Regulated proteolysis of the IFNaR2 subunit of the interferon-alpha receptor

Abstract

The type I interferons (IFNs) bind surface receptors, induce JAK kinases and activate STAT transcription factors to stimulate the transcription of genes downstream of IFN-stimulated response elements (ISREs). In this study, we demonstrate that IFNaR2, a subunit of the type I IFN receptor, is proteolytically cleaved in a regulated manner. Immunoblotting shows that multi-step cleavage occurs in response to phorbol ester (PMA) and IFN-a, generating both a transmembrane 'stub' and the intracellular domain (ICD), similar to Notch proteolysis. Isolated membrane fractions process IFNaR2 to release the ICD. A chimeric receptor construct is utilized to show that cleavage requires the presenilins and occurs in response to epidermal growth factor and protein kinase C-d overexpression, as well as PMA and type I IFNs. Fluorescence microscopy demonstrates that a green fluorescent protein-ICD fusion localizes predominantly to the nucleus. A fusion between the ICD and the Gal4 DNA-binding domain represses transcription, in a histone deacetylasedependent manner, of a Gal4 upstream activating sequence-regulated reporter, while overexpression of the ICD alone represses transcription of a reporter linked to an ISRE. Proteolytic cleavage events may facilitate receptor turnover or, more likely, function as a mechanism for signaling similar to that employed by Notch and the Alzheimer's precursor protein.

Intracellular domain of the IFNaR2 interferon receptor subunit mediates transcription via Stat2

“…While investigating the canonical JAK-STAT pathway for IFN signaling [25,33], we demonstrated that i) Stat2 can bind IFNaR2 constitutively; ii) this binding is s t r o n g e r t h a n t h e b i n d i n g o f phosphorylated-IFNaR1 to Stat2 that occurs following IFN-driven receptor dimerization; and iii) the IFNaR2-Stat2 interaction is not required for canonical JAK-STAT signaling/gene regulation. These findings suggested that IFNaR2 might signal by other, non-canonical mechanisms and, indeed, we subsequently found that this subunit of the IFN receptor is proteolytically cleaved in a regulated manner that resembles signaling by other RIP substrates [21]. We also showed that the receptor ICD can modulate transcription via the TAD of the bound Stat2 molecule [22].…”

“…and J.J.K.). The stub is subsequently cleaved in a constitutive manner by the g -secretase protease complex, containing PS1 and PS2 [21].…”

Nuclear transit of the intracellular domain of the interferon receptor subunit IFNaR2 requires Stat2 and Irf9

“…To test this possibility, we employed a construct in which GFP is fused to the IFNaR2-ICD, and a cell line (U6A) deficient in the expression of Stat2. Previously, we have shown that following transient transfection, GFP-ICD localized to the nucleus of U2OS cells [21]. In contrast, in U6A cells, GFP-ICD was predominantly cytoplasmic (Fig.…”

“…We have recently found that phorbol ester and type I IFNs induce proteolytic cleavage of the IFNaR2 receptor subunit in a manner that resembles the two-step RIP cleavage of Notch [21]. Subsequently, we demonstrated that the intracellular domain of IFNaR2 (IFNaR2-ICD) can mediate gene transcription in a Stat2 dependent manner [22].…”

“…Previously, we demonstrated that the IFNaR2 intracellular domain can translocate to the nucleus [21], suggesting that the ICD either contains an inherent NLS or binds to other proteins that facilitate nuclear translocation. In addition, we confirmed earlier reports [30][31][32] that the intracellular portion of IFNaR2 binds Stat2 in a constitutive, ligand-independent manner [25,33].…”