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D Cassel

Publications and source records attributed to D Cassel.

At least 19 recordsLinked to original sources
PubMed2000

Role of coatomer and phospholipids in GTPase-activating protein-dependent hydrolysis of GTP by ADP-ribosylation factor-1.

The binding of the coat protein complex, coatomer, to the Golgi is mediated by the small GTPase ADP-ribosylation factor-1 (ARF1), whereas the dissociation of coatomer, requires GTP hydrolysis on ARF1, which depends on a GTPase-activating protein (GAP). Recent studies demonstrate that when GAP activity is assayed in a membrane-free environment by employing an amino-terminal truncation mutant of ARF1 (Delta17-ARF1) and a catalytic fragment of the ARF GTPase-activating protein GAP1, GTP hydrolysis is strongly stimulated by coatomer (Goldberg, J., (1999) Cell 96, 893-902). In this study, we investigated the role of coatomer in GTP hydrolysis on ARF1 both in solution and in a phospholipid environment. When GTP hydrolysis was assayed in solution using Delta17-ARF1, coatomer stimulated hydrolysis in the presence of the full-length GAP1 as well as with a Saccharomyces cerevisiae ARF GAP (Gcs1) but had no effect on hydrolysis in the presence of the phosphoinositide dependent GAP, ASAP1. Using wild-type myristoylated ARF1 loaded with GTP in the presence of phospholipid vesicles, GAP1 by itself stimulated GTP hydrolysis efficiently, and coatomer had no additional effect. Disruption of the phospholipid vesicles with detergent resulted in reduced GAP1 activity that was stimulated by coatomer, a pattern that resembled Delta17-ARF1 activity. Our findings suggest that in the biological membrane, the proximity between ARF1 and its GAP, which results from mutual binding to membrane phospholipids, may be sufficient for stimulation of ARF1 GTPase activity.

ADP-Ribosylation Factor 1↗
PubMed2000

Efficacy of continuous sequential transdermal estradiol and norethindrone acetate in relieving vasomotor symptoms associated with menopause.

OBJECTIVE: This study was undertaken to evaluate the efficacy and tolerability of a combination estradiol plus norethindrone acetate transdermal delivery system given in a continuous sequential regimen with transdermal estradiol versus placebo in the treatment of vasomotor symptoms of menopause. STUDY DESIGN: This was a 12-week double-blind trial of 220 healthy postmenopausal women with > or = 8 moderate to severe hot flushes and sweating episodes per day. Women were randomly assigned to wear transdermal placebo patches or a transdermal patch releasing 50 microg/d 17beta-estradiol alone (Vivelle) for days 1 to 14 of each cycle and a combination patch releasing 50 microg/d 17beta-estradiol plus 1 of 3 dosage levels (140, 250, or 400 microg/d) of norethindrone acetate (CombiPatch) for days 15 through 28. RESULTS: There was a significant (P <.001) reduction by the second week in the mean number of daily hot flushes from baseline to end point with all 3 doses of estradiol plus norethindrone acetate compared with placebo. Significant (P <.001) reductions in the mean intensity of hot flushes and sweating were also noted with estradiol plus norethindrone acetate compared with placebo. The incidences of adverse events with all 3 doses of estradiol plus norethindrone acetate and with placebo were comparable. CONCLUSION: An estradiol plus norethindrone acetate transdermal delivery system administered in a continuous sequential regimen with transdermal estradiol was well tolerated and effective for the treatment of moderate to severe vasomotor symptoms in postmenopausal women.

Administration, Cutaneous↗
PubMed1999

The KDEL receptor regulates a GTPase-activating protein for ADP-ribosylation factor 1 by interacting with its non-catalytic domain.

