INTERACTING RESIDUES IN THE EXTRACELLULAR REGION OF THE COMMON BETA-SUBUNIT OF THE HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR, INTERLEUKIN (IL)-3, AND IL-5 RECEPTORS INVOLVED IN CONSTITUTIVE ACTIVATION

A previous study using random mutagenesis identified an activating mut ation in the common beta subunit (h beta c) of the human granulocyte-m acrophage colony stimulating factor, interleukin-3, and interleukin-5 receptors in which an isoleucine residue (Ile(374)) in the extracellul ar region of h beta c is replaced by asparagine (Jenkins, B. J., D'And rea, R., and Gonda, T. J. (1995) EMBO J 14, 4276-4287), To investigate the mechanism by which this mutation (I374N) acts, we employed site-d irected mutagenesis to explore predictions based on a structural model of h beta c, We focused on possible interactions between Ile(374) and other hydrophobic residues in its vicinity and found that replacement of two such residues, Leu(356) and Trp(358), With asparagine resulted in constitutive activation of h beta c, Hydrophilic substitutions at both of these positions and at position 374 resulted in the greatest d egree of activation, as measured by the growth rate of factor-independ ent cells, while hydrophobic substitutions had lesser or no effects, M oreover, these ''weak'' substitutions appeared to synergize, since fac tor-independent cells expressing the double mutants I374F/W358F and I3 74F/L356A showed substantially higher growth rates than the single mut ants, Taken together, these results suggest that Ile(374) normally int eracts with Leu(356) and Trp(358), and that disruption of these intera ctions results in a conformational change in h beta c that leads to co nstitutive activity, A model relating this notion to the predicted str ucture and to ligand- and alpha subunit-dependent activation of h beta c is proposed.