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competition studies, murine IFN-γ N-terminal peptide consisting of residues 1–39 [IFN-γ (1–39)]
blocked both binding to receptor and antiviral activity of IFN-γ [30]. Overlapping peptides of other
regions of the IFN-γ molecule failed to block binding and function of IFN-γ [31]. Thus the combination
of peptide mapping of epitope specificities and receptor competition using peptides has identified the Nterminus
as a structurally and functionally important region of the IFN-γ molecule. This region is
highlighted in the sequence of human IFN-γ found in Table 3.
Interestingly, site-specific antibodies to the C-terminus of murine IFN-γ, which were induced using the
peptide consisting of residues 95–133 [IFN-γ (95–133)], also neutralized IFN-γ activity, however IFN-γ
(95–133) failed to block binding of IFN-γ to receptor and IFN-γ activity simultaneously. Antibodies to
internal peptides failed to block both antiviral activity and binding of IFN-γ to receptor. In studies with
recombinant murine IFN-γ receptor, which consisted of the entire α chain except for the transmembrane
domain, the C-terminal peptide did block binding of IFN-γ to receptor [32]. Thus we have the interesting
paradox wherein the IFN-γ C-terminal peptide blocked binding of IFN-γ to the recombinant, soluble
receptor and yet did not block binding to the cell-surface receptor. One interpretation of these findings
has allowed us to formulate the “velcro-key” model of binding to receptor that involves both N- and Cterminal
domains of IFN-γ (Figure 5). The N-terminus binds in the “lock and key” manner characterized
by specific ligand-receptor binding. The hydrophilic C-terminus binds to a region of the receptor distinct
from that for the N-terminus, most likely through its polycationic region, which is conserved across
species barriers. Binding of this type would exhibit high affinity and low specificity, similar to a piece of
velcro. The C-terminal peptide of IFN-γ would therefore act as a poor competitor for cell-surface
binding due to its low specificity alone. This interaction becomes specific in the context of the whole
IFN-γ molecule and may increase the affinity of receptor binding.
An alternative explanation that may also account for the inability of the C-terminal peptide to compete
for cell-surface interactions is that its binding site is located not on the extracellular domain of the
receptor, but rather on the intracellular domain. The primary differences between the cell-surface form
of the IFN-γ receptor and (2) the accessibility of the recombinant receptor's cytoplasmic domain. A
synthetic peptide corresponding to the membrane proximal region of the cytoplasmic domain of the
murine IFN-γ receptor was able to bind IFN-γ and specifically compete with the binding of IFN-γ
(95–133) to fixed/permeabilized cells [33].
Studies by others have reaffirmed the importance of both the N- and C-terminal regions of IFN-γ in
function. Using recombinant DNA techniques, it has been shown that deletion of residues from the Nterminus
of the molecule
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