The heparan sulfate binding sequence of interferon-gamma increased the on rate of the interferon-gamma-interferon-gamma receptor complex formation.
Interferon-gamma (IFNgamma), in common with a number of growth factors, binds both to heparan sulfate or heparin-related molecules and to a specific high affinity receptor (IFNgammaR). Using surface plasmon resonance technology, kinetic analysis of the IFNgamma. IFNgammaR complex formation was performed with the extracellular part of IFNgammaR immobilized on a sensor chip. At the sensor chip surface, IFNgamma was bound by two IFNgammaR molecules with an affinity in the nanomolar range (0.68 nM). This binding was characterized by an important on rate, kon = 7.3 x 10(6) M-1.s-1, and an off rate, koff = 5 x 10(-3).s-1. This binding assay was used to investigate a possible role of heparin in the IFNgamma.IFNgammaR 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 IFNgamma 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 IFNgamma could be implicated in two discrete functions (i.e. binding to heparin and to IFNgammaR), we also analyzed in a detailed manner the role of the IFNgamma carboxyl-terminal sequence in receptor binding. Using forms of IFNgamma, 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 IFNgamma.IFNgammaR binding reaction but was not otherwise required for the stability of the complex. Interactions between the IFNgamma carboxyl-terminal domain and IFNgammaR 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 IFNgamma 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 IFNgamma with the IFNgammaR via competitive binding to the C1 domain.
Sadir
R
R
Institut Pasteur de Lyon, CNRS URA 1459, Avenue des Martyrs, 38027 Grenoble Cedex 01, France.
Forest
E
E
Lortat-Jacob
H
H
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D002268:Carboxypeptidases / Q000378:metabolism; D043402:Cathepsin A; D015951:Factor Xa / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D006868:Hydrolysis; D007371:Interferon-gamma / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D017471:Receptors, Interferon / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D000096743:Interferon gamma Receptor
D002268:Carboxypeptidases; D043402:Cathepsin A; D015951:Factor Xa; D006493:Heparin; D006497:Heparan Sulfate; D007371:Interferon-gamma; D017471:Receptors, Interferon; D012697:Serine Endopeptidases; D000096743:Interferon gamma Receptor; C044499:submandibular proteinase A