Abstract
The common gamma chain (gammac) is a shared subunit of the IL-2, IL-4, IL-7, IL-9, IL-15, and IL21 receptors and is essential for T cell development and function. gammac is required for cytokine signaling and also functions as a modulator of ligand binding. In order to determine how gammac contributes to the binding of multiple cytokines, we used two complementary approaches aimed at identifying potential gammac-ligand contact residues. In the first approach, detailed alanine scanning mutagenesis was performed in selected loops of the mouse gammac based on homology modeling predictions of gammac structure. Mutant gammac variants were tested for the binding of IL-2, IL-7, and anti-gammac monoclonal antibodies (mAbs), leading to the identification of 4 potential ligand binding residues (Y103, C161, C210, G211), 3 of which also contribute to the epitope of the antagonistic mAb TUGm2. In the second approach, further gammac residues were mutated near recognition sites of mAbs that selectively block gammac-cytokine interactions. Based on comparison of the rat and mouse gammac sequences, the epitopes of the rat anti-mouse gammac mAbs 4G3 and 3E12 have been mapped to residues V 121 and 8197 of the mouse gammac, respectively. Since 4G3 is a potent inhibitor of IL-7, IL-9, and IL-15 bioactivity, and directly interferes with interactions of IL-2 and IL-7 with the gammac, residues near the 4G3 epitope were mutated. A novel site, N128, was found to be most critical for the binding of IL-7. Thus, the cytokine binding surface of gammac includes at least 5 residues from 3 distinct loops and the linker region of the extracellular domain. Comparative analysis of IL-2, IL-4, IL7, and IL-15 binding to gammac mutants affected in these regions suggested that the binding of all 4 cytokines requires the presence of Y103 of the gammac, and the integrity of a putative disulfide bond between C161 and C210. N128 also contributes to the binding of IL-7, IL-2, and IL-15, but does not appear to be used by IL-4. Taken together, our data support a common mechanism for the binding of four different gammac-dependent cytokines, and a role of the gammac linker region in cytokine-specific interactions.