ADP-ribosylation factor 1 (ARF1) is a key regulator of transport in the secretory system. Like all small GTPases, deactivation of ARF1 requires a GTPase-activating protein (GAP) that promotes hydrolysis of GTP to GDP on ARF1. Structure-function analysis of a GAP for ARF1 revealed that its activity in vivo requires not only a domain that catalyzes hydrolysis of GTP on ARF1 but also a non-catalytic domain. In this study, we show that the non-catalytic domain of GAP is required for its recruitment from cytosol to membranes and that this domain mediates the interaction of GAP with the transmembrane KDEL receptor. Blocking its interaction with the KDEL receptor leaves the GAP cytosolic and prevents the deactivation in vivo of Golgi-localized ARF1. Thus, these findings suggest that the KDEL receptor plays a critical role in the function of GAP by regulating its recruitment from cytosol to membranes, where it can then act on its membrane-restricted target, the GTP-bound form of ARF1.

ADP-Ribosylation Factor 1↗
PubMed1999

Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function.

ARF proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the actions of GTPase-activating proteins (GAPs) for their function. The in vitro GTPase activity of the Saccharomyces cerevisiae ARF proteins Arf1 and Arf2 is stimulated by the yeast Gcs1 protein, and in vivo genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular transport. However, the Gcs1 protein is dispensable, indicating that additional ARF GAP proteins exist. We show that the structurally related protein Glo3, which is also dispensable, also exhibits ARF GAP activity. Genetic and in vitro approaches reveal that Glo3 and Gcs1 have an overlapping essential function at the endoplasmic reticulum (ER)-Golgi stage of vesicular transport. Mutant cells deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation of ER and are defective for protein transport between ER and Golgi. The glo3Delta and gcs1Delta single mutations each interact with a sec21 mutation that affects a component of COPI, which mediates vesicular transport within the ER-Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes encoding members of a v-SNARE family that functions within the ER-Golgi alleviates the effects of a glo3Delta mutation. An in vitro assay indicates that efficient retrieval from the Golgi to the ER requires these two proteins. These findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular transport from the Golgi to the ER.

ADP-Ribosylation Factor 1↗
PubMed1999

The ADP-ribosylation factor GTPase-activating protein Glo3p is involved in ER retrieval.

Retrograde transport of proteins from the Golgi to the endoplasmic reticulum (ER) has been the subject of some interest in the recent past. Here a new thermosensitive yeast mutant defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum was characterized. The ret4-1 mutant also exhibited a selective defect in forward ER-to-Golgi transport of some secreted proteins at the non-permissive temperature. The corresponding RET4 gene was found to encode Glo3p, a GTPase-activating protein (GAP) specific for ADP-ribosylation factor (ARF). In vitro, the Glo3 thermosensitive mutant showed a reduced ARF1-GAP activity. The Glo3 protein belongs to a family of zinc finger proteins that may include additional ARF-GAPs. Gene deletion experiments of other family members showed that only GLO3 deletion resulted in impaired retrieval of dilysine-tagged proteins back to the ER. These results demonstrate that Glo3p is the main ARF-GAP specifically involved in ER retrieval.

ADP-Ribosylation Factor 1↗
PubMed1998

Requirement for both the amino-terminal catalytic domain and a noncatalytic domain for in vivo activity of ADP-ribosylation factor GTPase-activating protein.

The small GTP-binding protein ADP-ribosylation factor-1 (ARF1) regulates intracellular transport by modulating the interaction of coat proteins with the Golgi complex. Coat protein association with Golgi membranes requires activated, GTP-bound ARF1, whereas GTP hydrolysis catalyzed by an ARF1-directed GTPase-activating protein (GAP) deactivates ARF1 and results in coat protein dissociation. We have recently cloned a Golgi-associated ARF GAP. Overexpression of GAP was found to result in a phenotype that reflects ARF1 deactivation (Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P. J., and Hsu, V. W. (1997) EMBO J. 16, 7305-7316). In this study, we used this phenotype to define domains in GAP that are required for its function in vivo. As expected, mutations in the amino-terminal part of GAP that were previously found to abolish ARF GAP catalytic activity in vitro abrogated ARF1 deactivation in vivo. Significantly, truncations at the carboxyl-terminal part of GAP that did not affect GAP catalytic activity in vitro also diminished ARF1 deactivation. Thus, a noncatalytic domain is required for GAP activity in vivo. This domain may be involved in the targeting of GAP to the Golgi membrane.

