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B F Dickey

Publications and source records attributed to B F Dickey.

At least 37 records · Page 2Linked to original sources
PubMed1996

Repetitive endocytosis and recycling of the beta 2-adrenergic receptor during agonist-induced steady state redistribution.

The human beta 2-adrenergic receptor (beta 2AR) rapidly internalizes after binding agonist, resulting in a dramatic redistribution of receptors from the plasma membrane and into endocytic vesicles. We sought to determine whether intracellular receptors constitute a static pool or represent a fraction of dynamically internalizing and recycling receptors. Using cells expressing a beta 2AR with an epitope tag at its amino-terminal ectodomain, changes in surface receptor levels were measured by flow cytometry and radioligand binding assays. The addition of a saturating level of a strong agonist (isoproterenol) caused the endocytosis of receptors with first-order kinetics (ke for naive cells, 0.222 min-1). After 10 min, the level of surface receptors remained stable at approximately 20% that of untreated cells, even though endocytosis continued with similar kinetics (ke for pretreated cells, 0.258 min-1), suggesting that internalized receptors were cycling in steady state with surface receptors. This prediction was confirmed directly by showing that internalized beta 2ARs recycled to the cell surface in the continued presence of agonist. The calculated transit times (1/k) in the presence of isoproterenol were 3.9 min for endocytosis and 11.2 min for recycling. The endocytic rate constant and the steady state redistribution to the internal pool were much lower after treatment with the partial agonist albuterol, suggesting a correlation between the efficiency of endocytosis and that of receptor coupling to the downstream signal transduction pathway. These findings indicate that in the presence of agonist, beta 2ARs are in a dynamic steady state between the plasma membrane and endosomes that is regulated principally by agonist efficacy.

Adrenergic beta-Agonists↗
PubMed1995

Functional and structural interactions of the Rab5 D136N mutant with xanthine nucleotides.

Rab5 is a Ras-related GTPase which regulates endosomal fusion. The D136N mutant of Rab5, which was predicted to switch specificity from guanine to xanthine nucleotides, was expressed in E. coli, extracted with urea, purified by column chromatography, and refolded by stepwise dialysis against buffer containing XDP. The purified protein bound xanthine nucleotides with considerably higher affinity than guanine nucleotides. In vitro prenylation of the mutant protein was highly dependent on xanthosine diphosphate. In contrast, both the wild type and mutant proteins were protected from proteolysis equally well by non-cognate and cognate triphosphate nucleosides at high concentration. The D136N Rab5 mutant appears to be a valuable reagent in conjunction with xanthine nucleotides for the study of protein-nucleotide interactions in systems in which multiple GTPases are active, although interactions with non-cognate nucleotides should be evaluated if they are present at high concentration.

Base Sequence↗
PubMed1995

Biochemical and functional characterization of a recombinant GTPase, Rab5, and two of its mutants.

Biochemical, structural, and functional properties of Rab5 wild-type (WT) protein were compared with those of Q79L and N133I mutants. The detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate increased guanine nucleotide binding to Rab5 WT approximately 10-fold. The single-step catalytic rate of Rab5 WT exceeded that of Q79L 12.2-fold, but the steady-state GTPase rate was only 2.8-fold greater because GDP dissociation was rate-limiting and GDP dissociation was 3.6-fold slower than for Q79L. In contrast, dissociation rates of GTP were indistinguishable. Binding to Rab5 N133I was not detectable. GTP protected Rab5 WT and Q79L from any apparent proteolysis by trypsin. A 20-kDa fragment was the major product of digestion in the presence of GDP, and 12- and 8-kDa fragments were the major products in the absence of added guanine nucleotides. Rab5 N133I underwent no apparent proteolysis with 10 mM GTP or GDP, suggesting a "triphosphate" conformation may be induced in Rab5 N133I by either GTP or GDP. Partially geranylgeranylated Rab5 WT stimulated endosome fusion in vitro, whereas unmodified Rab5 WT did not. Processed Rab5 Q79L failed to inhibit endosome fusion, and Rab5 N133I could not be geranylgeranylated. These findings identify biochemical and structural features of Rab5 proteins, providing data for the interpretation of functional assays.

