Biomedical subjects
Publications and source records attributed to B F Dickey.
Membrane proteins, membrane lipids, and luminal contents are exchanged among the intracellular compartments of eukaryotic cells by vesicular transport. This process must be highly ordered to maintain cellular architecture in the face of rapid membrane turnover. The Ras-related Rab GTPases play multiple roles in regulating this traffic.
Sec1 family proteins are regulators of diverse exocytic processes, from yeast to man. Three mammalian homologues, Munc18-1, -2, and -3 have been described. We have studied the structure and expression of the mouse Munc18-2 gene. The Munc18-2 gene comprises 19 exons whose sizes range from 50 to 158 bp, with a total gene size of approximately 11 kb. A single transcript of 2.1 kb is expressed in multiple non-neuronal murine tissues. Munc18-2 has a striking resemblance to Munc18-1 in structure despite only 60% sequence identity, suggesting a recent gene duplication event. Analysis of the region upstream of the transcription start site shows that Munc18-2 has a TATA-less promoter, with a consensus initiator (Inr) sequence at the start of transcription, several Sp1 binding sites, and strong promoter activity in RBL-2H3 mast cells. The region from +5 to -430 is more active than +5 to -800, suggesting upstream repressor elements.
The ras-related GTPase rab5 is rate-limiting for homotypic early endosome fusion. We used a yeast two-hybrid screen to identify a rab5 interacting protein, rab5ip. The cDNA sequence encodes a ubiquitous 75-kDa protein with an N-terminal transmembrane domain (TM), a central coiled-coil structure, and a C-terminal region homologous to several centrosome-associated proteins. rab5ip lacking the transmembrane domain (rab5ipTM(-)) had a greater affinity in vitro for rab5-guanosine 5'-O-2-(thio)diphosphate than for rab5-guanosine 5'-3-O-(thio)triphosphate. In transfected HeLa cells, rab5ipTM(-) was partly cytosolic and localized (by immunofluorescence) with a rab5 mutant believed to be in a GDP conformation (GFP-rab5(G78A)) but not with GFP-rab5(Q79L), a GTPase-deficient mutant. rab5ip with the transmembrane domain (rab5ipTM(+)) was completely associated with the particulate fraction and localized extensively with GFP-rab5(wt) in punctate endosome-like structures. Overexpression of rab5ipTM(+) using Sindbis virus stimulated the accumulation of fluid-phase horseradish peroxidase by BHK-21 cells, and homotypic endosome fusion in vitro was inhibited by antibody against rab5ip. rab5ipTM(-) inhibited rab5(wt)-stimulated endosome fusion but did not inhibit fusion stimulated by rab5(Q79L). rab5ip represents a novel rab5 interacting protein that may function on endocytic vesicles as a receptor for rab5-GDP and participate in the activation of rab5.
Rab proteins, members of the Ras superfamily of small GTPases, play regulatory roles in intercompartmental vesicular transport. Each step of traffic seems to require the participation of at least one distinct Rab, with the Rab3 subfamily involved in stimulated exocytosis. We report our studies on the murine rab3D gene, one of the four mammalian Rab3 isoforms. We located this gene on chromosome 13, region A(2-3). The rab3D gene consists of 5 exons spanning 10.6 kb, and the structural gene is contained in exons 2 through 5 with one canonical GTP-binding motif in each exon. Organization of the rab3D gene is identical to that of rab3A but different from other rab genes. Alternative poly-A(+) signals in the 3' untranslated region account for the identities of multiple transcripts detected by Northern blot analysis. Rab3D is expressed in all tissues studied, predominantly in heart, lung, and liver, and binding sites for multiple transcription factors are found in the TATA-less promoter region.
Synaptotagmins (Syts) I and II are believed to act as Ca2+ sensors in the control of neurotransmission. Here we demonstrate that mast cells express Syt II in their lysosomal fraction. We further show that activation of mast cells by either aggregation of FcepsilonRI or by Ca2+ ionophores results in exocytosis of lysosomes, in addition to the well documented exocytosis of their secretory granules. Syt II directly regulates lysosomal exocytosis, whereby overexpression of Syt II inhibited Ca2+-triggered release of the lysosomal processed form of cathepsin D, whereas suppression of Syt II expression markedly potentiated this release. These findings provide evidence for a novel function of Syt II in negatively regulating Ca2+-triggered exocytosis of lysosomes, and suggest that Syt II-regulated secretion from lysosomes may play an important role in mast cell biology.
