Abstract
A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells, denoted 2D1-IgM, was generated after fusion of immunized BALB/c mouse spleen cells with SP2/0-Ag14 myeloma cells. Specific binding of 2D1-IgM to the A431 cell-surface receptor for EGF was demonstrated by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Scatchard analysis of 125I-EGF binding to A431 cells demonstrated that 2D1-IgM treatment did not change the number of EGF receptors, but caused an increase in the affinity of EGF receptors from a population of low affinity to a uniform population of high affinity. Like EGF, 2D1-IgM induced phosphorylation of EGF receptors and EGF receptor clustering. As in the case of EGF, a biphasic growth response with stimulation of DNA synthesis at low and inhibition at high concentrations of 2D1-IgM was evident in A431 cells. The intrinsic "EGF-like" bioactivity of 2D1-IgM was enhanced by the presence of EGF. These results suggest that the binding of 2D1-IgM to the EGF receptor at a different site from that to which EGF binds can initiate an effective EGF-like biological response; and the EGF-like biological effects of 2D1-IgM may be mediated by a population of high affinity EGF receptors which may be involved in the control of cellular growth.
Highlights
This antibody, which sponse with stimulation of DNA synthesis at low and binds to a determinantat a site distincftrom the EGFbinding inhibition at high concentrations oZf D1-IgM was evi- site, (a) enhances the affinity of the EGFreceptor population dent inA43l cells
From the Immunoassay Section, Laboratory of Nuclear Medicine, Veterans Administration Medical Center and the Department of Medicine, St
A431 cells, suggesting reciprocal modulationof EGF receptor creasing the concentrations of EGF above 1nM resulted in a affinity
Summary
This antibody, which sponse with stimulation of DNA synthesis at low and binds to a determinantat a site distincftrom the EGFbinding inhibition at high concentrations oZf D1-IgM was evi- site, (a) enhances the affinity of the EGFreceptor population dent inA43l cells. Fig. 13Bshows that increasing the incubation experiments since the number of EGF receptors is increased times past 24 h resulted in a progressive concentration-deand their affinity is reduced as A431 cells reach confluence pendent inhibitionof thymidine incorporation.
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