Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.     

Functional selectivity of G protein signaling by agonist peptides and thrombin for the protease-activated receptor-1

Thrombin activates protease-activated receptor-1 (PAR-1) by cleavage of the amino terminus to unmask a tethered ligand. Although peptide analogs can activate PAR-1, we show that the functional responses mediated via PAR-1 differ between the agonists. Thrombin caused endothelial monolayer permeability and mobilized intracellular calcium with EC(50) values of 0.1 and 1.7 nm, respectively. The opposite order of activation was observed for agonist peptide (SFLLRN-CONH(2) or TFLLRNKPDK) activation. The addition of inactivated thrombin did not affect agonist peptide signaling, suggesting that the differences in activation mechanisms are intramolecular in origin. Although activation of PAR-1 or PAR-2 by agonist peptides induced calcium mobilization, only PAR-1 activation affected barrier function. Induced barrier permeability is likely to be Galpha(12/13)-mediated as chelation of Galpha(q)-mediated intracellular calcium with BAPTA-AM, pertussis toxin inhibition of Galpha(i/o), or GM6001 inhibition of matrix metalloproteinase had no effect, whereas Y-27632 inhibition of the Galpha(12/13)-mediated Rho kinase abrogated the response. Similarly, calcium mobilization is Galpha(q)-mediated and independent of Galpha(i/o) and Galpha(12/13) because pertussis toxin Y-27632 and had no effect, whereas U-73122 inhibition of phospholipase C-beta blocked the response. It is therefore likely that changes in permeability reflect Galpha(12/13) activation, and changes in calcium reflect Galpha(q) activation, implying that the pharmacological differences between agonists are likely caused by the ability of the receptor to activate Galpha(12/13) or Galpha(q). This functional selectivity was characterized quantitatively by a mathematical model describing each step leading to Rho activation and/or calcium mobilization. This model provides an estimate that peptide activation alters receptor/G protein binding to favor Galpha(q) activation over Galpha(12/13) by approximately 800-fold.     

A cline in frequency of autosomal males is not associated with insecticide resistance in house fly (Diptera: Muscidae)

Geographic variation in the chromosomal location of the male sex determining factor (M) was studied in four house fly, Musca domestica L., populations from the eastern United States. We found a strong clinal trend (29 degrees 41' latitude in Florida to 44 degrees 2' in Maine) in which the percentage of standard XY(M) males increased with increasing latitude. In Florida, 100% of the males possessed the M factor on the third autosome (III(M)). North Carolina had 20% III(M) males and 2.35% with both Y(M) and III(M). Fewer III(M) males were found in New York (4.35%). Populations from Maine contained 100% XY(M) males. In two of three standard laboratory-susceptible strains, all males carried M on an autosome ("autosomal males" or A(M)): CS (III(M)) and SRS (V(M)). Insecticide bioassays of four field-collected strains led us to conclude that resistance is not correlated with sex determination over a broad range of insecticides. For example, high levels of resistance to permethrin (86-99% survival at a diagnostic concentration) were found in all four field-collected strains. The five other insecticides evaluated showed varying levels of resistance among field strains. We conclude that a cline is present in house fly populations from the eastern United States with 100% III(M) males in the south and entirely Y(M) males in the north and that insecticide resistance is not a key factor influencing the evolution or linkage of M.     

A two-dimensional infrared study of localization, structure, and dynamics of a dipeptide in membrane environment

Using methods of time-resolved two-dimensional infrared (2D-IR) spectroscopy, we approach the problem of the structural characterization of small polypeptide systems in a membrane environment. The 2D-IR spectra recorded for a model dipeptide in different environments demonstrated a significant change in the homogeneous and inhomogeneous broadenings of the amide I resonances when the molecule inserts either into a surfactant or a phospholipid membrane. Besides the change in the diagonal features in the 2D-IR response, we observe both intramolecular and intermolecular crosspeaks between the carbonyls of the dipeptide and the phospholipid. Considering the character of the diagonal peaks and the presence of the crosspeaks, we discuss the localization of the dipeptide moieties in the membrane. Using both the anisotropy and relative intensity of the observed intramolecular crosspeaks between the two amide I modes, we provide observables that help to determine the phi/psi-dihedral angles for the backbone of the dipeptide. Time dependent studies revealed slower conformational fluctuations of the dipeptide backbone in a membrane as compared to that in an aqueous environment.     

Structural basis of function in heterotrimeric G proteins

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) act as molecular switches in signaling pathways by coupling the activation of heptahelical receptors at the cell surface to intracellular responses. In the resting state, the G-protein alpha subunit (Galpha) binds GDP and Gbetagamma. Receptors activate G proteins by catalyzing GTP for GDP exchange on Galpha, leading to a structural change in the Galpha(GTP) and Gbetagamma subunits that allows the activation of a variety of downstream effector proteins. The G protein returns to the resting conformation following GTP hydrolysis and subunit re-association. As the G-protein cycle progresses, the Galpha subunit traverses through a series of conformational changes. Crystallographic studies of G proteins in many of these conformations have provided substantial insight into the structures of these proteins, the GTP-induced structural changes in Galpha, how these changes may lead to subunit dissociation and allow Galpha and Gbetagamma to activate effector proteins, as well as the mechanism of GTP hydrolysis. However, relatively little is known about the receptor-G protein complex and how this interaction leads to GDP release from Galpha. This article reviews the structural determinants of the function of heterotrimeric G proteins in mammalian systems at each point in the G-protein cycle with special emphasis on the mechanism of receptor-mediated G-protein activation. The receptor-G protein complex has proven to be a difficult target for crystallography, and several biophysical and computational approaches are discussed that complement the currently available structural information to improve models of this interaction. Additionally, these approaches enable the study of G-protein dynamics in solution, which is becoming an increasingly appreciated component of all aspects of G-protein signaling.     

Myristoylation exerts direct and allosteric effects on Gα conformation and dynamics in solution

Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gβγ and activated receptors. N-Terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen-deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gα(i) functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.     

Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity.     

Combined in situ zymography, immunofluorescence, and staining of iron oxide particles in paraffin-embedded, zinc-fixed tissue sections

Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.