Amino acids conserved in interleukin-1 receptors (IL-1Rs) and the Drosophila toll protein are essential for IL-1R signal transduction.

A Heguy,C T Baldari,G Macchia,J L Telford,M Melli

doi:10.1016/s0021-9258(18)45924-8

Abstract

The cytoplasmic domain of the human T cell-type interleukin-1 receptor (hIL-1R) is not involved in the binding, internalization, or nuclear localization of interleukin-1 (IL-1), but is essential for signal transduction. We have previously localized a 50-amino acid region (residues 477-527) critical for IL-1-mediated activation of the interleukin-2 promoter in T cells. This region displays a striking degree of amino acid conservation in human, murine, and chicken IL-1Rs. Here we report the results of a site-directed mutational analysis of the cytoplasmic domain of the hIL-1R. We have introduced single-amino acid substitutions at positions conserved in all three receptors and at nonconserved positions and identified key amino acids for IL-1R function in signal transduction. Three basic (Arg431, Lys515, and Arg518) and 3 aromatic (Phe513, Trp514, and Tyr519) amino acids that are conserved in human, murine, and chicken IL-1Rs could not be replaced without abolishing IL-1R-mediated signal transduction. A substitution at another conserved position (Pro521) reduces significantly the ability of the IL-1R to transmit the IL-1 signal. Nonconserved residues could be replaced without affecting signal transduction. The cytoplasmic domain of the IL-1R is related to that of the Drosophila Toll protein, with a 26% identity and a 43% similarity in amino acid sequence. The amino acids shown to be essential for IL-1R function are conserved in the Toll protein. Our experimental data indicate that the amino acid sequence similarity between the IL-1R and the Drosophila toll protein reflects a functional homology between the two proteins.

Highlights

Terleukin- 1(IL-l),but is essential for signal transduc- Following IL-1 binding, the T cell-type IL-1R transduces tion
We tion of 213 amino acids not related to receptor kinases (Sims have introducedsingle-amino acid substitutionsat po- et al, 1989).The IL-1R is phosphorylated at serine/threonine sitions conserved in all three receptors anadt noncon- residues following IL-1 binding (Gallis et al, 1989), but the served positions and identifiedkey amino acids for IL-role of this phosphorylation in the signaling response is un
Tyr’l’) amino acidsthat are conserved in humanm, u- the murine IL-1R abolishes IL-1-mediated prostaglandin rerine, and chicken IL- c1oRulsd not be replaced without lease and colony-stimulating factor production in fibroblasts abolishing IL-la-mediated signal transduction

Summary

RESULTS

We have replaced several of these basic amino acids and all 3 layer chromatograms of the products of IL-2-CAT activity in Jurkat cell extracts following cotransfection of the reporter plasmid with the wild-type hIL-1R orpointmutants F513A,W514A, W514L, and Y519S. Transfected cells were treated polar amino acid Ser. A mutant receptor with the change with PHA (O), IL-1/PHA, or PMA/PHA as indicated. Residues have been shown to be important for the substrate murine IL-1Rs (Schneideret al., 1991),suggesting that these specificity of protein kinase C (House et al, 1987).A mutant two proteins may share similar pathways of signal transduccontaining the conservative change Lys4*' to Argwas fully tion These positions are outside of the region that we have functional (Fig. 4 andTableI), whereas mutant receptors mutagenized and may correspond to a functional domain carrying the change Lys4" to Ser or Glu showed a reduced different from the one described in this paper. Important in IL-1R signal transduction (Heguy et al, 1991)

DISCUSSION

Findings

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