Dioxin-inducible transactivation in a chromosomal setting. Analysis of the acidic domain of the Ah receptor

We analyzed the transactivation function of the acidic segment of the Ah receptor (amino acids 515-583) by reconstituting AhR-defective mouse hepatoma cells with mutants. Our data reveal that both hydrophobic and acidic residues are important for transactivation and that these residues are clustered in two regions of the acidic segment of AhR. Both regions are crucial for function, because disruption of either one substantially impairs transactivation of the chromosomal CYP1A1 target gene. Neither region contains an amino acid motif that resembles those reported for other acidic activation domains. Furthermore, proline substitutions in both regions do not impair transactivation in vivo, a finding that implies that alpha-helix formation is not required for function.     

Cytoplasmic domain of human Fcalpha/mu receptor is required for ligand internalization

The Fcalpha/mu receptor (Fcα/μR), a type I transmembrane protein, is an immunoglobulin Fc receptor for both IgA and IgM. Its functions in immune defense are not clear at present. In this work, human Fcα/μR was expressed in CHO, 293T, and COS-7 cells to study its biochemical functions. Fcα/μR expressed by CHO and 293T was only in monomer form in cytoplasma and the monomeric receptor could not bind IgA or IgM. In comparison, Fcα/μR expressed by COS-7 cells had both monomer and dimer forms. The binding assay showed that Fcα/μR expressed by COS-7 cells could bind IgM strongly and IgA weakly, implying that dimeric receptor could be expressed on cell membrane and functioned. The bound IgM could be internalized and the internalization was abolished when the cytoplasmic domain of Fcα/μR was truncated. Therefore, the cytoplasmic portion of human Fcα/μR is required in the internalization.     

The lid subdomain of DnaK is required for the stabilization of the substrate-binding site

We examined the effect of deletion of different segments in the helical subdomain (the so-called "lid") of the DnaK peptide-binding domain on peptide binding and protein stability. At 25 degrees C, wt DnaK and the deletion mutant proteins are able to stably bind peptides with similar affinity. However, at physiological (37 degrees C) and stress (42 degrees C) temperatures, removal of the N-terminal half of alphaB and the rest of the lid drastically decreases the ability of the protein to bind substrates. Differential scanning calorimetry and infrared spectroscopy show that this behavior is accompanied by destabilization of the peptide-binding domain. Our data suggest that the reversible interaction between the lid and beta-sandwich subdomains of DnaK peptide-binding domain is required for the stabilization of the loops that form the peptide-binding site, which in turn modulates the protein affinity for peptide substrates. This interaction might have functional implications because it could prevent rebinding of the peptide substrate, which would be forced to fold.     

Cytoplasmic juxtamembrane domain of the human EGF receptor is required for basolateral localization in MDCK cells

Although it is well established that epidermal growth factor receptors (EGFRs) are asymmetrically expressed at the basolateral plasma membrane in polarized epithelial cells, how this process is regulated is not known. The purpose of this study was to address the mechanism of directed EGFR basolateral sorting using the Madin-Darby canine kidney (MDCK) cell model. The first set of experiments established sorting patterns for endogenous canine EGFRs. The polarity of the canine EGFR was not quantitatively affected by differences in electrical resistance exhibited by the MDCK I and MDCK II cell strains. In both cases, greater than 90% of total surface EGFRs was localized to the basolateral surface. Canine EGFRs sort directly to the basolateral membrane from the trans-Golgi network with a halftime of approximately 45 min and have an approximate t_1/2 of 12.5 h once reaching the basolateral surface. Human holoreceptors expressed in stably transfected MDCK cells also localize to the basolateral membrane with similar efficiency. To identify EGFR sequences necessary for basolateral sorting, MDCK cells were transfected with cDNAs coding for cytoplasmically truncated human receptor proteins. Human EGFRs truncated at Arg-651 were localized predominantly at the apical surface of filter-grown cells, whereas receptors truncated at Leu-723 were predominantly basolateral. These results suggest that the cytoplasmic juxtamembrane domain contains a positive basolateral sorting determinant. Moreover, the EGFR ectodomain or transmembrane domain may possess a cryptic sequence that specifically interacts with the apical sorting machinery once the dominant basolateral sorting signal is removed. Further elucidation of the precise loacation of these signals will enhance our basic understanding of regulated plasma membrane sorting, as well as the functional consequences of inappropriate EGFR expression associated with certain pathophysiologic and malignant states. © 1995 Wiley-Liss, Inc.     

The ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

To examine the role of the ligand binding domain of epidermal growth factor receptor in its dimerization, we studied the dimerization of a truncated form of the receptor that resembles v-erbB in that it lacks a ligand binding domain. Receptor dimerization was determined by sedimentation analysis on sucrose density gradients at different concentrations of Triton X-100. At high concentrations of Triton X-100 (0.2%), the truncated receptor occurred as a monomer and displayed low basal autophosphorylation. By contrast, at low concentrations of Triton X-100 (0.01%), it existed as a dimer and exhibited high basal autophosphorylation. The ability of the truncated receptor to dimerize indicates that the ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.     

The N-terminal extracellular domain is required for polycystin-1-dependent channel activity

Autosomal dominant polycystic kidney disease (PKD) is caused by mutation of polycystin-1 or polycystin-2. Polycystin-2 is a Ca(2+)-permeable cation channel. Polycystin-1 is an integral membrane protein of less defined function. The N-terminal extracellular region of polycystin-1 contains potential motifs for protein and carbohydrate interaction. We now report that expression of polycystin-1 alone in Chinese hamster ovary (CHO) cells and in PKD2-null cells can confer Ca(2+)-permeable non-selective cation currents. Co-expression of a loss-of-function mutant of polycystin-2 in CHO cells does not reduce polycystin-1-dependent channel activity. A polycystin-1 mutant lacking approximately 2900 amino acids of the extracellular region is targeted to the cell surface but does not produce current. Extracellular application of antibodies against the immunoglobulin-like PKD domains reduces polycystin-1-dependent current. These results support the hypothesis that polycystin-1 is a surface membrane receptor that transduces the signal via changes in ionic currents.     

Mitochondrial function is required for hydrogen peroxide-induced growth factor receptor transactivation and downstream signaling

The transactivation of growth factor receptors is an early event in H(2)O(2)-induced signaling, although proximal targets in this process remain unclear. We found that inhibition of flavin- or heme-containing proteins eliminated H(2)O(2)-induced transactivation of the epidermal growth factor receptor and stimulation of its downstream targets, JNK and Akt. Inhibition of mitochondrial function with rotenone, antimycin A, KCN, carbonylcyanide-m-chlorophenylhydrazone, or oligomycin reproduced this effect, as did generation of mitochondrial DNA-deficient (pseudo-rho(0)) cells. Mitochondrial function had no role in JNK activation in response to UV irradiation or tumor necrosis factor-alpha. The impact of mitochondrial function on H(2)O(2)-induced growth factor transactivation was ubiquitous and applied to both the vascular endothelial growth factor (VEGF)-2 receptor and the platelet-derived growth factor-beta receptor in endothelium and fibroblasts, respectively. In contrast, ligand-induced growth factor activation was unrelated to mitochondrial function. Growth factor receptor transactivation and its downstream signaling in response to H(2)O(2) appeared to involve redox-sensitive mitochondrial events as they were abrogated by a mitochondrial-targeted antioxidants but not their nontargeted counterparts. Functionally, we found that mitochondrial-targeted antioxidants inhibited H(2)O(2)-induced apoptosis and cell death but had no effect with UV irradiation. These data establish a novel role for the mitochondrion as a proximal target specific to H(2)O(2)-induced signaling and growth factor transactivation.