TRANSLATIONAL REGULATION OF LIPOPROTEIN-LIPASE BY EPINEPHRINE INVOLVES A TRANS-ACTING BINDING-PROTEIN INTERACTING WITH THE 3'-UNTRANSLATED REGION

To better characterize the translational regulation of lipoprotein lip ase (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 t reatment with epinephrine, cell extracts from the adipocytes inhibited LPL translation in an in vitro translation assay, whereas extracts fr om the C2 cells and 3T3 fibroblasts did not affect LPL translation, To identify the region on the LPL mRNA that controlled translation in vi tro translation was carried out using constructs containing different LPL sequences, Specific deletion of the first 50 (1601-1650) nucleotid es of the 3' untranslated region (UTR) resulted in a loss of translati on inhibition, The addition of LPL 3' UTR to a heterologous reporter g ene construct resulted in an inhibition of translation Inhibition of t he reporter LPL 3' UTR translation was demonstrated by the addition of epinephrine-treated cell extracts to an in vitro translation assay, a s well as by transfection of this construct into 3T3-F442A cells, foll owed by treatment of the cells with epinephrine. Competition for a tra ns-acting binding protein was demonstrated by the addition of sense mR NA strands corresponding to the proximal 135 nucleotides of the 3' UTR of LPL, To identify a RNA-binding protein, adipocyte extracts were in cubated with P-32-labeled RNA sequences followed by RNase treatment, T he epinephrine-treated cell extract protected a fragment of RNA when t he RNA included sequences on the proximal 3' UTR of LPL, Cross-linking of this protected fragment and analysis by SDS-poIyacrylamide gel ele ctrophoresis revealed a protein that migrated at about 30 kDa, Thus, t he addition of epinephrine to 3T3 adipocytes results in an inhibition of translation through the production of a RNA-binding protein that bi nds to a region on the proximal 3' UTR of the LPL mRNA.