ADP-Ribosylation Factor 1↗
PubMed1998

cADP-ribose formation by blood platelets is not responsible for intracellular calcium mobilization.

Human platelet CD38 is a multifunctional ectoenzyme catalysing the synthesis and hydrolysis of cADP-ribose (cADPR), a recently identified calcium-mobilizing agent that acts independently of D-myo-inositol 1,4,5-trisphosphate and is known to be expressed by human platelets. The present work shows that ADP-ribosyl cyclase activity is exclusively a membrane activity, of which the major part is located in plasma membranes and a small part in internal membranes. In broken cells, cyclase activity was insensitive to the presence of calcium and was not modulated by agonists such as thrombin or ADP, whereas in intact cells thrombin increased cADPR formation by 30%, an effect due to fusion of granules with the plasma membrane. In order to assess the role of cADPR as a calcium-mobilizing agent, vesicles were prepared from internal membranes and loaded with 45CaCl2. These vesicles were efficiently discharged by IP3 in a dose-dependent manner, but were not responsive to cADPR or ryanodine in the presence or absence of calmodulin. Thus cADPR is unlikely to play a role in intracellular calcium release in human blood platelets.

ADP-ribosyl Cyclase↗
PubMed1997

The KDEL receptor, ERD2, regulates intracellular traffic by recruiting a GTPase-activating protein for ARF1.

The small GTPase ADP-ribosylation factor 1 (ARF1) is a key regulator of intracellular membrane traffic. Regulators of ARF1, its GTPase-activating protein (GAP) and its guanine nucleotide exchange factor have been identified recently. However, it remains uncertain whether these regulators drive the GTPase cycle of ARF1 autonomously or whether their activities can be regulated by other proteins. Here, we demonstrate that the intracellular KDEL receptor, ERD2, self-oligomerizes and interacts with ARF1 GAP, and thereby regulates the recruitment of cytosolic ARF1 GAP to membranes. Because ERD2 overexpression enhances the recruitment of GAP to membranes and results in a phenotype that reflects ARF1 inactivation, our findings suggest that ERD2 regulates ARF1 GAP, and thus regulates ARF1-mediated transport.

ADP-Ribosylation Factor 1↗
PubMed1997

Activation of ADP-ribosylation factor 1 GTPase-activating protein by phosphatidylcholine-derived diacylglycerols.

Disassembly of the coatomer from Golgi vesicles requires that the small GTP-binding protein ADP-ribosylation factor 1 (ARF1) hydrolyzes its bound GTP by the action of a GTPase-activating protein. In vitro, the binding of the ARF1 GTPase-activating protein to lipid vesicles and its activity on membrane-bound ARF1GTP are increased by diacylglycerols with monounsaturated acyl chains, such as those arising in vivo as secondary products from the hydrolysis of phosphatidylcholine by ARF-activated phospholipase D. Thus, the phospholipase D pathway may provide a feedback mechanism that promotes GTP hydrolysis on ARF1 and the consequent uncoating of vesicles.

ADP-Ribosylation Factor 1↗
PubMed1996

Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.

ADP-Ribosylation Factor 1↗
PubMed1995

The ARF1 GTPase-activating protein: zinc finger motif and Golgi complex localization.

Hydrolysis of guanosine triphosphate (GTP) by the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor-1 (ARF1) depends on a GTPase-activating protein (GAP). A complementary DNA encoding the ARF1 GAP was cloned from rat liver and predicts a protein with a zinc finger motif near the amino terminus. The GAP function required an intact zinc finger and additional amino-terminal residues. The ARF1 GAP was localized to the Golgi complex and was redistributed into a cytosolic pattern when cells were treated with brefeldin A, a drug that prevents ARF1-dependent association of coat proteins with the Golgi. Thus, the GAP is likely to be recruited to the Golgi by an ARF1-dependent mechanism.

ADP-Ribosylation Factor 1↗
PubMed1995

ADP-ribosylation factor-directed GTPase-activating protein. Purification and partial characterization.

