Characterization of a Dipeptide Motif Regulating IFN-γ Receptor 2 Plasma Membrane Accumulation and IFN-γ Responsiveness

Abstract

The IFN-γR complex is composed of two IFN-γR1 and two IFN-γR2 polypeptide chains. Although IFN-γR1 is constitutively expressed on all nucleated cells, IFN-γR2 membrane display is selective and tightly regulated. We created a series of fluorescent-tagged IFN-γR2 expression constructs to follow the molecule’s cell surface expression and intracellular distribution. Truncation of the receptor immediately upstream of Leu-Ile 255–256 (254X) created a receptor devoid of signaling that overaccumulated on the cell surface. In addition, this truncated receptor inhibited wild-type IFN-γR2 activity and therefore exerted a dominant negative effect. In-frame deletion (255Δ2) or alanine substitution (LI255–256AA) of these amino acids created mutants that overaccumulated on the plasma membrane, but had enhanced function. Single amino acid substitutions (L255A or I256A) had a more modest effect. In-frame deletions upstream (253Δ2), but not downstream (257Δ2), of Leu-Ile 255–256 also led to overaccumulation. A truncation within the IFN-γR2 Jak2 binding site (270X) led to a mutant devoid of function that did not overaccumulate and did not affect wild-type IFN-γR2 signaling. We have created a series of novel mutants of IFN-γR2 that have facilitated the identification of intracellular domains that control IFN-γR2 accumulation and IFN-γ responsiveness. In contrast to IFN-γR1, not only dominant negative, but also dominant gain-of-function, mutations were created through manipulation of IFN-γR2 Leu-Ile 255–256. These IFN-γR2 mutants will allow fine dissection of the role of IFN-γ signaling in immunity.