Interleukin-2 is a helical cytokine that has found wide spread clinical use in the treatment of some malignancies and for infectious diseases including HIV infection. The interleukin-2 receptor (IL-2R) is one of the most complex receptor systems of the large family of hematopoietin receptors. It may exist in a variety of affinity states depending upon the participation of different combinations of three separate cell surface subunits: IL-2Ralpha, beta and gamma. These subunits function in the form of two specific heterodimers (IL-2Ralpha beta) and (IL-2Rbeta gamma) and one heterotrimer (IL-2Ralpha beta gamma). Each of these cell surface assemblies performs a distinct physiologic function. It is the cooperativity of these subunits during ligand capture and signal transmission that provides the key to understanding exploiting the therapeutic potential of IL-2. This project was the first to propose and implement the use of coiled-coil recognition sequences (Leu Zippers) for the solution assembly of complex receptor systems. During the initial project period, the feasibility of this approach was established and methods were developed for the expression and characterization of soluble IL-2R coiled-coil complexes. Coiled-coil mediated solution assembly generated heteromeric IL-2R complexes that bound ligand in a fashion that resembled their cell surface counterparts. These complexes are already proving useful for IL-2 structure/function analysis. This proposal is a request for continuation of this work with the goals of stable solution assembly and complete characterization of all three of the functional heteromeric IL-2R complexes.
The Specific Aims of this project are: 1. To express, isolate and characterize the two functional IL-2 receptor heterodimers (IL-2Ralpha beta pseudo high affinity and IL-2R beta gamma intermediate affinity receptors) as stable coiled-coil complexes. 2. To express, isolate and characterize the presumed IL-2 receptor heterotrimeric high affinity receptor (IL-2Ralpha beta gamma) as a stable coiled-coil complex. 3. To determine the equilibrium and kinetic ligand binding characteristics of each receptor complex. 4. To obtain detailed structure-activity data on these complexes by co-crystallizing them with IL-2 and determining their X-ray structures to high resolution.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI034331-07
Application #
6149785
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Hackett, Charles J
Project Start
1994-05-01
Project End
2001-03-31
Budget Start
2000-02-01
Budget End
2001-03-31
Support Year
7
Fiscal Year
2000
Total Cost
$207,939
Indirect Cost
Name
Dartmouth College
Department
Pharmacology
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Cullen, Paul A; Haake, David A; Adler, Ben (2004) Outer membrane proteins of pathogenic spirochetes. FEMS Microbiol Rev 28:291-318
Liparoto, Stefano F; Myszka, David G; Wu, Zining et al. (2002) Analysis of the role of the interleukin-2 receptor gamma chain in ligand binding. Biochemistry 41:2543-51
Liparoto, S F; Ciardelli, T L (1999) Biosensor analysis of the interleukin-2 receptor complex. J Mol Recognit 12:316-21
Wu, Z; Goldstein, B; Laue, T M et al. (1999) Solution assembly of the pseudo-high affinity and intermediate affinity interleukin-2 receptor complexes. Protein Sci 8:482-9
Wang, R; Ciardelli, T L; Russell, J H (1997) Partial signaling by cytokines: cytokine regulation of cell cycle and Fas-dependent, activation-induced death in CD4+ subsets. Cell Immunol 182:152-60
Myszka, D G; Arulanantham, P R; Sana, T et al. (1996) Kinetic analysis of ligand binding to interleukin-2 receptor complexes created on an optical biosensor surface. Protein Sci 5:2468-78
Wu, Z; Johnson, K W; Choi, Y et al. (1995) Ligand binding analysis of soluble interleukin-2 receptor complexes by surface plasmon resonance. J Biol Chem 270:16045-51
Wu, Z; Johnson, K W; Goldstein, B et al. (1995) Solution assembly of a soluble, heteromeric, high affinity interleukin-2 receptor complex. J Biol Chem 270:16039-44
Nakarai, T; Robertson, M J; Streuli, M et al. (1994) Interleukin 2 receptor gamma chain expression on resting and activated lymphoid cells. J Exp Med 180:241-51