Amino Acid Sequence↗
PubMed1995

Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes.

The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor. Nearly all receptors were on the cell surface in the absence of agonist, but within ten minutes of agonist addition > 50% of receptors internalized and colocalized extensively with rab5. Hypertonic sucrose blocked beta 2-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediated process. In contrast, an inhibitor of potocytosis had little effect upon beta 2-adrenergic receptor internalization, suggesting that this process did not require active caveolae. Consistent with this finding, caveolin was not detectable in the 12 beta 6 line, as assessed by western blotting with a polyclonal anti-caveolin antibody. Stimulated receptor internalization did not affect the rate or capacity of the constitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of beta-agonist, nor was there a significant change in the amount of surface transferrin receptor. Altogether, these data suggest that beta 2-adrenergic receptors internalize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway. Further, agonist-stimulated receptor internalization has no detectable effect upon the function of this pathway.

Adrenergic beta-2 Receptor Agonists↗
PubMed1994

Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit.

Neutrophil infiltration is a prominent feature of Clostridium difficile-associated enteritis and colitis. The aim of this study was to examine the importance of neutrophil recruitment and neutrophil-mediated tissue damage in C. difficile toxin A-induced enteritis. Competitive binding experiments using purified 3H-toxin A demonstrated the presence of a single class of medium affinity receptors on rabbit neutrophils (Kd 7 x 10(-8) M). Pertussis toxin and the nonhydrolyzable GTP analog GTPgamma S both inhibited 3H-toxin A binding (by 56 and 65%, respectively), indicating that the rabbit neutrophil toxin A receptor is G protein linked. Toxin A elicited a dose-dependent (25-200 micrograms/ml) stimulation of neutrophil migration in vitro, and this functional effect was also pertussis toxin sensitive (69% inhibition). Treatment of neutrophils with R15.7, a blocking monoclonal antibody to the leuocyte adhesion molecule CD18, inhibited toxin A-stimulated neutrophil migration by 85% in vitro. Pretreatment of rabbits with R15.7 also prevented neutrophil infiltration of toxin A-exposed ileal loops in vivo as determined by histologic examination and by ileal tissue myeloperoxidase levels. Furthermore, R15.7 effected a substantial inhibition of fluid secretion (by 65%), mannitol permeability (by 66%), and histologic damage in toxin A-exposed ileal loops. Anti-CD18 (R15.7) had no inhibitory effect on cholera toxin enterotoxicity. These data demonstrate that C. difficile toxin A is a proinflammatory toxin whose enterotoxic effects are substantially dependent upon neutrophil recruitment.

Animals↗
PubMed1992

Identification of ras and ras-related low-molecular-mass GTP-binding proteins associated with rat lung lamellar bodies.

Recent evidence from genetic experiments in yeast and from studies using guanosine triphosphate (GTP) analogues in mammalian cells suggests a key role for low-molecular-mass GTP-binding proteins (LMM-GBPs) (Mr 19 to 28 kD) in processes of intracellular vesicular sorting and secretion. Assembly and exocytosis of the lamellar body (LB), the secretory organelle of the pulmonary alveolar type 2 pneumocyte, may be regulated by LMM-GBPs. We used [alpha-32P]GTP binding to Western blotted proteins, ultraviolet crosslinking of [alpha-32P]GTP to membrane proteins, immunoblotting with specific antisera, and botulinum exoenzyme C3-catalyzed ADP ribosylation to detect LMM-GBPs in LB. With the first two techniques, we have identified six LMM-GBPs of approximately 27, 25.5, 24.5, 23, 22, and 21 kD that are enriched in a highly purified LB fraction compared with type 2 pneumocyte homogenate, crude membranes, and cytosol. Further characterization of the LB LMM-GBPs by immunoblotting revealed that ras p21 is greatly enriched in the LB fraction compared with other type 2 pneumocyte fractions. In addition, botulinum exoenzyme C3 catalyzed the ADP ribosylation of 20- to 21-kD proteins that were similarly enriched in the LB fraction. In contrast, a monospecific antibody to ADP-ribosylation factor reacted with a 19-kD protein only in the type 2 pneumocyte homogenate and cytosol fractions. Monospecific antibodies to yeast Sec4 protein and to rab 3A did not react with any type 2 pneumocyte proteins. The LMM-GBPs specifically associated with LB may participate in intracellular events required for surfactant packaging and secretion.