Although mast cell secretion has been intensively studied because of its pivotal role in allergic reactions and its advantages as a physiologic model, the molecular composition of the secretory machine is virtually unknown. In view of the guanine-nucleotide dependency of mast cell exocytosis and the participation of Rab3 proteins in synaptic vesicle release, we hypothesized that a Rab3 isoform regulates mast cell secretion. Fragments of Rab3A, 3B, and 3D were cloned from RBL-2H3 mast cells by reverse transcription- polymerase chain reaction (RT-PCR). Northern blot analysis revealed Rab3D transcripts to be relatively abundant, Rab3B substantially less so, and Rab3A and 3C undetectable. By ribonuclease (RNase) protection assay, Rab3D transcripts were at least 10-fold more abundant than those of other isoforms, and by immunoblot analysis, Rab3D protein was at least 60-fold more abundant than that of Rab3B. Rab3D was more abundant in RBL cells than in brain, but the total mass of Rab3 proteins in RBL cells was 10-fold less than in brain. Rab3D only partly colocalized with secretory granules in RBL cells, but fully colocalized in mature peritoneal mast cells. There was a descending concentration gradient of Rab3D from peripheral to central granules, and no cytoplasmic pool was detectable in resting mast cells. Following exocytotic degranulation, Rab3D translocated to the plasma membrane and remained there for at least 15 min. These studies suggest that Rab3D is a component of the regulated exocytotic machine of mast cells, and identify differences between mast cells and neurons in Rab3 expression and trafficking.
During prolonged exposure to agonist, beta2-adrenergic receptors undergo downregulation, defined by the loss of radioligand binding sites. To determine the cellular basis for beta2-adrenergic receptor downregulation, we examined HEK293 cells stably expressing beta2-adrenergic receptors with an N-terminal epitope tag. Downregulation was blocked by leupeptin, a cysteine protease inhibitor, but not by pepstatin, an inhibitor of aspartate proteases. Immunofluorescence microscopy of cells treated with agonist for 3-6 hours in the presence of leupeptin showed beta2-adrenergic receptors, but not transferrin receptors, localizing with the lysosomal protease cathepsin D, and with lysosomes labeled by uptake of a fluorescent fluid-phase marker. No localization of beta2-adrenergic receptors with lysosomal markers was observed in the absence of leupeptin, most likely due to proteolysis of the epitope. The proton pump inhibitor, bafilomycin A1, significantly inhibited this agonist-induced redistribution of beta2-adrenergic receptors into lysosomes, causing receptors to accumulate in the rab11-positive perinuclear recycling compartment and slowing the rate of beta2-adrenergic receptor recycling. Control experiments showed that leupeptin had no nonspecific effects on the cellular trafficking of either beta2-adrenergic receptors or transferrin receptors. Although cAMP alone caused a small decline in receptor levels without redistributing beta2-adrenergic receptors from the plasma membrane, this effect was additive to that seen with agonist alone, suggesting that agonist-induced beta2-adrenergic receptor downregulation resulted largely from cAMP-independent mechanisms. These results indicate that during agonist-induced downregulation, a significant fraction of beta2-adrenergic receptors are specifically sorted to lysosomes via the endosomal pathway, where receptor degradation by cysteine proteases occurs. These results provide a cellular explanation for the loss of radioligand binding sites that occurs during prolonged exposure to agonist.
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Tryptic proteolysis of the small GTPases Rab4 and Rab5 is a multi-step, nucleotide-dependent process. Using N-terminal peptide sequencing, matrix-assisted laser desorption ionization-time-of-flight MS and molecular modelling, we identified the three initial sites of proteolysis in Rab5 as Arg-4, Arg-81 and Arg-197. Arg-4 and Arg-81 lie within regions previously implicated in Rab5 endocytic function, and Arg-197 lies in a region involved in membrane targeting. Topologically, Arg-81 lies within the conformationally variable Switch II region shown to be important for protein-protein interactions of other GTPases. Homology modelling studies on Rab5 indicate that the Arg-81 side chain is buried in the Rab5 GTP conformation, but is solvent-accessible in the GDP conformation, explaining the dependence of proteolysis on nucleotides. Peptide mapping of Rab4 was performed to take advantage of additional scissile bonds within Switch II to determine more precisely the limits of the nucleotide-dependent protease-accessible region. The Rab4 cleavage sites corresponded to Arg-81 and Pro-87 of Rab5, and taken together with the finding that Rab5 was not cleaved at Arg-91 this analysis defines an eight-residue surface-exposed conformationally variable region lying in the centre of Switch II. A sequence comparison of Rab proteins shows these eight residues to have a loosely conserved motif that we term Switch II(v) for its relative variability. C-terminal to Switch II(v) is a highly conserved Rab-specific YYRGA motif that we term Switch II(c) for its constant sequence. N-terminal to Switch II(v) is a sequence-invariant G-domain involved in nucleotide binding and hydrolysis. We propose that the Rab Switch II(v) region imparts specificity to nucleotide-dependent protein-protein interactions.
