Translational regulation of the vasopressin v1b receptor involves an internal ribosome entry site.
Posttranscriptional mechanisms play an important role regulating pituitary levels of vasopressin V1b receptors (V1bR) during adaptation to stress. This study investigates the involvement of an internal ribosome entry site (IRES) in the 5'untranslated region (5'UTR) on V1bR translation. Transfection of bicistronic luciferase constructs into MCF-7 cells showed marked increases in translation of the second cistron after insertion of a 499-bp fragment of the V1bR 5'UTR in the intercistronic region, independently of cap-mediated translation, indicating the presence of IRES activity. IRES-mediated translation was potentiated by the protein kinase C activators, 12-O-tetradecanoylphorbol 13-acetate (PMA) and bryostatin 1, and appears to involve phosphorylation of amino terminus of eIF4G. In Chinese hamster ovary cells transfected with pV1bR-green fluorescent protein (pV1bR-GFP), PMA increased V1bR-GFP protein levels when cap-mediated translation was inhibited by rapamycin. The effect of PMA was due to increased translation because it persisted under transcriptional blockade by actinomycin D, and it was completely abolished by cycloheximide. In addition, PMA stimulated [35S]methionine incorporation into V1bR-GFP but not beta-actin in the absence of mRNA changes. The data show that regulation of IRES activity in the 5'UTR of the V1bR mRNA probably through phosphorylation of eIF4G may serve as a mechanism for rapid changes in V1bR translation to meet physiological demands.
Rabadan-Diehl
Cristina
C
Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1862, USA.
Volpi
Simona
S
Nikodemova
Maria
M
Aguilera
Greti
G
D020121:5' Untranslated Regions; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D008156:Luciferases / Q000235:genetics; D010902:Pituitary Gland / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D017483:Receptors, Vasopressin / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D017483:Receptors, Vasopressin; D014157:Transcription Factors; D008156:Luciferases; D011493:Protein Kinase C