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

Abstract

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.

Ornithine Aminotransferase, an Important Glutamate-Metabolizing Enzyme at the Crossroads of Multiple Metabolic Pathways

“…Proteins involved, detected in a very large-scale co-precipitation study [95], with some of the interactions that were only predicted, are NME2 (nucleotide diphosphate kinase, involved in tumorigenesis), EZR (ezrin), the aspartate aminotransferase GOT1 (or more probably GOT2, its mitochondrial equivalent); ESD (esterase D, a marker of retinoblastoma), AKR1B1 (reducing aldehydes and ketones), ALDH1B1, CBS, SOD1 and SOD2, PDXK (pyridoxal kinase), UAP1L1, UQCRFS1, PREP, PGM1, PITPNB, PPID and ADSS, the biological relevance of which is still difficult to assess. Other studies have detected interactions with NTRK1 [96], HDAC5 (a histone deacetylase [97]), FAF2 [98], NUDT3 [99], SIRT-7 [100], ICT1 [101], SLC35F6 [102], OTUD4 [103], DMWD [103], CDK2 [104], UBQLN4 [105], HUWE1 [106], FUS1 [107], EGFR [108], ADRB2 [109], FBXO6 [110], CTSA [111], ATF2 [112]. However, to our knowledge, there is no experimental confirmation that these interactions occur in normal cells under physiological conditions.…”

A compendium of human genes regulating feeding behavior and body weight, its functional characterization and identification of GWAS genes involved in brain-specific PPI network

“…filesPutative homologs of human SNRPC and SNRPN were found interacting in yeast [106] ERBB4 Group enriched /TSEA[29]rs7599312 STAT3 / OMIM_all_text 10/3 Expressed in all /DEGMediumPutative homologs (ErbB-2 and Stat3) were found interacting in mice [107] PRKD1 Mixed /DEG[30][29]rs11847697rs12885454rs11847697MAPK9/ OMIM_all_text 4/9 Expressed in all /TSEAMediumHuman PRKD1 (=PKD) and MAPK8 (=JNK), which is MAPK9 homolog, were found interacting in humans [108]

a DEG, differentially expressed gene in mouse hypothalamic AGRP- or POMC-expressing neurons according to [49]

b TSEA gene. Revealed by TSEA (see Gene expression analysis section) as brain-specific at the pSI threshold = 0.05 c The rank was defined for each gene in the network Experimental_brain-specific on the base of the number of first neighbors d In [103] NDUFS3 was noted as a precursor of NADH-ubiquinone oxidoreductase flavoprotein 3 isoform a (see Supplemental data in [103])

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“…Revealed by TSEA (see Gene expression analysis section) as brain-specific at the pSI threshold = 0.05 c The rank was defined for each gene in the network Experimental_brain-specific on the base of the number of first neighbors d In [103] NDUFS3 was noted as a precursor of NADH-ubiquinone oxidoreductase flavoprotein 3 isoform a (see Supplemental data in [103])…”

“…d In [103] NDUFS3 was noted as a precursor of NADH-ubiquinone oxidoreductase flavoprotein 3 isoform a (see Supplemental data in [103])…”

Mass spectrometry analysis of PPIP5K1 interactions and data on cell motility of PPIP5K1-deficient cells

“…Tagged PPIP5K1 WT and PPIP5K1 ΔC constructs contained a hemagglutinin (HA) tag, streptavidin binding domain (SBD), TEV (tobacco etch virus) protease cleavage site, and a calmodulin-binding domain (CBD) at their N-termini. The purification protocol was adapted from previously published protocol from our group [2] . T175 flasks were placed on ice and cells were washed twice with ice-cold PBS.…”