Early endosome fusion, which has been extensively characterized using an in vitro reconstitution assay, is Rab5-dependent. To examine the requirement for Rab5 on both fusion partners, we prepared cytosol and endosomes depleted of Rab5. Unlike control cytosol, Rab5-depleted cytosol was only marginally active in the in vitro endosome fusion. However, fusion could be restored by the addition of wild-type Rab5 or Rab5 D136N, a mutant whose nucleotide specificity favors xanthine over guanine. The addition of Rab5 D136N restored fusion only in the presence of XTP. In the absence of XTP or in the presence of XDP, Rab5 D136N failed to restore fusion. When fusion was carried out with endosomal vesicles depleted of Rab GTPases (by preincubation of vesicles with GDP dissociation inhibitor), together with cytosol immunodepleted of Rab5, fusion was virtually absent. We then used immunodepleted cytosol and GDP dissociation inhibitor-treated vesicles to determine whether Rab5 is required by both fusion partners. Using separate sets of endosomal vesicles, we found that priming both sets of Rab5-depleted vesicles with Rab5 Q79L, a GTPase-defective mutant, substantially stimulated endosome fusion. Priming one set of vesicles with Rab5 Q79L and a second set of vesicles with Rab5 S34N failed to activate fusion. When both sets of Rab5-depleted vesicles were primed with Rab5 D136N supplemented with XTP, endosome fusion was stimulated, similar to that observed with Rab5 Q79L. However, when one set of vesicles was preincubated with Rab5 D136N plus XTP and the second set with Rab5 D136N and XDP, no stimulation of fusion was observed. We conclude that Rab5-GTP is required on both fusion partners for docking and fusion of early endosomes. To confirm the fusion of Rab5-GTP-positive vesicles in vivo, we expressed GFP-Rab5 Q79L in fibroblasts and observed fusion of Rab5-positive vesicles. We failed to record fusion of Rab5-positive vesicles with Rab5-negative vesicles. We conclude that Rab5-GTP is required on both sets of endosomes for fusion in vitro and in living cells.
Alveolar epithelial type 2 (T2) cells isolated from the lungs of adult rats responded to exogenous atrial natriuretic peptide (ANP) by two signalling mechanisms. First, ANP induced a dose-dependent reduction of ligand-stimulated adenylyl cyclase activity and cAMP accumulation. This effect was inhibited by the addition of GDPbetaS or by pretreatment with pertussis toxin (PT), consistent with mediation by a Gi protein(s). PT-catalyzed [32P]ADP-ribosylation, immunoblots with specific antisera, and Northern blot analysis demonstrated that T2 cells contain the G-proteins Gi2 and Gi3 which could transduce this signal. ANP also promoted PT-insensitive, dose-dependent accumulation of cGMP, consistent with activation of a receptor guanylyl cyclase. Isoproterenol-stimulated phosphatidylcholine secretion was markedly attenuated by ANP, and this effect was inhibited by PT pretreatment, consistent with mediation by a Gi protein(s). These data indicate that in addition to the lung being a major clearance organ for circulating ANP, lung parenchymal cells are targets of ANP action.
1. Partial agonists of the beta2-adrenoceptor which activate adenylyl cyclase are widely used as bronchodilators for the relief of bronchoconstriction accompanying many disease conditions, including bronchial asthma. The bronchodilator salmeterol has both a prolonged duration of action in bronchial tissue and the ability to reassert this activity following the temporary blockade of human beta2-adrenoceptors with antagonist. 2. We have compared the activation and desensitization of human beta2-adrenoceptor stimulation of adenylyl cyclase induced by salmeterol, adrenaline and salbutamol in a human lung epithelial line, BEAS-2B, expressing beta2-adrenoceptor levels of 40-70 fmol mg(-1), and in human embryonic kidney (HEK) 293 cell lines expressing 2-10 pmol mg(-1). The efficacy observed for the stimulation of adenylyl cyclase by salmeterol was only approximately 10% of that observed for adrenaline in BEAS-2B cells expressing low levels of beta2-adrenoceptor, but similar to adrenaline in HEK 293 cells expressing very high levels of receptors. Salmeterol pretreatment of these cells induced a rapid and stable activation of adenylyl cyclase activity which resisted extensive washing and beta2-adrenoceptor antagonist blockade, consistent with binding to a receptor exosite and/or to partitioning into membrane lipid. 3. The desensitization and internalization of beta2-adrenoceptors induced by the partial agonists salmeterol and salbutamol were considerably reduced relative to the action of adrenaline. Consistent with these observations, the initial rate of phosphorylation of the receptor induced by salmeterol and salbutamol was much reduced in comparison to adrenaline. 4. Our data suggest that the reduction in the rapid phase of desensitization of beta2-adrenoceptors after treatment with salmeterol or salbutamol is caused by a decrease in the rate of beta2-adrenoceptor kinase (betaARK) phosphorylation and internalization. In contrast, the rate of cyclic AMP-dependent protein kinase (PKA)-mediated phosphorylation by these partial agonists appears to be similar to adrenaline.
