Identification of protein binding factors to epidermal growth factor receptor gene mRNA
Defranco, Michael A.
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Past studies have shown that the EGF-receptor (EGF-R) mRNA is stabilized in cells incubated with EGF. These studies also indicate that the 3' untranslated region (3' UTR) of the EGF-R transcript contains sequences that regulate its stability. Work with other gene transcripts such as amyloid precursor protein and c-myc suggests that mRNA stability is often controlled by regulatory elements that bind to specific regions of the mRNA 3' UTR. The present study was performed to determine whether a similar mode of control exists for EGF-R mRNA. Radio-labeled transcripts of the EGF-R mRNA 3' UTR were synthesized in vitro and combined with cytosolic or ribosomal salt wash extracts (from human epidermal carcinoma cells; KB cells) in a gel shift assay. Initial binding was found for both cytosolic and ribosomal salt wadi extracts from both cells treated and not treated with EGF. Binding was more prevalent with extracts from cytosolic extracts and extracts from cells treated with EGF. Nonradio-labeled competition assays were also performed with intact 3' UTR transcripts as well as restriction fragments of the 3' UTR. Competition was confirmed indicating that binding is specific to the 3' UTR. Complete elimination of binding required larger doses of cold transcript in cells treated with EGF than in controls. Cytosolic extracts treated with heat and combined with radio-labeled 3' UTR EGF-R mRNA transcripts in gel shift assays showed no binding. This indicates that the putative binding factor present is heat labile and could be a protein. Gel shift assays separated on a ABSTRACT denaturing gel containing SDS revealed multiple bands of varying sizes for RSW and S- 130 cytosolic fractions.