Adenosine Diphosphate Ribose↗
PubMed1991

Mechanisms of mastoparan-stimulated surfactant secretion from isolated pulmonary alveolar type 2 cells.

Mastoparan, a tetradecapeptide component of wasp venom, is a potent activator of secretion in a variety of cell types, and has been shown to activate purified G-proteins reconstituted into phospholipid vesicles with a preferential activation of Gi over Gs (Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. R. (1988) J. Biol. Chem. 263, 6491-6494). To identify the biochemical activities of mastoparan in a cellular system, we characterized the effects of mastoparan on signal transduction pathways in rat pulmonary alveolar type 2 epithelial cells, which synthesize and secrete pulmonary surfactant. Mastoparan inhibited adenylylcyclase activity in a manner that was dose-dependent (IC50 = 30 microM), but sensitive to neither guanine nucleotide nor pertussis toxin (PT). Mastoparan induced a PT-sensitive increase in cellular inositol trisphosphate and a rapid rise in cytosolic calcium released from intracellular stores; the time to onset of the calcium rise, but neither the rate nor the amplitude of the rise, were PT-sensitive. Mastoparan also caused a dose- (EC50 = 16 microM) and time-dependent activation of arachidonic acid release that was completely insensitive to pretreatment with PT. Secretion of pulmonary surfactant was increased by mastoparan approximately 8-fold over constitutive levels at 1 h with an EC50 = 20 microM, and mastoparan-stimulated secretion was partially sensitive to PT at late time points and to inhibitors of arachidonic acid metabolism, but not to the protein kinase C inhibitor H7. These findings are consistent with the activation of Gi proteins in type 2 cells by mastoparan, although the lack of predicted triphosphoguanine nucleotide and PT sensitivity for some activities indicates that mastoparan does not act in a manner strictly analogous to liganded receptors or that some activities are not mediated by activation of Gi. While mastoparan is a potent secretagogue in several cell types, its secretory activity appears to have only a limited dependence on the activation of Gi proteins in type 2 cells.

Animals↗
PubMed1991

Modification of brain guanine nucleotide-binding regulatory proteins by tryptamine-4,5-dione, a neurotoxic derivative of serotonin.

We have recently characterized a novel oxidation product of serotonin (5-hydroxytryptamine, 5-HT), tryptamine-4,5-dione, which increases 5-HT efflux from striatum and hippocampus and causes selective neuronal death. Exposure of striatal synaptosomes or the major brain guanine nucleotide-binding regulatory proteins Gi and Go to [3H]tryptamine-4,5-dione resulted in the radiolabeling of a major band with an apparent molecular mass equivalent to that of the alpha subunits of Gi and Go (approximately 40,000). The binding of [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTP-gamma-S) to Gi and Go and pertussis toxin-catalyzed [32P]ADP-ribosylation of the G protein alpha subunits were both inhibited in a dose-dependent manner by tryptamine-4,5-dione. Thus, neurotoxins such as tryptamine-4,5-dione may exert their effects through specific interactions with G proteins.

Adenosine Diphosphate Ribose↗
PubMed1991

Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.

The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.

Animals↗
PubMed1991

Extrapulmonary thoracic restriction (hidebound chest) complicating eosinophilic fasciitis.

