The ubiquitin ligase RNF126 regulates the retrograde sorting of the cation-independent mannose 6-phosphate receptor

The ubiquitin proteasome system is central to the regulation of a number of intracellular sorting pathways in mammalian cells including quality control at the endoplasmic reticulum and the internalization and endosomal sorting of cell surface receptors. Here we describe that RNF126, an E3 ubiquitin ligase, is involved in the sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR). In cells transiently depleted of RNF126, the CI-MPR is dispersed into Rab4 positive endosomes and the efficiency of retrograde sorting is delayed. Furthermore, the stable knockdown of RNF126 leads to the lysosomal degradation of CI-MPR and missorting of cathepsin D. RNF126 specifically regulates the sorting of the CI-MPR as other cargo that follow the retrograde sorting route including the cholera toxin, furin and TGN38 are unaffected in the absence of RNF126. Lastly we show that the RING finger domain of RNF126 is required to rescue the decrease in CI-MPR levels, suggesting that the ubiquitin ligase activity of RNF126 is required for CI-MPR sorting. Together, our data indicate that the ubiquitin ligase RNF126 has a role in the retrograde sorting of the CI-MPR     

Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor

Disruption of latent TGF-beta binding protein (LTBP)-4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-beta, whereas secretion of latent TGF-beta was significantly increased. Expression and secretion of TGF-beta2 and -beta3 increased considerably. These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the -/- fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4-mediated targeting and activation of TGF-beta1 leads to enhanced BMP-4 signaling in mouse lung.     

Intracellular trafficking of LRP9 is dependent on two acidic cluster/dileucine motifs

LDL receptor-related protein 9 (LRP9) is a distant member of the low-density lipoprotein receptor (LDLR) superfamily. To date, there are no reports on the cellular distribution of LRP9 or the signals responsible for its localization. Here, we investigated the intracellular localization and trafficking of LRP9. Using confocal microscopy, we demonstrated that LRP9 was not present at the plasma membrane but co-localized with various markers of the trans-Golgi network (TGN) and endosomes. This co-localization was dependent on the presence of two acidic cluster/dileucine (DXXLL) motifs in the cytoplasmic tail of LRP9, which interact with GGA proteins, clathrin adaptors involved in transport between the TGN and endosomes. LRP9 is the first example of a transmembrane protein with an internal GGA-binding sequence in addition to the usual C-terminal motif. An inactivating mutation (LL --> AA) in both DXXLL motifs, which completely inhibited the interaction of LRP9 with GGA proteins, led to an intracellular redistribution of LRP9 from the TGN to early endosomes and the cell surface, indicating that the two DXXLL motifs are essential sorting determinants of LRP9. In conclusion, our results suggest that LRP9 cycles between the TGN, endosomes and the plasma membrane through a GGA dependent-trafficking mechanism.     

Selective targeting of avidin/mannose 6-phosphate receptor chimeras to early or late endosomes

In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulated within, early endosomes. This truncated chimera did not reach recycling early endosomes labelled with internalised transferrin, to any significant extent, but was accessible to biotinylated HRP internalised for 5 min (or for longer periods at 19 degrees C). Coinfection of these chimeras showed that they follow the same route from the TGN to the early endosomes. We conclude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results also suggest that the YKYSKV sequence close to the CI-MPR transmembrane segment is sufficient for targeting to sorting early endosomes.     

Role of CREB in the regulatory action of sarsasapogenin on muscarinic M1 receptor density during cell aging

This work studied the role of cyclic AMP responsive element binding protein (CREB) in the up-regulation of M₁ muscarinic acetylcholine receptor (M₁ receptor) density by sarsasapogenin (ZMS) in CHO cells transfected with M₁ receptor gene (CHOm1 cells). During cell aging, sarsasapogenin elevated M₁ receptor density as well as CREB and phosphor-CREB (pCREB) levels. CREB peaked earliest, followed by pCREB and M₁ receptor density peaked last. When CREB synthesis was blocked by antisense oligonucleotides, the elevation effect of sarsasapogenin on M₁ receptor density was abolished. These results suggest that sarsasapogenin up-regulates M₁ receptor density in aged cells by promoting CREB production and phosphorylation. Furthermore, the results support the hypothesis that pCREB regulates M₁ receptor gene expression through heterodimer formation.