Transmembrane Signaling by the α Subunit of the Type I Interferon Receptor Is Essential for Activation of the Jak Kinases and the Transcriptional Factor ISGF3

Oscar R Colamonici,Leonidas C Platanias,Paul Domanski,Raj Handa,Kimberly C Gilmour,Manuel O Diaz,Nancy Reich,Paula Pitha-Rowe

doi:10.1074/jbc.270.14.8188

Abstract

The Type I interferon (IFN) receptor has a multisubunit structure. The component of the receptor that has been most thoroughly studied is the alpha subunit. Expression of the alpha subunit in mouse L-929 cells confers antiviral response to human IFN alpha 8, but not to human IFN alpha 2 or IFN beta. This antiviral effect is observed without a significant increase in IFN binding. It has not been determined why mouse cells expressing the human alpha subunit show different response to the antiviral activity of distinct human Type I IFNs. In this report, we demonstrate that the response to human Type I IFNs in mouse cells expressing the alpha subunit is dependent on cross-binding to the mouse receptor. This is supported by the finding that human IFN alpha 8, but not human IFN alpha 2, cross-binds to the mouse receptor even in the absence of expression of the human alpha subunit. We also demonstrate that only mouse cells expressing the human alpha subunit are able to tyrosine-phosphorylate p135tyk2 and JAK-1 and to form the ISGF3 complex in response to human IFN alpha 8. These results demonstrate that the alpha subunit is essential for IFN alpha signaling through the JAK kinases and ISGF3.

Highlights

From the +Department of Pathology, University of Tennessee, Memphis, Tennessee 38163, the Wi vision of Hematology / Oncology, Loyola University of Chicago and Edward Hines Jr
Differences in the Antiviral Response to Human Typ e I IFN in Mouse Cells Tran sfected with the a Subunit of the Type I IFN receptor (IFN-R)-We have reported pr eviously that the cDNA cloned by Uze et al [7] corresponds to wh at we have previously designated as the a subunit of th e Typ e I IFN-R [9]
Mouse cells expressing the subunit of the Type I IFN-R cloned by Uze et al [7] are protected against vesicular stomatitis virus challenge by human IFNa8. This subunit of the receptor corresponds to a receptor component recognized by the IFNaR1, IFNaR2, and IFNaR3 monoclonal antibodies (mAb) that we previously designated as the a subunit [9]

Summary

EXPERIMENTAL PROCEDURES

Materials-Human recombinant IFNa2 was kindly providedby Drs M. Both cell lin es were gro wn in RPMI 1640 medium su ppleme nte d with 10% fet al calf serum and antibiotics (Life Tech nologies, In c.), Th e cell lin es SV. Im m unoblott ing - Cell s wer e treate d wit h differ ent concent ra tio ns of IFN 2 for the ind ica te d peri ods of time, ra pidly cent ri fuge d a t 2000 x g for 30 s in a n Eppendorf mi crocen trifuge, a nd su bse quently solu bilize d in lysi s buffer (1% Triton X-100 , 150 m xt NaCl, 25 mx; HEPES (pH 7.5), 1 rnxi EDTA , 20 0 J.l.~ 1 sodium or t hovanada te, 100 mx; Na F, 1 rnxt MgCI2, 1 ms: ph en ylm ethylsulfonyl flu oride, 10 J.l.g/ml a pro tinin , 10 J.l.g1ml leup eptin ) a t 4 °C for 30 min. Electr oph oreti c Mobility Shift A ssay-Nuclear a nd cytoplasmi c ext racts we re prep a r ed as describ ed a nd a nalyze d by elect r ophore tic mobilit y shi ft assays using an end lab eled oligo nu cleotide (5' -GATCGGGAAAGGGAAACCG AAACTGAAGCC-3' a nd 3' -CCC TTT CCC TTTGGCTTTGACTTCGGAG-5 ' ) to det ect ISGF 3 (25, 26 )

RESULTS

DISCUSSION

F NaR3