CHARACTERIZATION OF A LOW-MOLECULAR-WEIGHT ISOFORM OF IL-1 RECEPTOR ANTAGONIST

IL-1R antagonist (IL-1Ra) exists in two well-characterized forms, 17-k Da secretory IL-1Ra (sIL-1Ra) and 18-kDa intracellular IL-1Ra (icIL-1R a), that arise by alternative transcription of the same IL-1Ra gene. A third, lower molecular mass form (similar to 16 kDa) was detected by immunoblot within lysates of a variety of cells, including human monoc ytes and myelomonocytic cell lines. The 16-kDa isoform was designated icIL-1RaII, and the previously established 18-kDa form was designated icIL-1RaI, Intracellular IL-1RaII bound type I IL-1R up to fivefold le ss avidly than did sIL-1Ra and icIL-1RaI, Microsequencing of cyanogen bromide fragments of purified icIL-1RaII provided evidence consistent with initiation of protein translation at the second start site in eit her IL-1Ra mRNA, The results of site-directed mutation experiments est ablished that icIL-1RaII could be derived by alternative translation i nitiation. In vitro transcription and translation of intact sIL-1Ra cD NA in rabbit reticulocyte lysates led to both pro-sIL-1Ra and icIL-1Ra II proteins, whereas transcription and translation of icIL-1RaI cDNA p roduced both icIL-1RaI and icIL-1RaII proteins. Mutation of the first 5' ATG in sIL-1Ra cDNA led to translation of only icIL-1RaII, while on ly sIL-1Ra was observed after mutation of the second ATG. These result s indicate that icIL-1RaII is a third member of the IL-1Ra family and is a 16-kDa, 143-amino acid intracellular protein derived by alternati ve translation initiation from either sIL-1Ra mRNA or icIL-1Ra mRNA. T he role in biology of either intracellular form of IL-1Ra remains unkn own.