OBJECTIVE: To review the pharmacology of the long-acting inhaled beta2-agonists, salmeterol and formoterol, summarize results of their clinical trials, evaluate their safety records, and discuss their roles in the treatment of asthma. DATA SOURCES: Preclinical and clinical studies involving salmeterol or formoterol were identified by a MEDLINE search, weekly computerized literature updates, and manual searches. Studies of satisfactory quality were chosen for review. DATA SYNTHESIS: Salmeterol and formoterol are potent and selective beta2-adrenoceptor agonists with durations of action >12 h. Their major differences are that formoterol has a rapid onset of action and is a partial agonist of high intrinsic efficacy, whereas salmeterol has a delayed onset and is a partial agonist of low intrinsic efficacy. Twice daily use of either drug results in improved lung function, reduced symptoms, and a better quality of life. These agents protect against exercise-induced asthma for 12 h and eliminate nighttime awakening in most patients. Limited tolerance develops, especially to their bronchoprotective effects, but their improvement of lung function is sustained. CONCLUSIONS: Regular use of salmeterol or formoterol provides subjective and objective amelioration of asthma in patients experiencing excessive symptoms or physiologic impairment despite the regular administration of low doses of inhaled corticosteroids (equivalent to approximately 500 microg/d of beclomethasone). Intermittent use of either long-acting beta2-agonist can provide prolonged protection against exercise-induced asthma or nighttime symptoms. Patients should be instructed to continue taking inhaled steroids when long-acting beta2-agonists are administered on a regular schedule and to not take long-acting beta2-agonists between regularly scheduled doses. Used properly, they are effective and safe adjunctive agents in the treatment of asthma.
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G proteins are molecular switches that use a cycle of GTP binding and hydrolysis to regulate a wide variety of cellular biochemical processes. Because the functional state of these proteins is allosterically determined by bound guanine nucleotides, a nucleotide analogue with protein specificity might have pharmacological or biochemical value. The binding of [alpha-32P]GTP to four small G proteins immobilized on nitrocellulose was competed by a series of analogues with modifications at multiple sites. One analogue, N2-(p-n-butylphenyl)guanosine 5'-(beta,gamma-difluoromethylene)triphosphate, had a approximately 40-fold higher affinity for one small G protein than for two of the others. Systematic analysis of each modification in the synthetic nucleotide revealed that specificity was conferred by the carbon substitution in the beta,gamma-phosphoanhydride bond. These observations were then extended to purified proteins of known sequence in solution by filtration binding studies with H-ras and rab5. Ras was 9-fold more discriminant between guanosine-5'-(beta,gamma-difluoromethylene)triphosphate and guanosine-5'-O-(3-thiotriphosphate) than was rab5, and the Q79L GTPase-defective mutant of rab5 was 6-fold more discriminant than wild-type rab5. Guanosine-5'-(beta,gamma-difluoromethylene)triphosphate protected a 20-kDa fragment of rab5 from tryptic proteolysis with greater efficacy than guanosine-5'-O-(3-thiotriphosphate) or guanosine-5'-(beta,gamma-imido)triphosphate despite its lower affinity, and GMP stabilized a conformation indistinguishable from apo-rab5. These results identify a synthetic guanine nucleotide analogue with differential affinity for closely related G proteins, determine the atomic substitution in the analogue that confers specificity, demonstrate discrimination by the analogue between wild-type and a point-mutant G protein, and establish efficacy of the analogue in inducing conformational change of a target protein disproportionate to the affinity of the interaction.
We targeted a mutant p53 gene (val135), previously shown to cause tumors in transgenic mice, to the kidney and eye using a gamma-glutamyltranspeptidase promoter. Although transgene RNA was expressed in both tissues, and mutant protein could be detected at high levels in the kidney and was appropriately localized to the nuclei of proximal tubules, no gross or microscopic lesions developed, even when mice were held as long as 75 weeks. When these mice were crossed with transgenic mice carrying HrasT24 (containing a codon 12 mutation) driven by the same promoter, the p53val135 transgene partially suppressed the mutant ras phenotype (proximal tubular hyperplasia and adenomas and carcinomas of the ciliary body and retinal pigment epithelium). The kidneys of double transgenic mice younger than 25 weeks showed less tubular hyperplasia and cystic change than littermates carrying gamma-glutamyltranspeptidase(I)rasT24 alone. By 33 weeks, there was no difference in the severity of the kidney lesions. The eye lesions were less aggressive, and no malignant lesions were identified. Our findings are consistent with the work of others, indicating that p53val135 is not tumorigenic under all conditions; in fact, in some circumstances, it retains some of the suppressing activity of wild-type p53.
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.
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.