The heparan sulfate binding sequence of interferon-γ increased the on rate of the interferon-γ-interferon-γ receptor complex formation

Abstract

Interferon-γ (IFNγ), in common with a number of growth factors, binds both to heparan sulfate or heparin-related molecules and to a specific high affinity receptor (IFNγR). Using surface plasmon resonance technology, kinetic analysis of the IFNγ-IFNγR complex formation was performed with the extracellular part of IFNγR immobilized on a sensor chip. At the sensor chip surface, IFNγ was bound by two IFNγR molecules with an affinity in the nanomolar range (0.68 nM). This binding was characterized by an important on rate, k(on) = 7.3 x 106 M-1·s-1, and an off rate, k(off) = 5 x 10-3·s-1. This binding assay was used to investigate a possible role of heparin in the IFNγ-IFNγR complex formation. In contrast to growth factors for which binding to heparin is usually required for high affinity receptor interaction, we found in this study that IFNγ bound to heparin displayed a strongly reduced affinity for its receptor. This is consistent with the fact that a cluster of basic amino acids (KTGKRKR, called the C1 domain) in the carboxyl-terminal sequence of the cytokine was involved both in heparin and receptor recognition. To understand how a single domain of IFNγ could be implicated in two discrete functions (i.e. binding to heparin and to IFNγR), we also analyzed in a detailed manner the role of the IFNγ carboxyl-terminal sequence in receptor binding. Using forms of IFNγ, with carboxyl terminus truncations of defined regions of the heparin binding sequence, we found that the C1 domain functioned by increasing the on rate of the IFNγ-IFNγR binding reaction but was not otherwise required for the stability of the complex. Interactions between the IFNγ carboxyl-terminal domain and IFNγR could increased the association rate of the reaction either by increasing the number of encounters between the two molecules or by favoring productive collisions. The mechanisms by which heparan sulfate regulates IFNγ activity may thus include both control of selective protease cleavage events, which directly affect the cytokine activity, and also an ability to modulate the interaction of IFNγ with the IFNγR via competitive binding to the C1 domain.