We have previously demonstrated that important regulatory elements responsible for regulated expression of the human GLUT4 promoter are located between -1154 and -412 relative to transcription initiation (Olson, A. L., and Pessin, J. E. (1995) J. Biol. Chem. 270, 23491-23495). Through further analysis of this promoter regulatory region, we have identified a perfectly conserved myocyte enhancer factor 2 (MEF2)-binding domain (-CTAAAAATAG-) that is necessary, but not sufficient, to support tissue-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Biochemical analysis of this DNA element demonstrated the formation of a specific DNA-protein complex using nuclear extracts isolated from heart, hindquarter skeletal muscle, and adipose tissue but not from liver. DNA binding studies indicated that this element functionally interacted with the MEF2A and/or MEF2C MADS family of DNA binding transcription factors. MEF2 DNA binding activity was substantially reduced in nuclear extracts isolated from both heart and skeletal muscle of diabetic mice, which correlated with decreased transcription rate of the GLUT4 gene. MEF2 binding activity completely recovered to control levels following insulin treatment. Together these data demonstrated that MEF2 binding activity is necessary for regulation of the GLUT4 gene promoter in muscle and adipose tissue.
We have previously demonstrated that the important cis-acting elements regulating transcription of the human GLUT4 gene reside within 895 base pairs (bp) upstream of the transcription initiation site (Thai, M. V., Guruswamy, S., Cao, K. T., Pessin, J. E., and Olson, A. L. (1998) J. Biol. Chem. 273, 14285-14292). Our studies demonstrated that an MEF2 binding site within this region was necessary, but not sufficient, for GLUT4 promoter function in transgenic mice. We have identified a second regulatory element (Domain I) that functions cooperatively with the MEF2 domain in regulating GLUT4 transcription. Using a yeast-one hybrid screen, we obtained a partial cDNA and generated an antibody directed against a protein binding specifically to Domain I. Sequence analysis of the partial cDNA indicates that the protein binding to Domain I is a novel protein. The antibody specifically labels two proteins of approximately 70 and 50 kDa in Western blot analysis. These molecular masses correspond to Domain I binding proteins identified by UV-cross-linking nuclear extracts to a Domain I probe. The antibody raised against the Domain I binding protein inhibited formation of a Domain I-protein complex in electrophoretic mobility shift assays. We conclude that we have identified an authentic, novel, Domain I binding protein required for transcriptional regulation of the human GLUT4 promoter.
Rab proteins are small GTP-binding proteins of the Ras superfamily that function in the regulation of vesicle transport processes. The Rab4 isoform has been implicated in insulin action. For instance, overexpression of a prenylation-deficient form of Rab4 has been shown to inhibit insulin-dependent GLUT4 translocation. Other steps affected by Rab4 in the cascade of events resulting from insulin receptor activation have not been elucidated. In the present studies, we measured effects on insulin-signaling proteins in 3T3-L1 adipocytes transiently expressing cytoplasmic forms of Rab4 and Rab5. Expression of a mutant Rab4 lacking a prenylation site resulted in reduced insulin-dependent phosphorylation ofcytoplasmic and internal membrane-associated insulin receptor substrate-1, leading to decreased insulin receptor substrate-1-associated phosphatidylinositol 3'-OH kinase activation and decreased Akt activation. These effects were not observed upon introduction of a similar mutant form of Rab5. These data indicate that Rab4 or a Rab4-associated protein is involved at one or more steps in propagating the insulin signal, in addition to any role it may play in the regulation of GLUT4 vesicle translocation. Our results support models of insulin signaling in which regulation of internal membrane trafficking plays a role in transduction of the insulin signal.
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