Novel, Gel-free Proteomics Approach Identifies RNF5 and JAMP as Modulators of GPCR Stability
| Autor: | Christian Iorio-Morin, Sebastien Roy, Terence E. Hébert, Mélanie Robitaille, Phan Trieu, Chantal Binda, Louis Fréchette, Irina Glazkova, Jean-Luc Parent, Darlaine Pétrin, Stephane Angers |
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| Rok vydání: | 2013 |
| Předmět: |
Proteomics
Proteasome Endopeptidase Complex GTPase-activating protein Ubiquitin-Protein Ligases Receptors Prostaglandin Biology Endoplasmic Reticulum Cell Line Endocrinology Heterotrimeric G protein Humans 5-HT5A receptor RNA Small Interfering Receptors Immunologic Molecular Biology Original Research G protein-coupled receptor G alpha subunit Membrane Glycoproteins Ubiquitination General Medicine Cell biology Ubiquitin ligase DNA-Binding Proteins Protein Transport HEK293 Cells Membrane protein biology.protein RNA Interference Receptors Adrenergic beta-2 Signal transduction Carrier Proteins Signal Transduction |
| Zdroj: | Molecular Endocrinology. 27:1245-1266 |
| ISSN: | 1944-9917 0888-8809 |
| DOI: | 10.1210/me.2013-1091 |
| Popis: | The maturation and folding of G protein-coupled receptors are governed by mechanisms that remain poorly understood. In an effort to characterize these biological events, we optimized a novel, gel-free proteomic approach to identify partners of the β2-adrenergic receptor (β2AR). In addition to a number of known interacting proteins such as heterotrimeric G protein subunits, this allowed us to identify proteins involved in endoplasmic reticulum (ER) QC of the receptor. Among β2AR-associated proteins is Ring finger protein 5 (RNF5), an E3 ubiquitin ligase anchored to the outer membrane of the ER. Coimmunoprecipitation assays confirmed, in a cellular context, the interaction between RNF5 and the β2AR as well as the prostaglandin D2 receptor (DP). Confocal microscopy revealed that DP colocalized with RNF5 at the ER. Coexpression of RNF5 with either receptor increased levels of their expression, whereas small interfering RNA-mediated knockdown of endogenous RNF5 promoted the opposite. RNF5 did not modulate the ubiquitination state of β2AR or DP. Instead, RNF5 ubiquitinated JNK-associated membrane protein (JAMP), a protein that recruits the proteasome to the ER membrane and that is negatively regulated by RNF5-mediated ubiquitination. JAMP coimmunoprecipitated with both β2AR and DP and decreased total receptor protein levels through proteasomal degradation. Expression of DP, a receptor largely retained in the ER, promoted proteasome recruitment by JAMP. Degradation of both receptors via JAMP was increased when RNF5 was depleted. Our data suggest that RNF5 regulates the turnover of specific G protein-coupled receptors by ubiquitinating JAMP and preventing proteasome recruitment. |
| Databáze: | OpenAIRE |
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