Molecular characterization of an alpha interferon receptor 1 subunit (IFNaR1) domain required for TYK2 binding and signal transduction.
Binding of alpha interferon (IFNalpha) to its receptors induces rapid tyrosine phosphorylation of the receptor subunits IFNaR1 and IFNaR2, the TYK2 and JAK1 tyrosine kinases, and the Stat1 and Stat2 transcription factors. Previous studies have demonstrated that TYK2 directly and specifically binds to and tyrosine phosphorylates IFNaR1 in vitro. We now report a detailed analysis of the TYK2 binding domain on the IFNaR1 subunit. First, we used an in vitro binding assay to identify the TYK2 binding motif in IFNaR1 as well as the critical residues within this region. The most striking feature is the importance of a number of hydrophobic and acidic residues. A minor role is also ascribed to a region resembling the proline-rich "box 1" sequence. In addition, mutations which disrupt in vitro binding also disrupt the coimmunoprecipitation of the receptor and TYK2. We also provide direct evidence that the binding region is both necessary and sufficient to activate TYK2 in vivo. Specifically, mutations in the binding domain act in a dominant-negative fashion to inhibit the IFNalpha-induced tyrosine phosphorylation of TYK2 and Stat2. Further, introduction of dimerized glutathione S-transferase-IFNaR1 fusion proteins into permeabilized cells is sufficient to induce phosphorylation of TYK2 and the receptor, confirming the role of the binding domain in IFNalpha signal transduction. These studies provide clues to the sequences determining the specificity of the association between JAK family tyrosine kinases and cytokine receptors as well as the functional role of these kinases in cytokine signal transduction.
Yan
H
H
Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Krishnan
K
K
Lim
J T
JT
Contillo
L G
LG
Krolewski
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D002460:Cell Line; D005982:Glutathione Transferase / Q000096:biosynthesis; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D016898:Interferon-alpha / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011505:Protein-Tyrosine Kinases; D011506:Proteins / Q000378:metabolism*; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera; D053634:TYK2 Kinase; D014162:Transfection
D000906:Antibodies; D016898:Interferon-alpha; D010455:Peptides; D011506:Proteins; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D053633:Receptor, Interferon alpha-beta; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D053634:TYK2 Kinase; C070139:TYK2 protein, human