• hElp3 Directly Modulates the Expression of HSP70 Gene in HeLa Cells via HAT Activity

      Li, Fen; Ma, Jixian; Ma, Yu; Hu, Yanyan; Tian, Shujuan; White, Richard E.; Han, Guichun; Department of Pharmacology and Toxicology (2011-12-21)
      Human Elongator complex, which plays a key role in transcript elongation in vitro assay, is incredibly similar in either components or function to its yeast counterpart. However, there are only a few studies focusing on its target gene characterization in vivo. We studied the effect of down-regulation of the human elongation protein 3 (hELP3) on the expression of HSP70 through antisense strategy. Transfecting antisense plasmid p1107 into HeLa cells highly suppressed hELP3 expression, and substantially reduced expression of HSP70 mRNA and protein. Furthermore, chromatin immunoprecipitation assay (ChIP Assay) revealed that hElp3 participates in the transcription elongation of HSPA1A in HeLa cells. Finally, complementation and ChIP Assay in yeast showed that hElp3 can not only complement the growth and slow activation of HSP70 (SSA3) gene transcription, but also directly regulates the transcription of SSA3. On the contrary, these functions are lost when the HAT domain is deleted from hElp3. These data suggest that hElp3 can regulate the transcription of HSP70 gene, and the HAT domain of hElp3 is essential for this function. These findings now provide novel insights and evidence of the functions of hELP3 in human cells.
    • Internalization Dissociates b2-Adrenergic Receptors

      Lan, Tien-Hung; Kuravi, Sudhakiranmayi; Lambert, Nevin A.; Department of Pharmacology and Toxicology (2011-02-22)
      G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that b2-adrenergic receptors (b2ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β2ARs between subcellular compartments. BRET between b2ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between b2ARs and endosome markers increases. Energy transfer between b2ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled b2ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that b2ARs associate transiently with each other in the plasma membrane, or that b2AR dimers or oligomers are actively disrupted during internalization.