Down-regulation Of Receptor Activity Research Articles

Strategic Trastuzumab Mediated Crosslinking Driving Concomitant HER2 and HER3 Endocytosis and Degradation in Breast Cancer.

Efficacious anticancer therapies for targeting plasma membrane receptors with antibody based therapeutics are often contingent on sufficient endocytic delivery of receptor and conjugate to lysosomes. This results in downregulation of receptor activity and, in the case of antibody-drug conjugates (ADCs), intracellular release of a drug payload. The oncogenic receptor HER2 is a priority therapeutic target in breast cancer. Known as an “endocytosis resistant” receptor, HER2 thwarts the receptor downregulating efficiency of the frontline treatment trastuzumab and reduces the potential of trastuzumab-based therapies such as trastuzumab-emtansine. We previously demonstrated that strategically inducing trastuzumab and HER2 crosslinking in breast cancer cells promoted endocytosis and lysosomal delivery of the HER2-trastuzumab complex, stimulating downregulation of the receptor. Here we reveal that HER3, but not EGFR, is also concomitantly downregulated with HER2 after crosslinking. This is accompanied by strong activation of MEK/ERK pathway that we show does not directly contribute to HER2/trastuzumab endocytosis. We show that crosslinking induced trastuzumab endocytosis occurs via clathrin-dependent and independent pathways and is an actin-dependent process. Detailed ultrastructural studies of the plasma membrane highlight crosslinking-specific remodelling of microvilli and induction of extensive ruffling. Investigations in a cell model of acquired trastuzumab resistance demonstrate, for the first time, that they are refractory to crosslinking induced HER2 endocytosis and downregulation. This implicates further arrest of HER2 internalisation in developing trastuzumab resistance. Overall our findings highlight the potential of receptor crosslinking as a therapeutic strategy for cancer while exposing the ability of cancer cells to develop resistance via endocytic mechanisms.

Activated T lymphocytes suppress osteoclastogenesis by diverting early monocyte/macrophage progenitor lineage commitment towards dendritic cell differentiation through down-regulation of receptor activator of nuclear factor-kappaB and c-Fos

Activated T lymphocytes either stimulate or inhibit osteoclastogenesis from haematopoietic progenitors in different experimental models. To address this controversy, we used several modes of T lymphocyte activation in osteoclast differentiation--mitogen-pulse, anti-CD3/CD28 stimulation and in vivo and in vitro alloactivation. Osteoclast-like cells were generated from non-adherent immature haematopoietic monocyte/macrophage progenitors in murine bone-marrow in the presence of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and monocyte-macrophage colony-stimulating factor (M-CSF). All modes of in vivo and in vitro T lymphocyte activation and both CD4(+) and CD8(+) subpopulations produced similar inhibitory effects on osteoclastogenesis paralleled by enhanced dendritic cell (DC) differentiation. Osteoclast-inhibitory effect was associated with T lymphocyte activation and not proliferation, and could be replaced by their culture supernatants. The stage of osteoclast differentiation was crucial for the inhibitory action of activated T lymphocytes on osteoclastogenesis, because the suppressive effect was visible only on early osteoclast progenitors but not on committed osteoclasts. Inhibition was associated specifically with increased granulocyte-macrophage colony-stimulating factor (GM-CSF) expression by the mechanism of progenitor commitment toward lineages other than osteoclast because activated T lymphocytes down-regulated RANK, CD115, c-Fos and calcitonin receptor expression, and increased differentiation towards CD11c-positive DC. An activated T lymphocyte inhibitory role in osteoclastogenesis, confirmed in vitro and in vivo, mediated through GM-CSF release, may be used to counteract activated bone resorption mediated by T lymphocyte-derived cytokines in inflammatory and immune disorders. We also demonstrated the importance of alloactivation in osteoclast differentiation and the ability of cyclosporin A to abrogate T lymphocyte inhibition of osteoclastogenesis, thereby confirming the functional link between alloreaction and bone metabolism.

