Novel, Gel-free Proteomics Approach Identifies RNF5 and JAMP as Modulators of GPCR Stability

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 ...