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.
