Ubiquitination of the chemokine receptor CXCR4 serves as a targeting signal for lysosomal degradation, but the mechanisms mediating ubiquitination and lysosomal sorting remain poorly understood. Here we report that the Nedd4-like E3 ubiquitin ligase AIP4 mediates ubiquitination of CXCR4 at the plasma membrane, and of the ubiquitin binding protein Hrs on endosomes. CXCR4 activation promotes CXCR4 colocalization with AIP4 and Hrs within the same region of endosomes. Endosomal sorting of CXCR4 is dependent on Hrs as well as the AAA ATPase Vps4, the latter involved in regulating the ubiquitination status of both CXCR4 and Hrs. We propose a model whereby AIP4, Hrs, and Vps4 coordinate a cascade of ubiquitination and deubiquitination events that sort CXCR4 to the degradative pathway.

Ligand-induced trafficking plays an important role in the physiologic regulation of many G protein-coupled receptors (GPCRs). Although numerous GPCRs are sorted to a degradative pathway upon prolonged stimulation, the molecular events leading to degradation are poorly understood. Here we report that the human immunodeficiency virus co-receptor CXCR4 undergoes rapid agonist-promoted degradation by a process involving endocytosis via clathrin-coated pits and subsequent sorting to lysosomes. Studies analyzing the sorting of various CXCR4 mutants revealed the presence of a degradation motif (SSLKILSKGK) in the carboxyl terminus of CXCR4. The first two serines as well as the dileucine motif were critical for agonist-induced endocytosis, whereas all three serines but not the dileucine were important in mediating degradation. Mutation of the three lysine residues had no effect on CXCR4 endocytosis yet completely inhibited receptor degradation. Because lysine residues represent potential sites of ubiquitination, we also examined the ubiquitination of CXCR4. Interestingly, CXCR4 was shown to undergo rapid agonist-promoted ubiquitination that was attenuated by mutation of the lysine residues within the degradation motif. These studies implicate a specific role for ubiquitination in sorting endocytosed GPCRs to lysosomes.

The heptahelical G protein-coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine-and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs.

High expression of Notch-1 and Jagged-1 mRNA correlates with poor prognosis in breast cancer. Elucidating the cross-talk between Notch and other major breast cancer pathways is necessary to determine which patients may benefit from Notch inhibitors, which agents should be combined with them, and which biomarkers indicate Notch activity in vivo. We explored expression of Notch receptors and ligands in clinical specimens, as well as activity, regulation, and effectors of Notch signaling using cell lines and xenografts. Ductal and lobular carcinomas commonly expressed Notch-1, Notch-4, and Jagged-1 at variable levels. However, in breast cancer cell lines, Notch-induced transcriptional activity did not correlate with Notch receptor levels and was highest in estrogen receptor α–negative (ERα–), Her2/Neu nonoverexpressing cells. In ERα+ cells, estradiol inhibited Notch activity and Notch-1IC nuclear levels and affected Notch-1 cellular distribution. Tamoxifen and raloxifene blocked this effect, reactivating Notch. Notch-1 induced Notch-4. Notch-4 expression in clinical specimens correlated with proliferation (Ki67). In MDA-MB231 (ERα–) cells, Notch-1 knockdown or γ-secretase inhibition decreased cyclins A and B1, causing G2 arrest, p53-independent induction of NOXA, and death. In T47D:A18 (ERα+) cells, the same targets were affected, and Notch inhibition potentiated the effects of tamoxifen. In vivo, γ;-secretase inhibitor treatment arrested the growth of MDA-MB231 tumors and, in combination with tamoxifen, caused regression of T47D:A18 tumors. Our data indicate that combinations of antiestrogens and Notch inhibitors may be effective in ERα+ breast cancers and that Notch signaling is a potential therapeutic target in ERα– breast cancers.