Abstract
The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand–receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.
Highlights
The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes
To avoid artefacts caused by receptor overexpression, we have developed single molecule imaging techniques based on posttranslational labelling via strictly monomeric tags to visualize and quantify receptor dimerization in the plasma membrane of living cells at physiological receptor expression levels[23]
The ectodomains of IL-4Ra and IL-13Ra1 fused to a C-terminal decahistidine tag (IL-4Ra À EC and IL-13Ra1-EC, respectively) were tethered onto SSMs by means of a lipid analogue functionalized with tris-NTA36
Summary
The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. For systematic drug development, a quantitative understanding of the molecular and cellular determinants governing the receptor activation at the cell surface is required, which so far remained unresolved[4]: while a recent model proposed that dimerization of IL-4Ra and IL-13Ra1 requires uptake into endosomes[30,31], single molecule studies suggested substantial receptor dimerization by IL-13 at the plasma membrane[25]. Taking advantage of the versatile intrinsic and engineered features of this system, here we aimed to identify the molecular and cellular determinants governing the assembly and dynamics of the type II IL-4 signalling complex in a quantitative manner For this purpose, we quantified receptor dimerization in artificial membranes and in the plasma membrane of living cells. More detailed analysis of the spatiotemporal receptor dynamics suggests that plasma membrane microcompartmentation by the cortical actin cytoskeleton promotes efficient re-association of dissociated receptor dimers, ensuring robust maintenance of activated signalling complexes until endocytosis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
