Identification of primary structural features that define the differential actions of IL-3 and GM-CSF receptors.
Activation of human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors, ectopically expressed in FDCP-mix multipotent cells, stimulates self-renewal or myeloid differentiation, respectively. These receptors are composed of unique alpha subunits that interact with common beta(c) subunits. A chimeric receptor (hGM/beta(c)), comprising the extracellular domain of the hGM-CSF receptor alpha subunit (hGM Ralpha) fused to the intracellular domain of hbeta(c), was generated to determine whether hbeta(c) activation is alone sufficient to promote differentiation. hGM-CSF activation of hGM/beta(c), expressed in the presence and absence of the hbeta(c) subunit, promoted maintenance of primitive phenotype. This indicates that the cytosolic domain of the hGM Ralpha chain is required for differentiation mediated by activation of the hGM Ralpha, beta(c) receptor complex. We have previously demonstrated that the alpha cytosolic domain confers signal specificity for IL-3 and GM-CSF receptors. Bioinformatic analysis of the IL-3 Ralpha and GM Ralpha subunits identified a tripeptide sequence, adjacent to the conserved proline-rich domain, which was potentially a key difference between them. Cross-exchange of the equivalent tripeptides between the alpha subunits altered receptor function compared to the wild-type receptors. Both the mutant and the corresponding wild-type receptors promoted survival and proliferation in the short-term but had distinct effects on developmental outcome. The mutated hGM Ralpha promoted long-term proliferation and maintenance of primitive cell morphology, whereas cytokine activation of the corresponding hIL-3 Ralpha mutant promoted myeloid differentiation. We have thus identified a region of the alpha cytosolic domain that is of critical importance for defining receptor specificity.
Evans
Caroline A
CA
Leukaemia Research Fund Cellular Development Unit, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), Sackville Street, Manchester M60 1QD, UK.
Ariffin
Shahrul
S
Pierce
Andrew
A
Whetton
Anthony D
AD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006098:Granulocytes / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D016185:Receptors, Interleukin-3 / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014162:Transfection
D000943:Antigens, Differentiation; D007377:Interleukin-3; D021122:Protein Subunits; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D016185:Receptors, Interleukin-3; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor