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 mutation in the common beta subunit (hbetac) of the human granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5 receptors in which an isoleucine residue (Ile374) in the extracellular region of hbetac is replaced by asparagine (Jenkins, B. J., D'Andrea, R., and Gonda, T. J. (1995) EMBO J 14, 4276-4287). To investigate the mechanism by which this mutation (I374N) acts, we employed site-directed mutagenesis to explore predictions based on a structural model of hbetac. We focused on possible interactions between Ile374 and other hydrophobic residues in its vicinity and found that replacement of two such residues, Leu356 and Trp358, with asparagine resulted in constitutive activation of hbetac. Hydrophilic substitutions at both of these positions and at position 374 resulted in the greatest degree of activation, as measured by the growth rate of factor-independent cells, while hydrophobic substitutions had lesser or no effects. Moreover, these "weak" substitutions appeared to synergize, since factor-independent cells expressing the double mutants I374F/W358F and I374F/L356A showed substantially higher growth rates than the single mutants. Taken together, these results suggest that Ile374 normally interacts with Leu356 and Trp358, and that disruption of these interactions results in a conformational change in hbetac that leads to constitutive activity. A model relating this notion to the predicted structure and to ligand- and alpha subunit-dependent activation of hbetac is proposed.
Jenkins
B J
BJ
Hanson Centre for Cancer Research and Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia.
Bagley
C J
CJ
Woodcock
J
J
Lopez
A F
AF
Gonda
T J
TJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D002460:Cell Line; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007377:Interleukin-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018123:Receptors, Interleukin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053649:Receptors, Interleukin-5; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D007377:Interleukin-3; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D016178:Granulocyte-Macrophage Colony-Stimulating Factor