Aqueous and micelle-bound structural characterization of the epidermal growth factor receptor juxtamembrane domain containing basolateral sorting motifs.

The EGF receptor is the prototype for four highly related receptors constituting the ErbB family. The EGF receptor is normally targeted to the basolateral membrane in polarized epithelial cells, where it relays information from underlying tissues. Two basolateral sorting signals have been mapped to the cytoplasmic juxtamembrane region of the receptor, a dominant signal comprised of a polyproline core (667-PXXP) and a preceding basic residue (Arg662), and a consensus leucine-based signal (658-LL) responsible for residual sorting when the 667-PXXP signal is absent or defective. The goal of this study was to define the structure of these signals, and gain some insights into how these structures might be regulated by cellular microenvironment. Structural information was acquired for two peptides corresponding to EGF receptor residues Arg645 and Ala674 in aqueous solution or in the presence of membrane-mimicking dodecylphosphocholine micelles, using a variety of NMR and CD spectroscopic methods. Chemical shift data indicate that the 667-PXXP signal does not bind to the micelles and is in random coil state in both aqueous solution and a micellar environment, raising the possibility that 667-PXXP switches to an ordered structure during interaction with the basolateral sorting machinery. In contrast, the adjacent region including 658-LL does bind to micelles mediated by a highly positively charged region located between Arg645 and Arg656. The micelle-bound region also includes Thr654, a known substrate for PKC. This suggests a distinct mode of regulation for this signal involving membrane association and/or phosphorylation.

Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination.

Epithelia of the vertebrate intestinal tract characteristically maintain an inflammatory hyporesponsiveness toward the lumenal prokaryotic microflora. We report the identification of enteric organisms (nonvirulent Salmonella strains) whose direct interaction with model human epithelia attenuate synthesis of inflammatory effector molecules elicited by diverse proinflammatory stimuli. This immunosuppressive effect involves inhibition of the inhibitor kappaB/nuclear factor kappaB (IkappaB/NF-kappaB) pathway by blockade of IkappaB-alpha degradation, which prevents subsequent nuclear translocation of active NF-kappaB dimer. Although phosphorylation of IkappaB-alpha occurs, subsequent polyubiquitination necessary for regulated IkappaB-alpha degradation is completely abrogated. These data suggest that prokaryotic determinants could be responsible for the unique tolerance of the gastrointestinal mucosa to proinflammatory stimuli.

Regulation of EGF signaling by cell polarity in MDCK kidney epithelial cells.

Although the presence of a dominant basolateral sorting signal ensures that the majority of newly synthesized epidermal growth factor (EGF) receptors are delivered directly to the basolateral surface in polarized epithelial cells, a fraction of the receptors are also delivered to the apical surface. Similar to most basolateral membrane proteins, the EGF receptor has an additional signal(s) that selectively targets molecules lacking a dominant basolateral signal to the apical surface. Although the physiological relevance of signal hierarchy is not known, alternative targeting may occur in different epithelial cell types or during development. The goal of this study, therefore, was to determine the effect of membrane domain location on EGF receptor function, focusing on EGF-induced MAP kinase signaling and DNA synthesis. Whereas ligand responsiveness was restricted to the basolateral domain in Madin-Darby canine kidney (MDCK) cells expressing a normal complement of receptors, apical ligand was effective if apical receptor density was increased by overexpression of an exogenous wild-type human gene. Unexpectedly, cells expressing apically localized, cytoplasmically truncated receptors, which behave as dominant negative mutations in other cell types, were also responsive to apical EGF. The cytoplasmically truncated molecules appear to have at least two effects: first, to increase the local concentration of ligand at the apical cell surface; and second, to facilitate activation of the relatively few native EGF receptors normally located at the apical surface. These results indicate that cell context is a critical determinant of receptor mutant protein phenotype.

The cytoplasmic juxtamembrane domain of the epidermal growth factor receptor contains a novel autonomous basolateral sorting determinant.

The epidermal growth factor receptor (EGFR) is localized at the basolateral membrane of most epithelial cells in vivo and in cell lines used to study membrane protein sorting. The goal of this study was to define the molecular basis of polar EGFR membrane expression using the Madin-Darby canine kidney cell model. We have identified a 23-amino acid segment located near the cytoplasmic face of the membrane spanning domain (residues Lys-652 to Ala-674) that is necessary and sufficient for targeting EGFRs from the trans-Golgi network directly to the basolateral plasma membrane. Furthermore, the sequence between residues Lys-652 and Ala-674 is sufficient to direct the extracellular domain of an apical membrane protein, decay accelerating factor, to the basolateral membrane. In the absence of this cytoplasmic basolateral sorting signal, information within the extracellular ligand binding domain is sufficient to target EGFRs from the trans-Golgi network directly to the apical plasma membrane. The EGFR basolateral sorting determinant does not have sequence and structural requirements common to most basolateral membrane proteins and does not overlap any of the known EGFR endocytic signals. This 23-residue sequence lies in a predicted amphipathic helical structure, leading us to postulate that hydrophobic and/or electrostatic interactions may be important for activity of this autonomous basolateral sorting determinant.

On the mechanism of rectification of the isoproterenol-activated chloride current in guinea-pig ventricular myocytes.

The whole cell configuration of the patch clamp technique was used to investigate the mechanism underlying rectification of the isoproterenol-activated chloride (Cl-) current in isolated guinea pig ventricular myocytes. When extracellular Cl- was replaced with either bromide (Br-), glutamate (Glut), iodide (I-), isethionate (Iseth), or nitrate (NO3-), the magnitude of the shift in reversal potential of the macroscopic current suggested the following selectivity sequence: NO3- > Br- > or = Cl- > or = I- > Iseth > or = Glut. This information was used to investigate the role of permeant ions in rectification of this current. Consistent with previous observations, when the concentration of intracellular Cl- (Cli-) was less than the concentration of extracellular Cl- (Clo-) (40 mM Cli-/150 mM Clo-) the current exhibited outward rectification, but when Cli- was increased to equal that outside (150 Cli-/150 Clo-), the current no longer rectified. Rectification in the presence of asymmetrical concentrations of permeant ions on either side of the membrane is predicted by constant field theory, as described by the Goldman-Hodgkin-Katz current equation. However, when the Cl- gradient was reversed (150 Cli-/40 Clo-) the current did not rectify in the opposite direction, and in the presence of lower symmetrical concentrations of Cl- inside and out (40 Cli-/40 Clo-), outward rectification did not disappear. Reducing Cli- by equimolar replacement with glutamate caused a concentration dependent increase in the degree of rectification. However, when Cli- was replaced with more permeant anions (NO3- and Br-), rectification was not observed. These results can be explained by a single binding site model based on Eyring rate theory, indicating that rectification is a function of the concentration and the permeability of the anions in the intracellular solution.