Promiscuous binding of Janus kinases (JAKs) to class I/II cytokine receptors has been
reported, yet its role in signaling is unclear. To systematically explore JAK pairing in type
I interferon (IFN-I) signaling, we generated an artificial IFN-I receptor (AIR) by replacing
the extracellular domains of IFNAR1 and IFNAR2 with anti-mEGFP and mCherry
nanobodies. The heterodimeric AIR restored near-native IFN-I activity, while the
homomeric variant of IFNAR2 (AIR-dR2) initiated much weaker signaling despite
harboring docking sites for STAT proteins. To further investigate the roles of JAKs on the
receptors, knockout (KO) JAK1, JAK2, TYK2, and JAK2/TYK2 were generated. JAK1
KO led to a complete loss of IFN-I signaling, partially restored by TYK2 overexpression.
TYK2 KO cells retained partial activity, which was elevated by JAK1 overexpression,
suggesting both JAKs partially substitute each other.
Conversely, JAK2 KO only moderately impacted the biological activity of IFN-Is, even in
JAK2/TYK2 KO cells. Live cell micropatterning confirmed promiscuous binding of JAK1,
JAK2, and TYK2 to IFNAR1 and IFNAR2. Moreover, the identities of recruitment of
different JAKs on the ICDs were related to their respective concentrations in the cell, which
varies between cell lines. Yet, the efficiency of JAK cross-phosphorylation and
downstream signaling also depend on their identity. Promiscuity of JAK binding was also
observed for IFNL1, IL-10-Rbβ, TPOR, and GHR but not for EPOR, accompanied by
different downstream signaling activities. The competitive binding of JAKs to cytokine
receptors and the highly diverse absolute and relative JAK expression levels in different
cell types can account for cell type-dependent signaling pleiotropy observed for cytokine
receptors.
