Using a novel approach that detects changes in the conformation of ERalpha, we studied the efficacy of anti-estrogens to inactivate ERalpha under different experimental conditions. We show that phosphorylation of serine-305 in the hinge region of ERalpha by protein kinase A (PKA) induced resistance to tamoxifen. Tamoxifen bound but then failed to induce the inactive conformation, invoking ERalpha-dependent transactivation instead. PKA activity thus induces a switch from antagonistic to agonistic effects of tamoxifen on ERalpha. In clinical samples, we found that downregulation of a negative regulator of PKA, PKA-RIalpha, was associated with tamoxifen resistance prior to treatment. Activation of PKA by downregulation of PKA-RIalpha converts tamoxifen from an ERalpha inhibitor into a growth stimulator, without any effect on ICI 182780 (Fulvestrant).
MHC class I molecules usually present peptides derived from endogenous antigens that are bound in the endoplasmic reticulum. Loading of exogenous antigens on class I molecules, e.g., in cross-priming, sometimes occurs, but the intracellular location where interaction between the antigenic fragment and class I takes place is unclear. Here we show that measles virus F protein can be presented by class I in transporters associated with antigen processing-independent, NH 4 Clsensitive manner, suggesting that class I molecules are able to interact and bind antigen in acidic compartments, like class II molecules. Studies on intracellular transport of green fluorescent protein-tagged class I molecules in living cells confirmed that a small fraction of class I molecules indeed enters classical MHC class II compartments (MIICs) and is transported in MIICs back to the plasma membrane. Fractionation studies show that class I complexes in MIICs contain peptides. The pH in MIIC (around 5.0) is such that efficient peptide exchange can occur. We thus present evidence for a pathway for class I loading that is shared with class II molecules.MHC molecules display antigenic peptides on the cell surface for surveillance by T lymphocytes. MHC class I molecules present peptides to CD8 ϩ cytotoxic T cells, whereas MHC class II molecules present peptides to CD4 ϩ Th cells. The current dogma is that antigens from the extracellular fluid enter the exogenous processing pathway by endocytosis and are partially degraded in acidic endosomal or lysosomal structures to yield peptides that bind MHC class II molecules. This type of processing is inhibited by reagents that prevent endosomal acidification (chloroquine, NH 4 Cl) (1). In the endogenous processing pathway intracellular proteins are degraded in the cytosol by the proteasome complex, generating peptides that are transported from the cytoplasm into the lumen of the endoplasmic reticulum (ER) by the transporters associated with antigen processing (TAP), where they bind to nascent MHC class I heavy chain- 2 -microglobulin ( 2 m) heterodimers. Fully assembled class I͞peptide complexes exit the ER and are transported through the Golgi to the cell surface by the constitutive secretory route. This processing pathway can be blocked by proteasome inhibitors or Brefeldin A (BFA), an inhibitor of anterograde ER-Golgi transport, but not by lysosomotropic agents. Thus, in general endogenous antigens are presented by MHC class I molecules, and exogenous antigens are displayed at the cell surface by MHC class II molecules. However accumulating evidence has shown that this dichotomy in presentation of antigen from endogenous and exogenous origin is not absolute. It was demonstrated that cytotoxic T lymphocyte (CTL) responses can be primed in vitro and in vivo with exogenous antigen (reviewed in refs. 2 and 3). At least two fundamentally different pathways for presentation of exogenous antigens by MHC class I molecules in vitro have been described: one involving access of exogenous antigen to...
Abstract. Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in living cells. For this purpose we stably expressed a modified HLA-DR1 13 chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (13-GFP) in class II-expressing Mel JuSo cells. This modification of the class II 13 chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ 13-GFP chimera was studied in living cells at 37°C. We visualize rapid movement of acidic class II/13-GFP containing vesicles from lysosomal compartments to the plasma membrane and show that fusion of these vesicles with the plasma membrane occurs. Furthermore, we show that this transport route does not intersect the earlier endosomal pathway. MHC class II molecules present peptides to CD4 ÷ T cells. Most bound peptides are derived from antigens degraded in the endosomal pathway. To allow association with these peptides, class II molecules are targeted to endosomal compartments by the invariant chain (or Ii) 1 (7,8). Here, Ii is degraded and Ii-degradation products are exchanged with antigenic peptides, a process catalyzed by 41,47,48). The endosomal compartments where class II molecules are loaded with peptide may be considered "special" lysosomes with a multilamellar and/or multivesicular appearance and were originally termed MIIC for MHC class II--containing compartments (31). This "unique" morphology appears to be induced by the expression of class II molecules (4). Although earlier endosomal compartments have been noted as "specialized class II loading compartments" as well (1, 50), HLA-DM and class II molecules are generally located in compartments with lysosomal proteins like CD63, lamp-l, and cathepsin D (13,24,32,42).
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