Eosinophilic fasciitis (EF) is an unusual disorder characterized by diffuse skin thickening and induration due to inflammation within the deep fascia; visceral involvement is generally mild or absent. A patient with biopsy-proved EF developed progressive respiratory limitation. Physical examination revealed marked induration of the thoracic integument with a severely limited chest wall excursion. Total lung capacity was 62 percent of predicted with a normal corrected Dco and maximal inspiratory force; a chest computed tomogram with thin sections showed no evidence of parenchymal lung disease. Extrapulmonary thoracic restriction ("hidebound chest") has not been previously reported to complicate EF.

Aged↗
PubMed1990

Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules.

Neutrophils contain several distinct classes of secretory granules that may sequentially fuse with the phagosome after the ingestion of particulates, or that may be differentially exocytosed after cellular activation with soluble stimuli. The exocytosis of neutrophil secretory granules has been shown to be GTP-dependent at a step distal to activation of the transductional G proteins. Inasmuch as ras-related low molecular mass GTP-binding proteins have been shown to play regulatory roles in vesicle sorting in the secretory pathway in yeast, the differential mobilization of neutrophil granules might be regulated by distinct GTP-binding proteins. We therefore explored the distribution and identity of low molecular mass GTP-binding proteins in neutrophil secretory granules and other subcellular fractions. After lysis by nitrogen cavitation, four highly resolved fractions were harvested from discontinuous Percoll gradients: a microsomal fraction enriched for plasma membranes, specific granules, primary granules, and cytosol. At least seven bands of distinct Mr were detected by probing protein blots with [32P]GTP. Microsomes contained a prominent GTP-binding band at 26 kDa and weaker ones at 24 and 22.5 kDa; specific granules contained bands at 26, 24, 22, and 20 kDa; primary granules showed bands at 24 and 23 kDa; cytosol showed strong bands at 23.5 and 19 kDa and a weak band at 26 kDa. Antiserum against ADP-ribosylation factor reacted strongly with the 19-kDa band in cytosol but with none of the membrane fractions. None of these proteins was recognized by antibodies against ras or against Sec4p. Botulinum exoenzyme C3 labeled bands of molecular mass 20 and 21 kDa in cytosol and microsomes that have distinct mobilities from all the blotted [32P]GTP-binding proteins. The highly compartmentalized subcellular distribution of the blotted [32P]GTP-binding proteins in neutrophils is consistent with a regulatory role in the differential mobilization of granule compartments during cellular activation.

Adenosine Diphosphate Ribose↗
PubMed1990

Subcellular distribution and characterization of GTP-binding proteins in human neutrophils.

The subcellular distribution of GTP binding proteins in human neutrophils and their functional coupling to the N-formylmethionylleucylphenylalanine (FMLP) receptor was characterized to provide insight into mechanisms of cellular activation. Human neutrophils were nitrogen cavitated and fractionated on discontinuous Percoll gradients. Four subcellular fractions were obtained: cytosol, light membranes enriched for plasma membranes, specific granules and azurophilic granules. ADP-ribosylation catalyzed by pertussis toxin (PT) revealed a major substrate of 40 kDa only in plasma membrane and cytosol, and antiserum specific for Gi alpha confirmed the presence of neutrophil Gi alpha in plasma membrane and cytosol and its absence from specific granules. The cytosolic PT substrate was shown to be mostly in monomeric form by molecular sieve chromatography. The rate of the ribosyltransferase reaction was several-fold lower in cytosol compared to plasma membranes, and the extent of ADP-ribosylation was greatly augmented by supplementation with beta gamma subunits in cytosol. ADP-ribosylation catalyzed by cholera toxin (CT) revealed substrates of 52, 43 and 40 kDa in plasma membrane alone. FMLP receptors in plasma membrane were shown to be coupled to the 40 kDa substrate for CT by ligand-modulation of ADP-ribosylation, while FMLP added to specific granules did not induce ribosylation of this substrate even though FMLP receptors were found in high density in this compartment. Both 24 and 26 kDa [32P]GTP binding proteins were found to codistribute with FMLP receptors in specific granules and plasma membranes. Functional evidence for the coupling of GTP binding proteins to the FMLP receptor in specific granules was obtained by modulating [3H]FMLP binding with GTP gamma S, and by accelerating [35S]GTP gamma S binding with FMLP.

