Interleukin-1R3 mediates interleukin-1-induced potassium current increase through fast activation of Akt kinase

Abstract

Inflammatory cytokine interleukin-1 (IL-1) performs multiple functions in the central nervous system. The type 1 IL-1 receptor (IL-1R1) and the IL-1 receptor accessory protein (IL-1RAcP) form a functional IL-1 receptor complex that is thought to mediate most, if not all, IL-1-induced effects. Several recent studies, however, suggest the existence of a heretofore-unidentified receptor for IL-1. In this study, we report that the IL-1R1 gene contains an internal promoter that drives the transcription of a shortened IL-1R1 mRNA. This mRNA is the template for a unique IL-1R protein that is identical to IL-1R1 at the C terminus, but with a shorter extracellular domain at the N terminus. We have termed this molecule IL-1R3. The mRNA and protein for IL-1R3 are expressed in normal and two strains of commercially available IL-1R1 knockout mice. Western blot analysis shows IL-1R3 is preferentially expressed in neural tissues. Furthermore, IL-1β binds specifically to IL-1R3 when it is complexed with the newly discovered alternative IL-1 receptor accessory protein, IL-1RAcPb. Stimulation of neurons expressing both IL-1R3 and IL-1RAcPb with IL-1β causes fast activation of the Akt kinase, which leads to an increase in voltage-gated potassium current. These results demonstrate that IL-1R3/IL-1RAcPb complex mediates a unique subset of IL-1 activity that accounts for many previously unexplained IL-1 effects in the central nervous system.

Fig. 1.

Identification of a unique TSS in IL-1R1 gene. (A) Diagrammatic illustration of the half-nested PCR used to increase the sensitivity of 5′ RACE. The forward RACE primers were included in the CapFishing kit. The reverse primers for two rounds of PCR targeting different exons are indicated by arrows (purple, outer primers for first round of PCR; green, inner primers for second round of PCR). (B) Electrophoresis results of the nested PCR results targeting TSS downstream of exon 2. RNAs used in CapFishing were extracted from the following tissues: dentate gyrus (Dg), hypothalamus (Hy), liver (Liv), and lung (Lun). (C) Annotation of TSSs found in the context of known mIL-1R1 genomic sequence. TSSs are presented in green, purple, blue, and red to match Table 1.

Fig. 2.

IL-1R3 is detected both in vitro and in vivo. (A) Diagrammatic illustration of IL-1R1/R3 cloning into pcDNA6.2 vector. IL-1R1/R3 was inserted after CMV promoter and followed by an in-frame GFP (emerald GFP; EmGFP) protein or a Flag tag. (B) Expression of IL-1R3-Flag (R3F), IL-1R3-GFP (R3G), IL-1R1-Flag (R1F), and IL-1R1-GFP (R1G) detected by Western blot. (C) Immunofluorescence microscopy images of Neuro-2a cells transfected with C terminus GFP-tagged IL-1R1/IL-1R3. (D) Diagrammatic illustration of IL-1R1 KO designs from current IL-1R1 KO lines. The entire IL-1R3 OFR can be detected by RT-PCR in both strains of IL-1R1 KO mice (arrows denote PCR primers). (E) IL-1R3 is detected in various regions of the brain in both IL-1R1 KO and normal animal by Western blot. In contrast, IL-1R1 can only be detected in normal animals. HI, hippocampus; HY, hypothalamus; Cb, cerebellum; Br, brain; Li, Liver.

Fig. 3.

IL-1β binds IL-1R3. (A) Diagrammatic illustration of the domains of IL-1R1 and -1R3. TM, transmembrane domain; TIR, tToll/IL-1R domain. Each open box represents a specific domain with numbers indicating the number of amino acids. (B) Diagrammatic illustration of predicted IL-1R3 structure and its potential interaction with IL-1/IL-1ra in comparison with IL-1R1. IL-1R3 is predicted to interact with IL-1 because IL-1R3 retains one (Ig-like domain 3) of the two IL-1 binding sites of IL-1R1. IL-1R3 is unlikely to bind IL-1ra because it does not have the IL-1ra binding site (between the first and the second Ig-like domains of IL-1R1). (C) Detection of cell-bound His-tagged IL-1β with anti-His antibody to IL-1R1–transfected (Upper) or IL-1R3–transfected (Lower) cells. IL-1β binds both receptors specifically [compared with vector (V) group] in a concentration-dependent manner. (Upper) Lanes 1, 2, and 3 correspond to results generated from 0.5, 5, and 50 nM His-IL-1β, respectively. (Lower) Lanes 1, 2, and 3 correspond to results generated from 0, 50, and 250 nM HisIL-1β, respectively.

Fig. 4.

Role of IL-1R3 in mediating IL-1β–induced signaling. (A) IL-1β induces NF-κB activation. Fold increase in luciferase activity over the baseline obtained from cells cotransfected with NF-κB reporter plasmid and empty vector (V) are presented. The means ± SE are presented from three separate experiments. *P < 0.05. (B) Detection of IL-1β–induced phosphorylation of Akt and p38 in cells transfected with IL-1R1/IL-1R3 in combination with IL-1RAcP/IL-1RAcPb. *, indicates increased phosphor-protein. (C) Detection of IL-1β–induced phosphorylation of Akt in cells transfected with IL-1R3 in combination with IL-1RAcP/IL-1RAcPb. Quantification of blots from three different experiments is shown below the blot. Densitometric values are expressed as pAkt/total Akt and then normalized to untreated cells (0 min). *P < 0.05. (D) Time course of IL-1β–induced Akt phosphorylation in cells cotransfected with IL-1R3 and IL-1RAcPb.

Fig. 5.

IL-1β induces Kv current increases in cells cotransfected with IL-1R3/IL-1RAcPb. (A) Actual traces of Kv currents in an IL-1R3/IL-1RAcPb–transfected cell. (B) Representative Kv current amplitude increase after IL-1β stimulation in IL-1R3/IL-1RAcPb–cotransfected cells. (C) Representative null response after IL-1β stimulation in cells cotransfected with IL-1R1/IL-1RAcP.