Roy SJ, Glazkova I, Frechette L, Iorio-Morin C, Binda C, Petrin D, Trieu P, Robitaille M, Angers S, Hebert TE, Parent JL

The maturation and folding of G protein-coupled receptors (GPCRs) 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 ...

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as heterotrimeric G protein subunits, this allowed us to identify proteins involved in endoplasmic reticulum (ER) quality control 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. Co-immunoprecipitation 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 co-localized with RNF5 at the ER. Co-expression of RNF5 with either receptor increased levels of their expression, whereas siRNA-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 co-immunoprecipitated 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 GPCRs by ubiquitinating JAMP and preventing proteasome recruitment.

Date: Jun. 24, 2013

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