Homodimeric Murine Interleukin-3 Agonists Indicate that Ligand Dimerization is Important for High-Affinity Receptor Complex Formation

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Abstract
Homodimeric murine interleukin 3 (mIL-3) agonists were generated by intermolecular cystine-bonding. Steady-state binding assays and association kinetics performed at 4°C using these agonists revealed specific binding to both the high-and low-affinity receptor. DSS-mediated crosslinking studies performed at 4°C with agonist concentrations compatible with high-affinity receptor complex formation allowed to detect protein complexes of the α chain, the β chain(s) and the high-affinity receptor complex migrating with apparent molecular weights of 90 kDa, 140 kDa, and above 180 kDa, respectively. In contrast, monomeric mIL-3 was crosslinked to the α chain receptor only unless high concentrations were used. Binding studies performed at 4°C revealed a positive cooperative interaction of monomeric mIL-3 with the low-affinity receptor. Proliferation studies and association kinetics performed at 37°C showed that under physiological conditions these agonists were at least 2-to 3-fold more potent than monomeric mIL-3. We therefore propose that dimerization of mIL-3 may be involved in high-affinity receptor complex formation.