Synergistic activities of multiple phosphotyrosine residues mediate full signaling from the Drosophila Torso receptor tyrosine kinase.

Here, we identify four tyrosine residues (Y644, Y698, Y767, and Y772) that become phosphorylated after activation of the Torso (Tor) receptor tyrosine kinase. Previously, we characterized phosphotyrosine sites (P-Y630 and P-Y918). Of the six P-Y sites identified, three (Y630, Y644, and Y698) are located in the kinase domain insert region, one (Y918) is located in the C-terminal tail region, and two (Y767 and Y772) are located in the activation loop of the kinase domain. To investigate the function of each P-Y residue in Tor signaling, we have generated transgenic Drosophila embryos expressing mutant Tor receptors containing either single or multiple tyrosine to phenylalanine substitutions. Single P-Y mutations were found to have either positive, negative, or no effect on the signaling activity of the receptor. Elimination of all P-Y sites within the kinase insert region resulted in the complete loss of receptor function, indicating that some combination of these sites is necessary for Tor signaling. Mutation of the C-terminal P-Y918 site revealed that this site is responsible for negative signaling or down-regulation of receptor activity. Mutation of the P-Y sites in the kinase domain activation loop demonstrated that these sites are essential for enzymatic activity. Our analysis provides a detailed in vivo example of the extent of cooperativity between P-Y residues in transducing the signal received by a receptor tyrosine kinase and in vivo data demonstrating the function of P-Y residues in the activation loop of the kinase domain.

Apolipoprotein E polymorphism and the distribution profile of very low density lipoproteins; an influence of the E4 allele on large (Sf>60) particles

Very low density lipoprotein (VLDL) distribution and composition have been examined as a function of apo E genotype (E2/2+E2/3 vs. E3/3 vs. E3/4+E4/4) in healthy, normolipaemic subjects. Apo E genotype had a marked impact on plasma concentrations of apo E rich VLDL, but no influence on concentrations of apo E free particles. Thus, there was a trend to lower concentrations of apo E rich total VLDL in apo E4 carriers (mg/dl; E2, 49.1±35.2; E3, 52.5±30.9; E4 35.2±22.3; ANOVA P=0.16; when comparing E4 with E2+E3, P=0.06). Consequently, there were highly significant differences between apo E-defined subgroups in terms of the percentage distribution of bound and non-bound fractions (% total VLDL non-bound to apo E: E2, 44.0±12.7%; E3, 39.7±8.7%; E4 51.0±12.2%; ANOVA P=0.007). Subfractionation of VLDL into density subclasses revealed that genotype differences were restricted to large VLDL (Sf>60). Significantly lower concentrations of apo E-rich particles were observed in E4 carriers for VLDL-1 Sf 400–100 (ANOVA P=0.004) and VLDL-2 ( P=0.009) but not for small VLDL-3 Sf 60–20 ( P=0.34). No differences in plasma concentrations of apo E free VLDL were observed between genotype subclasses across the density spectrum. Compositional differences between the apo E defined VLDL were also evident for the core lipids. Apo E containing VLDL was enriched in esterified cholesterol and depleted in triglycerides compared to apo E poor VLDL: the difference became more marked with increasing density of the particles. Lipoprotein composition was not modulated to any great extent by apo E genotype. In patients with familial hypercholesterolaemia, relative concentrations of apo E rich, large VLDL were significantly higher than in controls. Treatment lowered concentrations of both apo E rich and apo E free VLDL but led to a greater relative enrichment of large VLDL in apo E containing particles. Apo E polymorphism appears to influence plasma concentrations of VLDL particles. The data are consistent with more pronounced receptor-mediated elimination of apo E4 containing VLDL. This may be a contributory factor to the down regulation of receptor activity which is suggested to be of major importance in provoking higher cholesterol levels associated with the apo E4 isoform.