Translational regulation of lipoprotein lipase by epinephrine involves a trans-acting binding protein interacting with the 3' untranslated region

Gouri Ranganathan, Diane Vu, Philip A. Kern

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

To better characterize the translational regulation of lipoprotein lipase (LPL) by epinephrine, cytoplasmic extracts were prepared from 3T3-L1 adipocytes, 3T3-F442A adipocytes, and other nonadipocyte cell lines (C2 cells, 3T3 fibroblasts, and Chinese hamster ovary cells). After treatment with epinephrine, cell extracts from the adipocytes inhibited LPL translation in an in vitro translation assay, whereas extracts from the C2 cells and 3T3 fibroblasts did not affect LPL translation. To identify the region on the LPL mRNA that controlled translation, in vitro translation was carried out using constructs containing different LPL sequences. Specific deletion of the first 50 (1601-1650) nucleotides of the 3' untranslated region (UTR) resulted in a loss of translation inhibition. The addition of LPL 3' UTR to a heterologous reporter gene construct resulted in an inhibition of translation. Inhibition of the reporter LPL 3' UTR translation was demonstrated by the addition of epinephrine-treated cell extracts to an in vitro translation assay, as well as by transfection of this construct into 3T3-F442A cells, followed by treatment of the cells with epinephrine. Competition for a trans-acting binding protein was demonstrated by the addition of sense mRNA strands corresponding to the proximal 135 nucleotides of the 3' UTR of LPL. To identify a RNA-binding protein, adipocyte extracts were incubated with 32P- labeled RNA sequences followed by RNase treatment. The epinephrine-treated cell extract protected a fragment of RNA when the RNA included sequences on the proximal 3' UTR of LPL. Cross-linking of this protected fragment and analysis by SDS-polyacrylamide gel electrophoresis revealed a protein that migrated at about 30 kDa. Thus, the addition of epinephrine to 3T3 adipocytes results in an inhibition of translation through the production of a RNA- binding protein that binds to a region on the proximal 3' UTR of the LPL mRNA.

Original languageEnglish
Pages (from-to)2515-2519
Number of pages5
JournalJournal of Biological Chemistry
Volume272
Issue number4
DOIs
StatePublished - 1997

Funding

FundersFunder number
National Institute of Diabetes and Digestive and Kidney DiseasesR29DK039176

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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