Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.

Hyaluronan is a polyanionic, megadalton-scale polysaccharide, which initiates cell signaling by interacting with several receptor proteins including CD44 involved in cell-cell interactions and cell adhesion. Previous studies of the CD44 hyaluronan binding domain have identified multiple widespread residues to be responsible for its recognition capacity. In contrast, the X-ray structural characterization of CD44 has revealed a single binding mode associated with interactions that involve just a fraction of these residues. In this study, we show through atomistic molecular dynamics simulations that hyaluronan can bind CD44 with three topographically different binding modes that in unison define an interaction fingerprint, thus providing a plausible explanation for the disagreement between the earlier studies. Our results confirm that the known crystallographic mode is the strongest of the three binding modes. The other two modes represent metastable configurations that are readily available in the initial stages of the binding, and they are also the most frequently observed modes in our unbiased simulations. We further discuss how CD44, fostered by the weaker binding modes, diffuses along HA when attached. This 1D diffusion combined with the constrained relative orientation of the diffusing proteins is likely to influence the aggregation kinetics of CD44. Importantly, CD44 aggregation has been suggested to be a possible mechanism in CD44-mediated signaling.