Subcellular localization of the enzymes that dephosphorylate <i>myo</i>-inositol polyphosphates in human platelets

Vedia, L Molina y; Nolan, Roger D.; Lapetina, Eduardo G.

doi:10.1042/bj2550795

Cited by 19 publications

(10 citation statements)

References 42 publications

(35 reference statements)

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“…6). A similar observation has been reported (15,21) on the decrease of ADP-ribosylation of the membrane-bound substrate of cholera toxin after 24 hr of treatment with iloprost. The cholera toxin-induced ADP-ribosylation of the cytosolic 44-kDa peptide was also decreased by pretreatment with iloprost, and this change, observed at 20 min, was more evident at 1 hr (Fig.…”

Section: Resultssupporting

confidence: 66%

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein.

Vedia

Reep²,

Lapetina

1988

Proc. Natl. Acad. Sci. U.S.A.

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Communicated by George H. Hitchings, May 20, 1988 ABSTRACT ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADPribosylates a 41-kDa polypeptide. Two antisera against the a subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42-and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with

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Section: Resultssupporting

confidence: 66%

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein.

Vedia

Reep²,

Lapetina

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…Antiserum A-572 recognizes the peptide Lys-Gln-LeuGln-Lys-Asp-Lys-Gln-Val-Tyr-Arg-Ala-Thr-His-Arg, which corresponds to residues 28-42 in the amino-terminal region of Gs,. In previous studies using immunoblot techniques, we showed that when platelets were incubated with iloprost under conditions in which cholera toxin-induced ADPribosylation was decreased, there was no loss of Gs, from the membrane (6). We now show that a 23-kDa peptide that is recognized by antiserum 584, but not by antiserum A-572, is lost from the membrane during treatment with iloprost and is recovered in the platelet cytosol.…”

mentioning

confidence: 62%

Iloprost-induced translocation of a 23-kDa protein that is recognized by a Gs alpha antiserum.

Vedia

Lapetina²

1989

Proc. Natl. Acad. Sci. U.S.A.

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We have observed that ioprost, a prostacyclin analog, causes the translocation of a 23-kDa protein from the membrane to the cytosol of human platelets. This 23-kDa protein is recognized by a G.. (a subunit ofstimulatory guanine nucleotide binding regulatory protein) antiserum but is not ADP-ribosylated by cholera toxin. The translocation of the G,.-related protein might be an important part of the complex chain of events resulting in agonist-induced desensitization.Iloprost is a stable prostacyclin analog that stimulates platelet adenylyl cyclase and inhibits platelet responses (1). This inhibition is thought to result from increased levels of cyclic AMP (2) but the precise molecular mechanism of the inhibition is not understood. Prolonged treatment of platelets with iloprost decreases the cholera toxin-induced ADP-ribosylation of the a subunit of the stimulatory guanine nucleotide binding regulatory protein (Gs,,,) (1,3). Iloprost also decreases cholera toxin-induced ADP-ribosylation of a cytosolic 44-kDa protein (3).We wanted to know if the decrease of cholera toxininduced ADP-ribosylation of G,, was due to a loss of Gsa from platelet membranes. To investigate this, we first used two Gsa antibodies that were raised against synthetic peptides (4, 5). Antiserum 584 recognizes the peptide Thr-Pro-Glu-Pro-GlyGlu-Asp-Pro-Arg-Val-Thr-Arg-Ala-Lys-Tyr, which corresponds to residues 325-339 of the carboxyl-terminal region of Gsa. Antiserum A-572 recognizes the peptide Lys-Gln-LeuGln-Lys-Asp-Lys-Gln-Val-Tyr-Arg-Ala-Thr-His-Arg, which corresponds to residues 28-42 in the amino-terminal region of Gs,. In previous studies using immunoblot techniques, we showed that when platelets were incubated with iloprost under conditions in which cholera toxin-induced ADPribosylation was decreased, there was no loss of Gs, from the membrane (6). We now show that a 23-kDa peptide that is recognized by antiserum 584, but not by antiserum A-572, is lost from the membrane during treatment with iloprost and is recovered in the platelet cytosol.EXPERIMENTAL PROCEDURES Materials. Affinity-purified goat antibody to rabbit IgG [F(ab')2 fragment] labeled with 125I was from NEN. Gsa 584 and Gs, A-572 antisera were kindly provided by Susanne Mumby and Alfred Gilman (University of Texas, Dallas). AS7 antiserum was provided by Allen Spiegel (National Institutes of Health) and iloprost was a gift from ClausSteffen Sturzebecher (Schering).Preparation of Platelets. Platelet membranes were prepared as described (1, 3). Briefly, platelets from 5 ml of platelet-rich plasma were disrupted in 5 ml of 5 mM Tris HCI buffer (pH 7.4) containing 0.25 mM EDTA, using a Dounce homogenizer. The membranes and supernatant were separated after centrifugation at 100,000 x g for 40 min, and the membrane pellet was washed twice by resuspension in 5 ml of the same Tris-HCl/EDTA buffer. The washes were discarded. The pellet was finally resuspended in 200 ,l of 50 mM Tris HCl buffer (pH 7.4) containing 0.25 mM EDTA.[a-32P]ADP-Ribosylation of Proteins by Cholera ...

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“…Another possibility, not addressed in this study, is that protein kinase C may be activated differently by the three agonists and that this determines the direction of the metabolism of 145IP3. In some cell types 145IP35' phosphatase has been shown to be localized at the cytoplasmic membrane (Molina et al 1988) and sensitive to phosphorylation by protein kinase C (Connolly et al 1987). However, this is not a universal finding (Biden et al 1988).…”

Section: Discussionmentioning

confidence: 98%

Mechanisms Involved in the Stimulation of Aldosterone Production by Angiotensin Ii, Vasopressin and Endothelin

Woodcock

Tanner

Caroccia

et al. 1990

Clin Exp Pharma Physio

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1. Endothelin (ET), vasopressin (VP) and angiotensin II (AII) all stimulate aldosterone production in adrenal glomerulosa cells but the response to AII is greater than that to either ET or VP. 2. Total inositol phosphate responses to AII and ET were similar but the response to VP was lower. 3. Cytosolic free Ca2+ responses to AII were higher than to either of the other peptides. 4. Metabolism of 145IP3 was different under stimulation by the three different peptides. 5. Adrenal glomerulosa cells can distinguish between three different agonists which stimulate phosphatidylinositol turnover and produce a selective response to each peptide.

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Subcellular localization of the enzymes that dephosphorylate myo-inositol polyphosphates in human platelets

Cited by 19 publications

References 42 publications

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein.

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein.

Iloprost-induced translocation of a 23-kDa protein that is recognized by a Gs alpha antiserum.

Mechanisms Involved in the Stimulation of Aldosterone Production by Angiotensin Ii, Vasopressin and Endothelin

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