Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes.

The fluorescence recovery after photobleaching (FRAP) method is a straightforward means of assessing the diffusional mobility of membrane-associated proteins that is readily performed with current confocal microscopy instrumentation. We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility.

Metabolically Biotinylated Reporters for Electron Microscopic Imaging of Cytoplasmic Membrane Microdomains.

The protein and lipid substituents of cytoplasmic membranes are not in general homogeneously distributed across the membrane surface. Many membrane proteins, including ion channels, receptors, and other signaling molecules, exhibit a profound submicroscopic spatial organization, in some cases clustering in submicron membrane subdomains having a protein and lipid composition distinct from that of the bulk membrane. In the case of membrane-associated signaling molecules, mounting evidence indicates that their nanoscale organization, for example the colocalization of differing signaling molecules in the same membrane microdomains versus their segregation into distinct microdomain species, can significantly impact signal transduction.

Coarse-grained molecular simulation of epidermal growth factor receptor protein tyrosine kinase multi-site self-phosphorylation.

Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites.

A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains.

The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties.

Cbl controls EGFR fate by regulating early endosome fusion.

Amino acid residues 1 to 434 of the E3 ubiquitin ligase Cbl control signaling of the epidermal growth factor receptor (EGFR) by enhancing its ubiquitination, down-regulation, and lysosomal degradation. This region of Cbl comprises a tyrosine kinase-binding domain, a linker region, a really interesting new gene finger (RF), and a subset of the residues of the RF tail. In experiments with full-length alanine substitution mutants, we demonstrated that the RF tail of Cbl regulated biochemically distinct checkpoints in the endocytosis of EGFR.

Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain.

The C-terminal phosphorylation domain of the epidermal growth factor receptor is believed to regulate protein kinase activity as well as mediate the assembly of signal transduction complexes. The structure and dynamics of this proposed autoregulatory domain were examined by labeling the extreme C terminus of the EGFR intracellular domain (ICD) with an extrinsic fluorophore. Fluorescence anisotropy decay analysis of the nonphosphorylated EGFR-ICD yielded two rotational correlation times: a longer time, consistent with the global rotational motion of a 60- to 70-kDa protein with an elongated globular conformation, and a shorter time, presumably contributed by segmental motion near the fluorophore.

Conformational changes accompany phosphorylation of the epidermal growth factor receptor C-terminal domain.

The precise regulation of epidermal growth factor receptor (EGFR) signaling is crucial to its function in cellular growth control. Various studies have suggested that the C-terminal phosphorylation domain, itself a substrate for the EGFR kinase activity, exerts a regulatory influence upon it, although the molecular mechanism for this regulation is unknown. The fluorescence resonance energy transfer (FRET) technique was employed to examine how C-terminal domain conformational changes in the context of receptor activation and autophosphorylation might regulate EGFR enzymatic activity.

Activation of the epidermal growth factor receptor by respiratory syncytial virus results in increased inflammation and delayed apoptosis.

Respiratory syncytial virus (RSV) preferentially infects lung epithelial cells. Infection by RSV leads to an extended inflammatory response, characterized by the release of interleukin-8 (IL-8). Activation of ERK MAP kinase is required for both RSV-induced inflammation and the extended survival of infected cells.

Roles of mitogen-activated protein kinase and phosphoinositide 3'-kinase in ErbB2/ErbB3 coreceptor-mediated heregulin signaling.

ErbB2/HER2 and ErbB3/HER3, two members of the ErbB/HER family, together constitute a heregulin coreceptor complex that elicits a potent mitogenic and transforming signal. Among known intracellular effectors of the ErbB2/ErbB3 heregulin coreceptor are mitogen-activated protein kinase (MAPK) and phosphoinositide (PI) 3-kinase. Activation of the distinct MAPK and PI 3-kinase signaling pathways by the ErbB2/ErbB3 coreceptor in response to heregulin and their relative contributions to the mitogenic and transformation potentials of the activated coreceptor were investigated here.

Expression of inducible nitric oxide synthase in the lower esophageal sphincter of the endotoxemic opossum.

Background: Endotoxin modulates esophageal motor function by increasing nitric oxide (NO) production. The aims of this study were to examine inducible nitric oxide synthase (iNOS) induction in the lower esophageal sphincter (LES) of endotoxemic opossums and to investigate the effects of aminoguanidine (AG), a selective inhibitor of iNOS, on plasma nitrite/nitrate levels and on iNOS protein and mRNA expression after exposure to lipopolysaccharide (LPS).

Methods: Before and 12 h after the intravenous administration of LPS and/or AG, plasma nitrite/nitrate levels were determined.

Tyrosine phosphorylation of maspin in normal mammary epithelia and breast cancer cells.

Maspin is a 42kDa tumor suppressor protein that belongs to the serine protease inhibitor (serpin) family. It inhibits cell motility and invasion in vitro, and tumor growth and metastasis in nude mice; however, maspin's molecular mechanism of action has remained elusive. Maspin contains several tyrosine residues and we hypothesized that phosphorylation of maspin could play a role in its biological function.