internalization of the chemokine. We used site-directed mutagenesis to disrupt AP-2-binding motifs, singly or in combination, which resulted in diminished internalization of CX(3)CL1. Although CX(3)CL1 was present in both superficial and endomembrane compartments, ADAM10 (a disintegrin and metalloprotease 10) and tumor necrosis factor-converting enzyme, the two metalloproteases that cleave CX(3)CL1, localized predominantly to the plasmalemma. Inhibition of endocytosis using the dynamin inhibitor, Dynasore, promoted rapid metalloprotease-dependent shedding of CX(3)CL1 from the cell surface into the surrounding medium. These findings indicate that the cytoplasmic tail of CX(3)CL1 facilitates its constitutive clathrin-mediated endocytosis. Such regulation enables intracellular storage of a sizable pool of presynthesized CX(3)CL1 that protects the chemokine from degradation by metalloproteases at the plasma membrane.

Highlights

Among the 40 chemokines identified so far, CX3CL1 is one of only two that have a transmembrane structure [1, 2]
We recently demonstrated that cell surface CX3CL1 rapidly recycles to and from a specialized endocytic compartment, raising the possibility that the intracellular pool serves as a storage depot and that dynamic equilibrium between the endocytic compartment and the plasma membrane determines the availability and processing of transmembrane CX3CL1 [19]
Our data suggest that plasmalemmal CX3CL1 undergoes constitutive clathrin-mediated endocytosis (CME), facilitating storage of an intracellular pool of chemokine that is protected from cell surface metalloproteases

Summary

Introduction

Among the 40 chemokines identified so far, CX3CL1 is one of only two that have a transmembrane structure [1, 2]. CX3CL1 possesses a cytoplasmic tail 37 amino acids in length. Cell surface expression of CX3CL1 is known to be regulated by proteolytic cleavage, or shedding, from the plasma membrane (16 –18). Constitutive cleavage of CX3CL1 occurs at low levels and is mediated by ADAM10 (a disintegrin and metalloprotease 10) [17]. In addition to proteolytic cleavage, surface expression of CX3CL1 is regulated by subcellular trafficking. We explored whether the unique cytoplasmic tail of CX3CL1 is important for this novel mode of regulation of the chemokine. Endocytosis of CX3CL1 and whether it affects susceptibility of the chemokine to surface proteases. Our data suggest that plasmalemmal CX3CL1 undergoes constitutive clathrin-mediated endocytosis (CME), facilitating storage of an intracellular pool of chemokine that is protected from cell surface metalloproteases

Objectives

Results

Conclusion