Investigation of G-Protein Coupled Receptor Folding and Maturation by LC–MS/MS

The process by which G-protein coupled receptors (GPCRs) mature and fold is poorly understood. Previous research has demonstrated that this happens during the biosynthesis in the endoplasmic reticulum (ER).¹ As subunits are combined within the ER, they are accompanied by various proteins that direct them either toward the plasma membrane or into proteasomes for destruction as faulty assemblages. As discussed in a previous blog article, Validating a Novel Gel-free Method for Investigating G-Protein Coupled Receptor Proteomic Partners, structural integrity must be maintained in order to reveal the pathways involved and the receptor-protein interactions and in order to obtain meaningful proteomics results.

Recently, Roy et al. (2013) validated a novel gel-free proteomics method that avoids ligand/receptor disruption and allows researchers to investigate the GPCR interactome.² The researchers used liquid chromatography–tandem mass spectrometry (LC-MS/MS) to screen for and identify novel protein interactions.

Using a gel-free extraction method, they purified human β2-adrenergic (β2-AR) and prostaglandin D₂ (DPR) receptors from transfected human embryonic kidney (HEK) cells to investigate the proteomics of GPCR maturation and degradation pathways in the ER. The purified digests were then assayed using an LTQ XL Linear Ion Trap mass spectrometer (Thermo Scientific) to identify protein-protein interactions and reveal new binding partners.

Once the assay method was validated, Roy and co-workers used co-immunoprecipitation protocols to confirm the association between the ER quality control system (ERQC), ER-associated degradation (ERAD) and the receptors. ERAD detects mis-folded proteins and targets them for removal. Results showed that RING finger protein 5 (RNF5), an E3 ubiquitin ligase located in the ER and associated with GPCR turnover, associates with both β2-AR and DPR. Confocal fluorescence microscopy confirmed that these interactomes were located within the ER.

Additional experiments showed that, when RNF5 was co-expressed in the HEK cells, the total protein levels of β2-AR and DPR increased, whereas transfection with an RNF5 mutant form decreased the GPCR levels. This effect was not directly modulated through β2-AR or DPR ubiquitination, as shown by western-ligand blotting.

RNF5 did, however, increase ubiquitination of JNK-associated membrane protein (JAMP), another factor responsible for coordinating degradation of mis-folded proteins by the ERAD. Western-ligand blotting confirmed β2-AR and DPR association with JAMP, and confocal microscopy was used to show correct ER cellular localization. 

JAMP regulates proteolytic degradation by promoting protein targeting into proteasomes.¹ Incubating cells co-transfected with DPR and JAMP overnight in the presence of epoxomicin, an irreversible proteasome inhibitor, blocked the JAMP-associated decrease in GPCRs.

Further experiments confirmed that ubiquitination of JAMP by RNF5 was associated with increased levels of β2-AR and DPR, whereas decreased expression of RNF5 increased the β2-AR and DPR proteasomal degradation initiated by JAMP. Interestingly, the researchers also found that increased levels of β2-AR and DPR themselves seemed to protect RNF5 from proteasomal degradation, suggesting that GPCR interaction is protective.

In summary, using a structurally protective, gel-free purification method to prepare samples for proteomic analysis, researchers were able to discover novel ER binding partners for two GPCRs. From these data, they showed that RNF5 regulates β2-AR and DPR turnover by ubiquitinating JAMP and preventing proteasome uptake.

References

1. Tcherpakov, M., et al. (2009) “Regulation of endoplasmic reticulum-associated degradation by RNF5-dependent ubiquitination of JNK-associated membrane protein (JAMP),” Journal of Biological Chemistry, 284(18) (pp. 12099–109), doi: 10.1074/jbc.M808222200.

2. Roy, S.J., et al. (2013) “Novel, Gel-free Proteomics Approach Identifies RNF5 and JAMP as Modulators of GPCR Stability,” Molecular Endocrinology, 27(8) (pp. 1245–66).

Post Author: Amanda Maxwell. Mixed media artist; blogger and social media communicator; clinical scientist and writer; SAHM and expat trailing spouse. A digital space explorer, engaging readers by translating complex theories and subjects creatively into everyday language.