Structure-function relationships of interleukin-3. An analysis based on the function and binding characteristics of a series of interspecies chimera of gibbon and murine interleukin-3.

Abstract

IL-3 is a glycoprotein cytokine involved in the hematopoietic response to infectious, immunologic, and inflammatory stimuli. In addition, clinical administration of recombinant IL-3 augments recovery in states of natural and treatment-related marrow failure. IL-3 acts by binding to high affinity cell surface receptors present on hematopoietic cells. To determine the site(s) at which IL-3 binds to its receptor, we analyzed a series of interspecies chimera of the growth factor for species-specific receptor binding and biological activity. The results suggest that IL-3 binds to its receptor and triggers a proliferative stimulus through two noncontiguous helical domains located near the amino terminus and the carboxy terminus of the molecule. To corroborate these findings, we have also mapped the binding epitopes of 10 mAb of human or murine IL-3, and have defined four distinct epitopes. Two of these epitopes comprise the amino-terminal receptor binding domain. A third epitope corresponds to the carboxy-terminal receptor interactive domain, and the fourth epitope, apparently not involved in the interaction of IL-3 and its receptor, lies between these sites. And on the basis of sandwich immunoassays using pairs ofthese mAbs, the two receptor interactive regions appear to reside in close juxtaposition in the tertiary structure of the molecule. These results provide a correlation of the structure-function relationships of IL-3 that should prove useful in evaluating the details of IL-3-IL-3 receptor interaction and in the rational design of clinically useful derivatives of this growth factor. (J.

A Discontinuous Eight-Amino Acid Epitope in Human Interleukin-3 Binds the α-Chain of Its Receptor

“…Structure-function studies using site-directed mutagenesis have revealed that residues 21, 22, 27, 33, 43, 44, 48, 53, 58, 61, 64, 94, 101, 110, 111, 115, and 116 are important for activity (9 -13), although several of these residues may be buried in the IL-3 structure. In addition, mouse-gibbon chimeras (14) and site-directed mutagenesis (15) of IL-3 identified two noncontiguous regions near the N and C termini that are required for biological activity.…”

Differences among nonhuman primates in susceptibility to bone marrow progenitor transduction with retrovirus vectors

“…Under typical conditions, 5 × 10 4 CD34-enriched marrow cells were suspended per milliliter of virus supernatant supplemented with 4 g/ml protamine sulfate (Sigma), 50 ng/ml recombinant human SCF (a gift from Amgen, Thousand Oaks, CA, USA), 100 ng/ml recombinant human IL-6 (a gift from Sandoz Pharmaceuticals, Hanover, NJ, USA), and 5% v/v conditioned medium from BHKIL-3 cells (a gift from K Kaushansky, University of Washington, Seattle, WA, USA) 51 containing gibbon IL-3. Cultures were incubated in tissue culture-treated multiwell dishes at 33°C, 5% CO 2 .…”

The Receptor Binding Site of Human Interleukin-3 Defined by Mutagenesis and Molecular Modeling

“…This study identified several discrete regions of hIL-3 that are important for activity. To date, the relationships of these discontinuous regions to one another could only be inferred on the basis of secondary structure prediction and homology modeling of hIL-3 (3,12,17,19). The recently available three-dimensional structure of an hIL-3 variant (7) provides for the first time an experimentally derived model to understand the effect of these mutations.…”

“…Following the formation of the high affinity IL-3⅐IL-3R complex, signal transduction is mediated by the ␤ chain. Although the precise stoichiometry of the IL-3 receptor has not been established, we refer to the receptor as a heterodimer for simplicity.Several approaches have been undertaken to define the regions of hIL-3 that interact with the receptor, including constructing interspecies chimeras (12, 13), mapping neutralizing and non-neutralizing anti-IL-3 monoclonal antibody epitopes (12,14), and site-specific mutagenesis (14 -20). These studies identified sequences important for activity in both N-and Cterminal regions of IL-3 and revealed many specific sites where amino acid substitutions lead to significant changes in receptor binding affinity and growth-promoting activity.…”