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.
Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network therefore controls IFN signaling, which in turn interferes with medical interventions. The JAK-STAT signaling pathway transmits the IFN extracellular signal to the nucleus for alterations of gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized crucial component of the USP18-mediated negative feedback control in both, human and murine cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2 binding site. This mechanistic coupling of effector and negative feedback functions of STAT2 provides novel strategies in treatment of IFN signaling related human diseases.
The use of micropatterned surfaces that bind HaloTag fusion proteins allows spatial organization of plasma membrane proteins for efficient visualization and quantification of protein–protein interactions in live cells.
We developed in situ single cell pull-down (SiCPull) of GFP-tagged protein complexes based on micropatterned functionalized surface architectures. Cells cultured on these supports are lysed by mild detergents and protein complexes captured to the surface are probed in situ by total internal reflection fluorescence microscopy. Using SiCPull, we quantitatively mapped the lifetimes of various signal transducer and activator of transcription complexes by monitoring dissociation from the surface and defined their stoichiometry on the single molecule level.
Unraveling the spatiotemporal organization of signaling complexes within the context of plasma membrane nanodomains has remained a highly challenging task. Here, we have applied super-resolution image correlation based on tracking and localization microscopy (TALM) for probing transient confinement as well as ligand binding and intracellular effector recruitment of the type I interferon (IFN) receptor in the plasma membrane of live cells. Ligand and receptor were labeled with monofunctional quantum dots, thus allowing long-term tracking with very high spatial and temporal resolution without an artificial receptor cross-linking at the cell surface. Dual-color TALM was employed for visualizing protein-protein interactions involved in IFN signaling at both sides of the plasma membrane with high spatial and temporal resolution. By pair correlation analyses based on time-lapse TALM images (pcTALM), complex assembly within dynamic submicroscopic zones was identified. Strikingly, recruitment of the IFN effector protein signal transducer and activator of transcription 2 (STAT2) into these dynamic signaling zones could be observed. The results suggest that confined diffusion zones in the plasma membrane are employed as transient platforms for the assembly of signaling complexes.
The alkali cation potassium is the main osmolyte in the cytoplasm of prokaryotes (1). It binds close to the active center of the ribosome (2) and participates actively in pH homeostasis (3) and osmoadaptation (4, 5) by transport across the cell membrane. Hence, prokaryotes regulate their K ϩ contents and adapt them rapidly in response to changes in the environment. For this purpose, they possess a number of K ϩ channels, pumps, and transporters (1, 6). Several of the K ϩ -uptake systems contain a K ϩ -translocating subunit belonging to the superfamily of K ϩ transporters (7, 8) (termed SKT proteins (9)). These proteins may have evolved from simple K ϩ channels of the M 1 PM 2 type, like KcsA (10, 11) or Kir (12), by multiple gene duplications and gene fusions (7). Whereas the channels form homotetramers from four identical M 1 PM 2 subunits, SKT proteins consist of four covalently linked M 1 PM 2 motifs connected by cytoplasmic loops. The four p-loops (P) are thought to fold back from the external medium to the middle of the membrane, where they form a part of the permeation pathway for K ϩ through the channel center (7-9, 13). Within each p-loop, most SKT proteins contain one conserved glycine residue, which is part of their K ϩ selectivity filter (9, 14 -17). With single conserved glycine residues in SKT proteins, this filter appears to have a simpler structure than in K ϩ channels, in which the filter is formed by the well conserved p-loop sequence TVGYG from each subunit (18).The SKT-protein KtrB forms the K ϩ -translocating subunit of the Na ϩ -dependent K ϩ -uptake system KtrAB from bacteria (9, 14, 19 -22). KtrA, the other subunit from KtrAB, is located at the cytoplasmic side of the membrane and is a member of the RCK/KTN protein family (1, 23). KtrA may regulate K ϩ transport by binding ATP (24,25). It confers velocity, Na ϩ dependence, and K ϩ selectivity to the complex (9). KtrB alone transports K ϩ slowly in a process that is independent of Na ϩ . In addition, it transports Na ϩ with relatively low affinity (K m value of ϳ3 mM Na ϩ (9)). The exact structure of KtrB is unknown, but it has been modeled based on the structure of KcsA (11,13). Most of the KtrB structure was similar to that of KcsA. However, in particular, the C termini from the membrane spans M 2C and M 2D deviated from that of KcsA-M 2 . This may reflect the difference in function between the channel KcsA and the transporter KtrB (13). Subsequent cross-linking studies showed that the external half of KtrB is very similar to that of the KcsA tetramer, whereas its cytoplasmic half deviates. In addition, KtrB may form dimers (26). In their modeling studies, Durell and Guy (13) focused on membrane span M 2C . They divided it into three regions, from M 2C1 to M 2C3 (see Fig. 1A). M 2C1 and M 2C3 can form hydrophobic ␣-helices. However, M 2C2 contains many conserved small and polar residues (Ala, Gly, and Ser, Thr, Lys, respectively; see Fig. 1B). It may form a random coil or -turn structure (13). According to the first Durell and Guy model ...
Die Funktionalisierung von Quantenpunkten (QDs) gelang, wenn die Oberflächendichte an funktionellen Gruppen durch elektrostatische Abstoßung eingestellt wurde. Solche QDs konjugierten in vitro und in lebenden Zellen durch Selbstorganisation mit His‐markierten Proteinen und konnten dann ohne weitere Fraktionierung zur Zweifarbenbildgebung von Zelloberflächenrezeptoren eingesetzt werden (siehe Schema).
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