Chemokine signaling specificity: essential role for the N-terminal domain of chemokine receptors

Abstract

Chemokine IL-8 (CXCL8) binds to its cognate receptors CXCR1 and CXCR2 to induce inflammatory responses, wound healing, tumorogenesis, and neuronal survival. Here we identify the N-loop residues in IL-8 (H18 and F21) and the receptor N-termini as the major structural determinants regulating the rate of receptor internalization, which in turn controlled the activation profile of ERK1/2, a central component of the receptor/ERK signaling pathway that dictates signal specificity. Our data further support the idea that the chemokine receptor core acts as a plastic scaffold. Thus, the diversity and intensity of inflammatory and noninflammatory responses mediated by chemokine receptors appear to be primarily determined by the initial interaction between the receptor N-terminus and the N-loop of chemokines.

FIGURE 1

ELR chemokines induce internalization of CXCR2. COS-7 cells expressing CXCR2 were treated with saturating concentrations (100 nM) of IL-8 (●), MGSA (▼), or GCP-2 (O) at 37 °C for various amounts of time. After an acid wash, the surface receptors were assessed by [¹²⁵I]IL-8 binding at 4 °C (25). Sequence alignments of IL-8, MGSA, and GCP-2 are shown with their corresponding secondary structure domains; the thick red line identifies the conserved ELR triad, and the thick blue line identifies the divergent N-loop sequences.

FIGURE 2

N-Loop IL-8 mutants fail to trigger internalization of CXCR1 and CXCR2. COS-7 cells expressing CXCR1 (a) or CXCR2 (b) were treated with saturating concentrations (100 nM) of IL-8 (●), H18A (○), F21A (▼), or H18A/F21A (▽) at 37 °C for different amounts of time. After an acid wash, the surface IL-8 receptors were assessed by [¹²⁵I]IL-8 binding (25).

FIGURE 3

IL-8 and the ELR mutant (R6A) induce internalization of CXCR2. COS-7 cells expressing CXCR2 were treated with 100 nM IL-8 or 1 µM R6A for different amounts of time. After an acid wash, the cell surface receptors were assessed by [¹²⁵I]IL-8 binding (25).

FIGURE 4

Receptor N-termini determine the rate of receptor internalization. COS-7 cells expressing CXCR1 (●), CXCR2 (○), N1R2 (▼), and N2R1 (△) were treated with 100 nM IL-8 for various amounts of time. After an acid wash, the cell surface receptors were assessed by [¹²⁵I]IL-8 binding (25).

FIGURE 5

Receptor N-termini determine the activation profile of ERK1/2. COS-7 cells transfected with CXCR1, CXCR2, N1R2, or N2R1 were treated with 100 nM IL-8 for different amounts of time. The phosphorylated ERK1/2 was analyzed by Western blot analysis (a). The levels of phospho-ERK1/2 relative to the total ERK were plotted (b). Data are representative of three independent experiments.

FIGURE 6

Hypothetical model of binding of IL-8 to its cognate receptor. The putative interacting surfaces of the receptor and IL-8 (sites 1 and 2) are encircled. Site 1 represents the interaction of the N-terminus of the receptor with the N-loop of IL-8, whereas site 2 represents the interaction of the extracellular loops of the receptor with the ELR triad of IL-8.