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A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the a and P subunits. The a subunit alone is sufficient for ligand binding, but association with the P subunit leads to a 2-to 3-fold increase in binding affinity. The P chain is shared with the receptors for IL1 and granulocyte/macrophage-colonystimulating factor-hence the descriptor pc (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5Ra interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor ai subunit were clustered in a loop connecting 13-strand 1 and helix B (mutants H38A, K39A, and H41A), in P-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, WillS, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.Human interleukin 5 (hIL5) is a disulfide-linked homodimeric glycoprotein with 115 aa per monomer (1) and has been produced in various heterologous systems (2-4). Analysis of the glycosylation pattern of Chinese hamster ovary (CHO) cell-derived IL5 revealed an antiparallel dimer linkage. Also, hIL5 was found to have 0-linked glycosylation at and N-linked glycosylation at Asn-28 (N28) (5), but deglycosylation did not affect the biological activity (2). The structure of IL5 purified from Escherichia coli (6) and Sf9 cells (ref. 7; see Fig. 5) has been determined. hIL5 adopts the typical four a-helical bundle "cytokine fold" which has also been described for other cytokines, including granulocyte/macrophagecolony-stimulating factor (GM-CSF), IL2, IL4, macrophagecolony-stimulating factor, and growth hormone (GH). This fold consists of a bundle of four a-helices in an up-up, down-down array. Unique to 1L5, however, is the phenomenon of D-helix swapping, whereby one bundle is built up of three helices coming from one monomer and a fourth helix which is contributed by the second monomer. In addition to the four a-helical bundle, hIL5 also contains two short antiparallel (3-strands located between helices A and B and helices C and D.The human as well as the mouse IL5 receptor (IL5R) consists of two different chains, the a and 3 subunits (8-13). hIL5 binds to the a subunit with intermediate affinity (Kd = The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solel...

By use of a 3' extension PCR strategy, cDNA clones were isolated spanning the transmembrane region and a complete cytoplasmic domain of the human interleukin 5 receptor a subunit (hIL5Ra). These cDNAs differ from previously isolated clones encoding a soluble hIL5Ra form by a sequence switch at position 1243. When expressed in COS-1 cells, only low-affinity binding of 2'sI-labeled human interleukin 5 was observed. Coexpression of the hIL5Rfi chain led to a 2-fold increase in binding affinity. In addition, this same cloning strategy allowed us to identify a putative second soluble isoform of hIL5Ra. Genomic data revealed that the two soluble variants arise from either a "normal" splicing event or from the absence of splicing, whereas synthesis of the membraneanchored form requires alternative splicing.

The molecular basis for the commitment of multipotential myeloid progenitors to the eosinophil lineage, and the transcriptional mechanisms by which eosinophil-specific genes are subsequently expressed and regulated during eosinophil development are currently unknown. Interleukin-5 (IL-5) is a T cell and mast cell-derived cytokine with actions restricted to the eosinophil and closely related basophil lineages in humans. The high affinity receptor for IL-5 (IL-5R) is composed of an alpha subunit (IL-5R alpha) expressed by the eosinophil lineage, that associates with a beta c subunit shared with the receptors for IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF). As a prerequisite to studies of the transcriptional regulation of the IL-5R alpha subunit gene, we used three different methods, including primer extension, RNase protection, and 5'-RACE to precisely map the transcriptional start site to a position 15 base pairs (bp) upstream of the 5' end of the published sequence of IL-5R alpha exon 1. To initially identify the IL-5R alpha promoter, 3.5 kilobases (kb) and 561 bp of the 5' sequence flanking the transcriptional start site were subcloned into the promoterless pXP2-luciferase vector. Transient transfection of these constructs into an eosinophil-committed HL-60 subline, clone HL-60-C15, induced the expression of approximately 240-fold greater luciferase activity than the promoterless vector, identifying a strong functionally active promoter region within the 561 bp of sequence proximal to the transcriptional start site and with activity equivalent to pXP2 constructs containing the entire 3.5 kb of upstream sequence. To more precisely localize the cis-acting regulatory elements in this region important for promoter activity, a series of 5' deletion mutants of the 561-bp region were generated in the pXP2-luciferase vector. Deletion of the region between bp -432 and -398 reduced promoter activity by more than 80% in the HL-60-C15 cell line. Further analyses of the activity of the IL-5R alpha promoter constructs in various other eosinophil, myeloid, and non-myeloid cell lines indicated that the promoter was relatively myeloid and eosinophil lineage-specific in its expression. Consensus sequences for known transcription factor binding sites were not present in the 34-bp region of the promoter required for maximal activity, suggesting unique myeloid- and possibly eosinophil-specific regulatory elements.(ABSTRACT TRUNCATED AT 400 WORDS)

SummaryThe T cell product interleukin 5 (IL 5) has been shown to be a key factor in the development and the maturation of the eosinophilic cell lineage . We report here on the detection of human IL5 receptors on eosinophilic sublines of the promyelocytic leukemia HL-60. Sodium butyrate, which initiates differentiation to mature eosinophils, also induces the appearance of high affinity (Kd 1-5 x 10 -11 M) IL5 binding sites on these cells. The receptors are specific for 11,5, since binding of radiolabeled ligand can only be inhibited with homologous or murine IL-5 and not by other cytokines. We further show that the receptors are functional, since 116 can stimulate the proliferation of these cells. Affinity crosslinking of surface-bound 1251 human IL-5 or 35S mouse IL5 identified two membrane polypeptides of -60 and -130 kD to which 1175 is closely associated. The presence of granuloryte/macrophage-colony-stimulating factor or tumor necrosis factor during butyrate induction decreased the expression of 11,5 binding sites compared with control cultures. The identification and characterization of human 1175 receptors on HL60 sublines should provide new insight into the role of this cytokine in eosinophil differentiation .