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Interaction of mouse interferon-[beta] with mouse L929 cells: receptor binding, antiviral induction, internalization and degradation
Dissertation

Interaction of mouse interferon-[beta] with mouse L929 cells: receptor binding, antiviral induction, internalization and degradation

Michael Kulka
Doctor of Philosophy (Ph.D.), Medical College of Pennsylvania
Jun 1986
DOI:
https://doi.org/10.17918/00008148
pdf
Kulka_Michael_19869.14 MB
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Abstract

The purpose of this thesis was twofold: 1) to determine the kinetic relationship between mouse interferon-b receptor binding and mouse interferon-b induced antiviral response in mouse L929 suspension cells and 2) to examine the extent of internalization and degradation of interferon during induction of antiviral activity using a monoclonal antibody purified mouse interferon b (specific activity 1-2x10⁸ U/mg). L929 cells were incubated with radiolabeled interferon, then separated from their incubation media for subsequent analysis of degraded and internalized interferon, respectively. For the binding studies, nonspecific binding of ¹²⁵I-interferon-b was determined in the presence of 100-fold excess nonlabeled interferon-b. Under identical experimental conditions, labeled and nonlabeled interferon-b were capable of inducing similar antiviral responses. Antiviral induction and receptor binding, examined in identical cell samples using ¹²⁵I-interferon-b, exhibit similar time dependent responses at 4°C and dissimilar responses at 37°C. Scatchard analysis of binding data for 1 hour incubation revealed 2x10⁵ receptors with 0.37 nM Kd at 4°C and 4x10⁵ receptors with 2.1 nM Kd at 37°C. Graphical analysis of antiviral induction estimated 0.23 nM Kd at 4°C and 0.28 nM at 37°C. The concentration of ¹²⁵I-interferon-b necessary to half-saturate antiviral induction is less than the concentration required to half saturate the available receptor sites. The results suggest that only a small fraction of available receptors need to be bound by interferon-b in order to initiate the antiviral response in L929 suspension cells. In addition, the nearly 8-fold difference in the Kd's at 37°C suggests a temperature dependent shift in receptor affinity that probably requires the presence of ligand. Finally, there is no kinetic evidence to support the existence of specifically internalized receptor-bound ligand. Extremely low levels of internalization and degradation do occur but are suggestive of a nonspecific mechanism usually associated with absorptive endocytosis or pinocytosis.

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