# Characterization of fish‐specific IFNγ‐related binding with a unique receptor complex and signaling through a novel pathway

## TL;DR

This study identifies a unique signaling pathway for a fish-specific interferon, IFNγrel 1, which activates STAT6 through a novel receptor pair, suggesting it is a new type of interferon in vertebrates.

## Contribution

The discovery of a novel interferon signaling pathway in fish, involving unique receptor interactions and STAT6 activation, expands understanding of interferon evolution.

## Key findings

- IFNγrel 1 activates STAT6 through binding to Crfb5 and Crfb17 receptors.
- Crfb5 and Crfb17 dimerize and are essential for antiviral gene Isg15 expression.
- The signaling pathway of IFNγrel 1 is distinct from known interferons in other vertebrates.

## Abstract

Unlike mammals, fish express two type II interferons, IFNγ and fish‐specific IFNγ (IFNγ‐related or IFNγrel). We previously reported the presence of two IFNγrel genes, IFNγrel 1 and IFNγrel 2, which exhibit potent antiviral activity in the Ginbuna crucian carp, Carassius auratus langsdorfii. We also found that IFNγrel 1 increased allograft rejection; however, the IFNγrel 1 receptor(s) and signaling pathways underlying this process have not yet been elucidated. In this study, we examined the unique signaling mechanism of IFNγrel 1 and its receptors. The phosphorylation and transcriptional activation of STAT6 in response to recombinant Ginbuna IFNγrel 1 (rgIFNγrel 1) was observed in Ginbuna‐derived cells. Binding of rgIFNγrel 1 to Class II cytokine receptor family members (Crfbs), Crfb5 and Crfb17, which are also known as IFNAR1 and IFNGR1‐1, respectively, was detected by flow cytometry. Expression of the IFNγrel 1‐inducible antiviral gene, Isg15, was highest in Crfb5‐ and Crfb17‐overexpressing GTS9 cells. Dimerization of Crfb5 and Crfb17 was detected by chemical crosslinking. The results indicate that IFNγrel 1 activates Stat6 through an interaction with unique pairs of receptors, Crfb5 and Crfb17. Indeed, this cascade is distinct from not only that of IFNγ but also that of known IFNs in other vertebrates. IFNs may be classified by their receptor and signal transduction pathways. Taken together, IFNγrel 1 may be classified as a novel type of IFN family member in vertebrates. Our findings provide important information on interferon gene evolution in bony fish.

Unique signaling mechanisms of fish‐specific isoforms of IFNγ, IFNγrel 1, from the Ginbuna crucian carp have been identified. Monomeric ligand phosphorylates STAT6 and induces transcriptional activation by binding to the unique pair of receptors, Crfb5 (IFNAR1) and Crfb17 (IFNGR1‐1). These suggest IFNγrel 1 may be classified as a novel type of IFN family member in vertebrates.