Volume 271, Number 47, Issue of November 22, 1996 pp. 29707-29714
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Interacting Residues in the Extracellular Region of the Common  Subunit of the Human Granulocyte-Macrophage Colony-stimulating Factor, Interleukin (IL)-3, and IL-5 Receptors Involved in Constitutive Activation

(Received for publication, June 17, 1996, and in revised form, September 3, 1996)

From the Hanson Centre for Cancer Research and Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia

A previous study using random mutagenesis identified an activating mutation in the common  subunit (hc) 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 hc 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 hc. 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 hc. 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 hc that leads to constitutive activity. A model relating this notion to the predicted structure and to ligand- and  subunit-dependent activation of hc is proposed.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				T. J. Blake, B. J. Jenkins, R. J. D'Andrea, and T. J. Gonda Functional cross-talk between cytokine receptors revealed by activating mutations in the extracellular domain of the {beta}-subunit of the GM-CSF receptor J. Leukoc. Biol., December 1, 2002; 72(6): 1246 - 1255. [Abstract] [Full Text] [PDF]

				I. Wolf, B. J. Jenkins, Y. Liu, M. Seiffert, J. M. Custodio, P. Young, and L. R. Rohrschneider Gab3, a New DOS/Gab Family Member, Facilitates Macrophage Differentiation Mol. Cell. Biol., January 1, 2002; 22(1): 231 - 244. [Abstract] [Full Text] [PDF]

				J. Rossjohn, W. J. McKinstry, J. M. Woodcock, B. J. McClure, T. R. Hercus, M. W. Parker, A. F. Lopez, and C. J. Bagley Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common beta -chain bound to an antagonist Blood, April 15, 2000; 95(8): 2491 - 2498. [Abstract] [Full Text] [PDF]

				M. P. McCormack and T. J. Gonda Novel murine myeloid cell lines that exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the GM-CSF receptor beta subunit Blood, January 1, 2000; 95(1): 120 - 127. [Abstract] [Full Text] [PDF]

				R. C. H. Lee, J. A. Walters, M. E. Reyland, and S. M. Anderson Constitutive Activation of the Prolactin Receptor Results in the Induction of Growth Factor-independent Proliferation and Constitutive Activation of Signaling Molecules J. Biol. Chem., April 9, 1999; 274(15): 10024 - 10034. [Abstract] [Full Text] [PDF]

				B. J. Jenkins, F. Le, and T. J. Gonda A Cell Type-specific Constitutive Point Mutant of the Common beta -Subunit of the Human Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF), Interleukin (IL)-3, and IL-5 Receptors Requires the GM-CSF Receptor alpha -Subunit for Activation J. Biol. Chem., March 26, 1999; 274(13): 8669 - 8677. [Abstract] [Full Text] [PDF]

				B. J. Jenkins, T. J. Blake, and T. J. Gonda Saturation Mutagenesis of the beta  Subunit of the Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor Shows Clustering of Constitutive Mutations, Activation of ERK MAP Kinase and STAT Pathways, and Differential beta  Subunit Tyrosine Phosphorylation Blood, September 15, 1998; 92(6): 1989 - 2002. [Abstract] [Full Text] [PDF]

				M. D. Edgerton, P. Graber, D. Willard, T. Consler, M. McKinnon, I. Uings, C. Y. Arod, F. Borlat, R. Fish, M. C. Peitsch, et al. Spatial Orientation of the alpha  and beta c Receptor Chain Binding Sites on Monomeric Human Interleukin-5 Constructs J. Biol. Chem., August 15, 1997; 272(33): 20611 - 20618. [Abstract] [Full Text] [PDF]

				C. J. Bagley, J. M. Woodcock, F. C. Stomski, and A. F. Lopez The Structural and Functional Basis of Cytokine Receptor Activation: Lessons From the Common beta  Subunit of the Granulocyte-Macrophage Colony-Stimulating Factor, Interleukin-3 (IL-3), and IL-5 Receptors Blood, March 1, 1997; 89(5): 1471 - 1482. [Full Text] [PDF]