Leptin (L) and its receptor (L-R) are key players in the regulation of energy homeostasis and body weight. Complex formation between leptin and the extracellular portion of L-R results in the activation of Janus kinase 2 (JAK2) that is constitutively bound on the intracellular regions of the receptor. Leptin-instigated JAK2 signaling in hypothalamic nuclei reduces food intake and stimulates energy expenditure, while functional defects in either leptin or L-R result in morbid obesity, hyperglycemia, decreased insulin sensitivity, and hyperlipidemia. Despite the crucial impact of the leptin system on body weight and other physiological responses, little is known about the structure of the L/L-R complex and its association with JAK2. One of the reasons for this lack of insight is that both L-R and JAK2 have a relatively long and flexible multi-domain arrangement that has proved to be very challenging for both large-scale purification and implementation of X-ray crystallography. The present proposal aims to overcome these limitations in addressing the architectural prerequisites of L-R signaling by applying single- particle cryo-electron microscopy (cryo-EM) to characterize the holo-complex of full-length L/L-R and JAK2. Single-particle EM has emerged as a very powerful tool for the characterization of dynamic protein assemblies in relatively small concentrations and without the need for crystallization. We anticipate that the application of single-particle EM techniques on this system will reveal the architecture of the L/L-R assembly and JAK2 independently, and in complex. Given the underlying importance of this membrane-localized signaling complex in obesity, energy metabolism, and heart disease, the structural results obtained will be of very broad biomedical interest. Considering the current lack of structural information on any receptor/JAK complex, our studies will provide the general architectural framework for understanding how extracellular ligand binding on cytokine receptors results in intracellular JAK activation.

Public Health Relevance

Leptin signaling is key to the regulation of mammalian energy homeostasis and body weight. Extracellular binding of leptin to its receptor results in the intracellular activation of Janus kinase 2 (JAK2) which, in turns, regulates a number of physiologically crucial signaling cascades. We propose to apply molecular electron microscopy techniques to characterize the structure of the signaling complex between leptin, its receptor, and JAK2. The elucidation of this architecture will provide a mechanistic understanding of JAK2 activation via extracellular receptor engagement by leptin. Since deficient leptin signaling is associated with severe pathologies, such as obesity and diabetes, this knowledge will be ultimately used for the design of therapeutic strategies that are based on targeting the leptin receptor complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK090165-02
Application #
8223124
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Sechi, Salvatore
Project Start
2011-04-01
Project End
2017-07-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$342,327
Indirect Cost
$118,327
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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