3 RESIDUES IN THE COMMON BETA-CHAIN OF THE HUMAN GM-CSF, IL-3 AND IL-5 RECEPTORS ARE ESSENTIAL FOR GM-CSF AND IL-5 BUT NOT IL-3 HIGH-AFFINITY BINDING AND INTERACT WITH GLU21 OF GM-CSF

The beta subunit (beta(c)) of the receptors for human granulocyte macr ophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and in terleukin-5 (IL-5) is essential for high affinity ligand-binding and s ignal transduction. An important feature of this subunit is its common nature, being able to interact with GM-CSF, IL-3 and IL-5. Analogous common subunits have also been identified in other receptor systems in cluding gp130 and the IL-2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site-dir ected mutagenesis and binding assays with radiolabelled GM-CSF, IL-3 a nd IL-5 to identify residues in the beta(c) subunit involved in affini ty conversion for each ligand. Alanine substitutions in the region Tyr 365-ILe368 in beta(c) showed that Tyr365, His367 and Ile368 were requi red for GM-CSF and IL-5 high affinity binding, whereas Glu366 was unim portant. In contrast, alanine substitutions of these residues only mar ginally reduced the conversion of IL-3 binding to high affinity by bet a(c). To identify likely contact points in GM-CSF involved in binding to the 365-368 beta(c) region we used the GM-CSF mutant eco E21R which is unable to interact with wild-type beta(c) whilst retaining full GM -CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chai ns Y365A, H367A and I368A than to those composed of wild-type beta(c) or mutant E366A. These results (i) identify the residues Tyr365, His36 7 and Ile368 as critical for affinity conversion by beta(c), (ii) show that high affinity binding of GM-CSF and IL-5 can be dissociated from IL-3 and (iii) suggest that Tyr365, His367 and Ile368 in beta(c) inte ract with Glu21 of GM-CSF.