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A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide
Published on: October 18, 2014
Epidermal growth factor binding and stimulation of inositol trisphosphate production in basolateral membranes from
1Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Abstract:
To ascertain whether epidermal growth factor (EGF) receptors are present in proximal tubular basolateral membranes from dog kidney and whether the growth factor activates phospholipase C at this site, we measured binding of 125I-EGF in isolated membranes and determined whether incubation of the membranes with EGF results in generation of inositol trisphosphate. Specific binding of 125I-EGF to basolateral membranes was observed that was half-maximally inhibited by approximately 7 x 10(-10) M EGF. The specific binding capacity of basolateral membranes for EGF averaged 45.9 fmol/mg protein. 125I-EGF was crosslinked to 2 proteins in basolateral membranes with M(r)s of 170,000 and 150,000. EGF stimulated phosphorylation of a 170,000 M(r) protein. Incubation of basolateral membranes with EGF stimulated inositol trisphosphate production in a concentration-dependent manner. We conclude that EGF activates a phospholipase C activity that cleaves phosphatidylinositol 4,5-bisphosphate within the basolateral membrane.
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