Cell Membrane↗
PubMed1990

GTP-binding proteins in rat liver nuclear envelopes.

Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membrane vesicles required for reassembly of the nucleus after mitosis.

Adenosine Triphosphate↗
PubMed1989

Low molecular weight GTP-binding proteins are associated with neuronal organelles involved in rapid axonal transport and exocytosis.

Recent evidence suggests that low molecular weight GTP-binding proteins may play important roles in a variety of membrane transport processes. In order to address the question of whether these proteins are involved in transport processes in the nerve axon, we have assessed their presence in rapid transport membranes from rabbit optic nerve. We report the characterization of a group of low molecular weight GTP-binding proteins which are constituents of rapid transport vesicles. Although these proteins are components of rapid transport vesicles, they are apparently not major rapidly transported species. They are localized in cytosolic as well as in membrane fractions of axons, and the membrane-associated form behaves as an integral membrane protein(s). These proteins are also found in association with a variety of vesicular and organellar components of neurons including coated vesicles, synaptic vesicles, synaptic plasma membranes, and mitochondria. We discuss the possible roles of these proteins in rapid axonal transport and exocytosis.

Animals↗
PubMed1988

Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein.

We describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [3H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [3H]fMet-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, we also demonstrated fMet-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils.

Adenine Nucleotides↗
PubMed1987

Purification and characterization of the rat liver vasopressin (V1) receptor.

Utilizing a proteoliposome reconstitution system, we have purified the rat liver V1 vasopressin receptor to near homogeneity. The receptor was purified approximately 21,000-fold from rat liver membranes, using differential detergent solubilization, size exclusion gel filtration, lectin affinity, and ion-exchange chromatography. The purified receptor exhibits a Kd of 6 nM, when, prior to solubilization, the membranes were exposed to 1 microM vasopressin. This resulted in the association of a pertussis toxin-insensitive guanine nucleotide-binding protein with the receptor during most of the purification procedure. In the absence of this association, the receptor had a Kd of approximately 30 nM. Association of the receptor with a G-protein was confirmed by the ability of vasopressin to stimulate the hydrolysis of [gamma-32P]GTP. The specific activity of the vasopressin-stimulated hydrolysis was 25 nmol/min/mg, approximately 8,000-fold higher than values obtained with crude reconstituted receptor preparations. Cross-linking of 125I-vasopressin to a partially purified preparation of receptor demonstrated that the receptor had a molecular weight of approximately 68,000 under reducing conditions, and 58,000 under nonreducing conditions. The purification procedure may prove useful in purifying a number of small peptide hormone receptors (e.g. bradykinin, angiotensin II) and perhaps their associated G-proteins as well.

Animals↗
PubMed1987

Identification and purification of a novel G protein from neutrophils.

A novel G protein which appears to couple chemotactic peptide receptors to a polyphosphoinositide phospholipase C has been purified from rabbit neutrophils. Neutrophil membranes were solubilized with sodium cholate and fractionated by successive anion exchange, gel filtration and hydrophobic chromatography. Guanosine-5'-(3-O-thio)triphosphate binding activity was purified 170-fold from the soluble extract. The alpha-subunit of the purified G protein was identified by pertussis toxin-catalyzed ADP-ribosylation, and found to have an Mr of 40,000. The beta-subunit (Mr 36,000) comigrated on SDS-polyacrylamide gel electrophoresis with the beta-subunits of bovine brain Gi and Go. The neutrophil pertussis toxin substrate is highly unstable in cholate solution unless 30% ethylene glycol is added. Structural and functional analysis of this novel G protein will advance our understanding of the molecular mechanisms of coupling of receptors to phospholipase C.

Animals↗