The AB loop and D-helix in binding site III of human Oncostatin M (OSM) are required for OSM receptor activation

Abstract

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are closely related members of the interleukin-6 (IL-6) cytokine family. Both cytokines share a common origin and structure, and both interact through a specific region, termed binding site III, to activate a dimeric receptor complex formed by glycoprotein 130 (gp130) and LIF receptor (LIFR) in humans. However, only OSM activates the OSM receptor (OSMR)-gp130 complex. The molecular features that enable OSM to specifically activate the OSMR are currently unknown. To define specific sequence motifs within OSM that are critical for initiating signaling via OSMR, here we generated chimeric OSM-LIF cytokines and performed alanine-scanning experiments. Replacement of the OSM AB loop within OSM's binding site III with that of LIF abrogated OSMR activation, measured as STAT3 phosphorylation at Tyr-705, but did not compromise LIFR activation. Correspondingly, substitution of the AB loop and D-helix in LIF with their OSM counterparts was sufficient for OSMR activation. The alanine-scanning experiments revealed that residues Tyr-34, Gln-38, Gly-39, and Leu-45 (in the AB loop) and Pro-153 (in the D-helix) had specific roles in activating OSMR but not LIFR signaling, whereas Leu-40 and Cys-49 (in the AB loop), and Phe-160 and Lys-163 (in the D-helix) were required for activation of both receptors. Because most of the key amino acid residues identified here are conserved between LIF and OSM, we concluded that comparatively minor differences in a few amino acid residues within binding site III account for the differential biological effects of OSM and LIF.

The AB loop of oncostatin M (OSM) determines species-specific signaling in humans and mice

“…We reason that the generation of mouse OSM variants essentially owning the same functional features as human OSM will facilitate future preclinical studies in mice. Our results also offer an explanation for the potential evolutionary path of this cytokine, in which the spatial rearrangements that resulted in promiscuous OSMR and LIFR activation (24) are followed by amino acid mutations promoting receptor specialization.…”

“…1C) based on data from the published hOSM structure (Protein Data Bank code 1EVS) (13). We recently reported the existence of two specific regions within binding site III of hOSM, the AB loop and N-terminal helix D, determining hOSMR interaction (24). Reasoning that the same regions might also influence species-specific receptor recognition, we aligned the amino acid sequences of mouse and human OSM using ClustalW (25) and compared the AB loop and helix D, selecting two different replacement lengths for each region.…”

“…Because we knew from previous experience that bacterial expression of hOSM-based chimeras is not efficient (24), we employed a mammalian expression system (FreeStyle 293-F cells) for all experiments.…”

“…S2). Human receptor activation was measured using a similar approach, employing different cell lines (A375 cells for hOSMR, JAR cells for hLIFR) and previously established short-and long-term readout systems, such as the assessment of STAT3 phosphorylation, TIMP1 expression, and inhibition of A375 cell proliferation after hOSMR activation (24).…”

“…Here we investigated the molecular determinants of mouse and human OSM for controlling receptor recognition and activation. Chimeric cytokines were created to assess the relevance of binding site III region of mouse OSM for receptor interactions, as components of this region are crucial for initiation of hOSMR signaling (24). Replacement of the AB loop in mOSM by the corresponding hOSM amino acid sequence resulted in a chimera that initiates mLIFR as well as hOSMR and hLIFR signaling.…”