STAT RECRUITMENT BY TYROSINE-PHOSPHORYLATED CYTOKINE RECEPTORS - AN ORDERED REVERSIBLE AFFINITY-DRIVEN PROCESS

Herein, we demonstrate that purified Stat1 binds to its tyrosine-phosp horylated docking site on the IFN gamma receptor alpha chain in a dire ct, specific, and reversible manner. Using surface plasmon resonance, we determine the affinity(K-D = 137 nM) and specificity of the interac tion and define the minimum affinity needed for receptor-mediated Stat 1 activation. In addition, we quantitate the relative ability of purif ied Stat1 to interact with tyrosine-phosphorylated binding sites on ot her Stat proteins. Finally, we describe experiments that Imply that th e unidirectional release of activated Stat1 from the IFN gamma recepto r reflects the preference of free tyrosine-phosphorylated Stat1 monome rs to form high avidity reciprocal homodimers rather than reassociatin g with the receptor binding site. Our results demonstrate that IFN gam ma-induced Stat1 activation is an ordered and affinity-driven process and we propose that this process may serve as a paradigm for Stat acti vation by other cytokine receptors.