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Direct interaction between Kit and the interleukin-7 receptor.

ABSTRACT: In vivo analyses of thymopoiesis in mice defective in signaling through Kit and gammac or Kit and IL-7Ralpha demonstrate synergy and partial complementation of gammac or IL-7-mediated signaling by the Kit signaling pathway. Our molecular analysis in T-lymphoid cells as well as in nonhematopoietic cells shows that Kit and IL-7R signaling pathways directly interact. KL-mediated activation of Kit induced strong tyrosine phosphorylation of gammac and IL-7Ralpha in the absence of IL-7. Activated Kit formed a complex with either IL-7Ralpha or gammac, and tyrosine phosphorylation of both subunits occurred independently of Jak3, suggesting that gammac and IL-7Ralpha are each direct substrates of Kit. Kit activated Jak3 in an IL-7R-dependent manner. Moreover, deficient Stat5 activation of the Kit mutant YY567/569FF lacking intrinsic Src activation capacity was partially reconstituted in the presence of IL-7R and Jak3. Based on the molecular data, we propose a model of Kit-mediated functional activation of gammac-containing receptors such as IL-7R, similar to the interaction between Kit and Epo-R. Such indirect activation of the Jak-Stat pathway induced by the interaction between an RTK and type I cytokine receptor could be the underlying mechanism for a context-specific signaling repertoire of a pleiotropic RTK-like Kit.

SUBMITTER: Jahn T

PROVIDER: S-EPMC1976346 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Direct interaction between Kit and the interleukin-7 receptor.

Jahn Thomas T Sindhu Simran S Gooch Stacie S Seipel Petra P Lavori Philip P Leifheit Erica E Weinberg Kenneth K

Blood 20070606 6

In vivo analyses of thymopoiesis in mice defective in signaling through Kit and gammac or Kit and IL-7Ralpha demonstrate synergy and partial complementation of gammac or IL-7-mediated signaling by the Kit signaling pathway. Our molecular analysis in T-lymphoid cells as well as in nonhematopoietic cells shows that Kit and IL-7R signaling pathways directly interact. KL-mediated activation of Kit induced strong tyrosine phosphorylation of gammac and IL-7Ralpha in the absence of IL-7. Activated Kit ...[more]

PMID: 17554063

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Project description:Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and ?(2)-adrenergic receptor (?(2)AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on ?(2)AR properties, we showed that IR coexpression had no effect on the ligand binding properties of ?(2)AR; however, IR reduced ?(2)AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between ?(2)AR and IR, we employed quantitative BRET(2) saturation and competition assays. Saturation assay data suggested constitutive ?(2)AR and IR homo- and heteromerization. Calculated acceptor/donor (AD50) values as a measure of the relative affinity for homo- and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET(2) signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET(2) data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of ?(2)AR and IR identified intracellular domains encompassing residues at the end of the 7th TM domain and C-terminal tail of ?(2)AR and a cytoplasmic part of the IR ? chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order ?(2)AR:IR oligomer formation, likely comprising heteromers of homodimers.

Project description:The interaction between interleukin-7 (IL-7) and its α-receptor, IL-7Rα, plays fundamental roles in the development, survival, and homeostasis of B- and T-cells. N-Linked glycosylation of human IL-7Rα enhances its binding affinity for human IL-7 300-fold versus that of the nonglycosylated receptor through an allosteric mechanism. The N-glycans of IL-7Rα do not participate directly in the binding interface with IL-7. This biophysical study involves dissection of the properties of binding of IL-7 to both nonglycosylated and glycosylated forms of the IL-7Rα extracellular domain (ECD) as functions of salt, pH, and temperature using surface plasmon resonance (SPR) spectroscopy. Interactions of IL-7 with both IL-7Rα variants display weaker binding affinities with increasing salt concentrations primarily reflected by changes in the first on rates of a two-step reaction pathway. The electrostatic parameter of the IL-7-IL-7Rα interaction is not driven by complementary charge interactions through residues at the binding interface or N-glycan composition of IL-7Rα, but presumably by favorable global charges of the two proteins. van't Hoff analysis indicates both IL-7-IL-7Rα interactions are driven by large favorable entropy changes and smaller unfavorable (nonglycosylated complex) and favorable (glycosylated complex) enthalpy changes. Eyring analysis of the IL-7-IL-7Rα interactions reveals different reaction pathways and barriers for the transition-state thermodynamics with the enthalpy and entropy changes of IL-7 binding to nonglycosylated and glycosylated IL-7Rα. There were no discernible heat capacity changes for the equilibrium or transition-state binding thermodynamics of the IL-7-IL-7Rα interactions. The results suggest that the unbound nonglycosylated IL-7Rα samples an extensive conformational landscape relative to the unbound glycosylated IL-7Rα, potentially explaining the switch from a "conformationally controlled" reaction (k(1) ∼ 10(2) M(-1) s(-1)) for the nonglycosylated interaction to a "diffusion-controlled" reaction (k(1) ∼ 10(6) M(-1) s(-1)) for the glycosylated interaction. Thus, a large favorable entropy change, a global favorable electrostatic component, and glycosylation of the receptor, albeit not at the interface, contribute significantly to the interaction between IL-7 and the IL-7Rα ECD.

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Direct interaction between Kit and the interleukin-7 receptor.

Publications

Direct interaction between Kit and the interleukin-7 receptor.

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