A receptor binding domain of mouse interleukin-4 defined by a solid-phase binding assay and in vitro mutagenesis.
Interleukin 4 (IL-4) is a potent, pleiotropic lymphokine that affects a variety of cells, especially those of hematopoietic origin. Although murine and human IL-4 are homologous proteins, they display a species specificity in which murine IL-4 acts only upon mouse cells, and human IL-4 only upon human cells. We have used a mutagenesis strategy to define both the structural determinants of this specificity and a receptor binding domain of murine IL-4. To do this, we developed convenient solid-phase binding assays for mouse and for human IL-4, each utilizing receptor-immunoglobulin fusion proteins and alkaline phosphatase-tagged ligands. These were employed to assess the receptor binding activities of wild type and mutant forms of IL-4. In a separate biological assay, we measured the ability of each version of IL-4 to induce proliferation of a cultured mouse T-cell line. By replacing regions of mouse IL-4 with homologous segments of human IL-4, we found that the amino-terminal 16 residues and the carboxyl-terminal 20 residues of murine IL-4 are required for species-specific receptor binding as well as for T-cell proliferation. A major portion of the amino acid sequence between these regions can be substituted between mouse and human without loss of receptor binding or biological activity. Further, alanine-scanning mutagenesis revealed specific residues in the amino- and carboxyl-terminal regions (Glu-12, Ile-14, Leu-104, Asp-106, Phe-107, and Leu-111) that bear side chains critical for function. An analysis of the carboxyl-terminal region of murine IL-4 and its comparison with carboxyl-terminal regions of other related cytokines suggest an evolutionary conservation of structural and functional features.
Morrison
B W
BW
Department of Genetics, Harvard Medical School, Howard Hughes Medical Institute, Boston, Massachusetts 02115.
Leder
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002455:Cell Division; D004247:DNA / Q000235:genetics; D006801:Humans; D015847:Interleukin-4 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism
D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen; D011993:Recombinant Fusion Proteins; D015847:Interleukin-4; D004247:DNA