Stat recruitment by tyrosine-phosphorylated cytokine receptors: An ordered reversible affinity-driven process

• Published in: Immunity (Cambridge, Mass.) Vol. 2; no. 6; pp. 677 - 687
• Main Authors: Greenlund, Andrew C., Morales, Mary O., Viviano, Beth L., Yan, Hai, Krolewski, John, Schreiber, Robert D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.1995
• Subjects: Amino Acid Sequence; Binding, Competitive; Biosensing Techniques; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Humans; Interferon gamma Receptor; Interferon-gamma - physiology; Molecular Sequence Data; Phosphopeptides - metabolism; Phosphorylation; Protein Binding - physiology; Protein-Tyrosine Kinases - metabolism; Receptors, Cytokine - metabolism; Receptors, Interferon - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Signal Transduction; STAT1 Transcription Factor; Trans-Activators - chemistry; Trans-Activators - isolation & purification; Trans-Activators - metabolism
• ISSN: 1074-7613; 1097-4180
• DOI: 10.1016/1074-7613(95)90012-8

Herein, we demonstrate that purified Stat1 binds to its tyrosine-phosphorylated docking site on the IFNγ receptor α chain in a direct, specific, and reversible manner. Using surface plasmon resonance, we determine the affinity (K D = 137 nM) and specificity of the interaction and define the minimum affinity needed for receptor-mediated Stat1 activation. In addition, we quantitate the relative ability of purified Stat1 to interact with tyrosine-phosphorylated binding sites on other Stat proteins. Finally, we describe experiments that imply that the unidirectional release of activated Stat1 from the IFNγ receptor reflects the preference of free tyrosine-phosphorylated Stat1 monomers to form high avidity reciprocal homodimere rather than reassociating with the receptor binding site. Our results demonstrate that IFNγ-induced Stat1 activation is an ordered and affinity-driven process and we propose that this process may serve as a paradigm for Stat activation by other cytokine receptors.