The small GTP-binding protein ARF plays an established role in the control of vesicular traffic and in the regulation of phospholipase D activity. Like other GTP binding proteins, ARF becomes activated upon the binding of GTP, whereas GTP hydrolysis acts as a turn-off signal. The fact that purified ARF proteins have negligible GTPase activity has suggested that GTP hydrolysis by ARFs is dependent on a GTPase-activating protein (GAP). Here we report the complete purification of an ARF GAP from rat liver cytosol. Advanced stages in the purification were carried out in the presence of denaturing agents, making use of an unusual conformational stability, or refolding capacity, of the GAP. The GAP was purified about 15,000-fold and was identified as a protein of 49 kDa. Partial amino acid sequence analysis showed that the GAP is a previously uncharacterized protein. Both crude and purified GAP migrated on a Superdex 200 column as a 200-kDa complex, suggesting a tetrameric structure. The purified ARF GAP was stimulated by phosphoinositides and was inhibited by phosphatidylcholine, similar to the results previously reported for a preparation from brain (Randazzo, P. A., and Kahn, R. A. (1994) J. Biol. Chem. 269, 10758). The availability of the ARF GAP molecule will advance the understanding of the regulation of the cellular processes in which ARF proteins participate.

ADP-Ribosylation Factors↗
PubMed1995

Use of a tantalum-178 generator and a multiwire gamma camera to study the effect of the Mueller maneuver on left ventricular performance: comparison to hemodynamics and single photon emission computed tomography perfusion patterns.

During the Mueller maneuver, there is a decrease in intrathoracic pressure and an increase in transmural left ventricular pressure. The changes in loading conditions cause transient left ventricular dysfunction. This study examined the effects of the Mueller maneuver on left ventricular performance using tantalum (Ta)-178 (half-life 9.3 min) and a multiwire gamma camera. First-pass radionuclide angiograms were obtained at baseline and during Mueller maneuver in 41 patients aged 58 +/- 10 years. In 34 patients, stress single photon emission computed tomography (SPECT) myocardial perfusion imaging with thallium-201 or sestamibi was also performed. Hemodynamic measurements during the Mueller maneuver (n = 10) showed a decrease in systemic pressure (139 +/- 25 mm Hg vs 123 +/- 24 mm Hg, p < 0.001) and pulmonary artery pressure (24 +/- 6 mm Hg vs 14 +/- 12 mm Hg, p = 0.01) and an increase in heart rate (67 +/- 10 bpm vs 75 +/- 14 beats/min, p = 0.001). Among the 34 patients who had perfusion imaging, the left ventricular ejection fraction remained unchanged or increased in 17 patients (group 1) (48% +/- 19% vs 49% +/- 21%, p not significant) and decreased (> or = 5%) in 17 patients (group 2) (55% +/- 13% vs 40% +/- 16%, p = 0.001). The stress SPECT images showed no or only fixed defects in 11 (65%) patients in group 1 and 3 (18%) patients in group 2 (p = 0.02), and reversible defects in 6 (35%) patients in group 1 and 14 (82%) patients in group 2 (p = 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1995

Results of adenosine single photon emission computed tomography thallium-201 imaging in hemodynamic nonresponders.

Systolic blood pressure typically decreases during adenosine infusion because of stimulation of A2b receptors, resulting in systemic vasodilation. This study examined the results of adenosine single photon emission computed tomography (SPECT) thallium-201 imaging in patients who did not show such a decrease in blood pressure during peak adenosine effect (nonresponders). The 102 nonresponders and 341 responders had no significant differences in age, gender, history of diabetes mellitus, hypertension, or previous myocardial infarction. The extent of coronary artery disease (CAD) by angiography was also similar. The sensitivity of SPECT thallium-201 imaging in patients with one-vessel disease was 82% in nonresponders and 84% in responders (p value not significant [NS]); in patients with multivessel disease, it was 90% in nonresponders and 94% in responders (p = NS) and for all CAD, it was 87% in non-responders and 91% in responders (p = NS). Thus lack of hemodynamic systemic response during adenosine infusion does not affect sensitivity for detecting CAD.

Adenosine↗