Abstract
Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20-80 microm) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including alpha-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.
Highlights
EXPERIMENTAL PROCEDURESPeptide Extraction—Crude peptide extracts from rat brains and CHO-S and HEK293 cells were prepared as previously described with some modifications [35]
Our results show that such peptides can efficiently interfere with G protein-coupled receptor (GPCR) signal transduction in CHO-S cells expressing AT1 receptors (AT1R-CHO-S) stimulated with angiotensin II and in HEK293 cells stimulated with isoproterenol
Alteration in Extracellular acidification rates (ECAR) following angiotensin II (ang II) stimulation was monitored for 10 min, after which running medium was replaced by running medium plus peptides; this mixture was used to perfuse the cells for 30 min before the second stimulation with AT1 receptor agonist
Summary
Peptide Extraction—Crude peptide extracts from rat brains and CHO-S and HEK293 cells were prepared as previously described with some modifications [35]. Alteration in ECAR following ang II stimulation was monitored for 10 min, after which running medium was replaced by running medium plus peptides; this mixture was used to perfuse the cells for 30 min before the second stimulation with AT1 receptor agonist. In this way, each well was used as its own control, and results are expressed as the percentage of the first response. The relative luciferase activity was calculated by normalizing firefly luciferase activity to that of Renilla luciferase This same protocol was used to analyze the effect of peptides in another signaling pathway, that of the -adrenergic receptor, using HEK293 cells stimulated with isoproterenol (10 M). Statistical analyses of data were generated using GraphPad Prism, version 4.02 (GraphPad)
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