scholarly journals A novel regulatory mechanism for trimeric GTP-binding proteins in the membrane and secretory granule fractions of human and rodent β cells

1996 ◽  
Vol 313 (1) ◽  
pp. 97-107 ◽  
Author(s):  
Anjaneyulu KOWLURU ◽  
Scott E. SEAVEY ◽  
Christopher J. RHODES ◽  
Stewart A. METZ
Keyword(s):  
Molecular Mass ◽  
Secretory Granule ◽  
Membrane Fraction ◽  
Binding Proteins ◽  
Β Subunit ◽  
Β Cells ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Granule Fraction ◽  
Normal Rat

Recently we described roles for heterotrimeric and low-molecular-mass GTP-binding proteins in insulin release from normal rat islets. During these studies, we observed that a protein with an apparent molecular mass (37 kDa) similar to that of the β subunit of trimeric GTP-binding proteins underwent phosphorylation in each of five classes of insulin-secreting cells. Incubation of the β cell total membrane fraction or the isolated secretory granule fraction (but not the cytosolic fraction) with [γ-32P]ATP or [γ-32P]GTP resulted in the phosphorylation of this protein, which was selectively immunoprecipitated by an antiserum directed against the common β subunit of trimeric G-proteins. Disruption of the αβγ trimer (by pretreatment with either fluoroaluminate or guanosine 5ʹ-[γ-thio]triphosphate) prevented β subunit phosphorylation. Based on differential sensitivities to pH, heat and the histidine-selective reagent diethyl pyrocarbonate (and reversal of the latter by hydroxylamine), the phosphorylated amino acid was presumptively identified as histidine. Incubation of pure β subunit alone or in combination with the exogenous purified α subunit of transducin did not result in the phosphorylation of the β subunit, but addition of the islet cell membrane fraction did support this event, suggesting that membrane localization (or a membrane-associated factor) is required for β subunit phosphorylation. Incubation of phosphorylated β subunit with Gα·GDP accelerated the dephosphorylation of the β subunit, accompanied by the formation of Gα·GTP. Immunoblotting detected multiple α subunits (of Gi, Go and Gq) and at least one β subunit in the secretory granule fraction of normal rat islets and insulinoma cells. These data describe a potential alternative mechanism for the activation of GTP-binding proteins in β cells which contrasts with the classical receptor-agonist mechanism: Gβ undergoes transient phosphorylation at a histidine residue by a GTP-specific protein kinase; this phosphate, in turn, may be transferred via a classical Ping-Pong mechanism to Gα·GDP (inactive), yielding the active configuration Gα·GTP in secretory granules (a strategic location to modulate exocytosis).

Low molecular mass GTP-binding proteins in subcellular fractions of the pancreas: regulated phosphoryl G proteins

1992 ◽  
Vol 262 (2) ◽  
pp. C493-C500 ◽  
Author(s):  
B. Goke ◽  
J. A. Williams ◽  
M. J. Wishart ◽  
R. C. De Lisle
Keyword(s):  
Molecular Mass ◽  
Membrane Fraction ◽  
Binding Proteins ◽  
Total Cell ◽  
Cell Protein ◽  
Zymogen Granule ◽  
Total Cell Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Granule Membrane

Low molecular mass guanine nucleotide-binding proteins [small guanosine 5'-triphosphate (GTP)-binding proteins] and phosphoproteins of the pancreatic acinar cell were compared by two-dimensional gel electrophoresis. [35S]GTP alpha S blotting analysis of the total cell protein revealed 20 GTP-binding proteins ranging in molecular mass from 20 to 28 kDa and pI of 4.8-6.4. Analysis of 32P-labeled total cell protein revealed over 300 phosphoproteins. The subcellular distribution of the small GTP-binding proteins was examined: 17 were located in the rough endoplasmic reticulum (RER) fraction, 19 in the smooth microsome fraction, 14 in the zymogen granule membrane fraction, and 11 in the cytosolic fraction, with overlap between fractions. Of the GTP-binding proteins, two were also found to be phosphoproteins, one located on the RER and one on the zymogen granule membrane. The phosphorylation of both small GTP-binding proteins was increased by secretagogue stimulation of the cells but with different time courses. The RER small GTP-binding protein demonstrated a rapid and transient increase in 32P labeling, whereas the granule membrane small GTP-binding protein showed an increase at longer times (30 min). Two of the cytosolic small GTP-binding proteins were also seen in particulate fractions, especially in the zymogen granule membrane fraction, suggesting the possibility of cycling between cytosolic and membrane-associated forms.

scholarly journals Identification of the ral and rac1 Gene Products, Low Molecular Mass GTP-binding Proteins from Human Platelets

1989 ◽  
Vol 264 (28) ◽  
pp. 16383-16389
Author(s):  
P G Polakis ◽  
R F Weber ◽  
B Nevins ◽  
J R Didsbury ◽  
T Evans ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Binding Proteins ◽  
Human Platelets ◽  
Gene Products ◽  
Gtp Binding Proteins ◽  
Gtp Binding

scholarly journals Modulation of insulin secretion from normal rat islets by inhibitors of the post-translational modifications of GTP-binding proteins

1993 ◽  
Vol 295 (1) ◽  
pp. 31-40 ◽  
Author(s):  
S A Metz ◽  
M E Rabaglia ◽  
J B Stock ◽  
A Kowluru
Keyword(s):  
Insulin Secretion ◽  
Insulin Release ◽  
Binding Proteins ◽  
Mevalonic Acid ◽  
Post Translational Modifications ◽  
Gtp Binding Proteins ◽  
Carboxyl Methylation ◽  
Gtp Binding ◽  
Protein Palmitoylation ◽  
Normal Rat

Many GTP-binding proteins (GBPs) are modified by mevalonic acid (MVA)-dependent isoprenylation, carboxyl methylation or palmitoylation. The effects of inhibitors of these processes on insulin release were studied. Intact pancreatic islets were shown to synthesize and metabolize MVA and to prenylate several candidate proteins. Culture with lovastatin (to inhibit synthesis of endogenous MVA) caused the accumulation in the cytosol of low-M(r) GBPs (labelled by the [alpha-32P]GTP overlay technique), suggesting a disturbance of membrane association. Concomitantly, lovastatin pretreatment reduced glucose-induced insulin release by about 50%; co-provision of 100-200 microM MVA totally prevented this effect. Perillic acid, a purported inhibitor of the prenylation of small GBPs, also markedly reduced glucose-induced insulin secretion. Furthermore, both N-acetyl-S-trans, trans-farnesyl-L-cysteine (AFC), which inhibited the base-labile carboxyl methylation of GBPs in islets or in transformed beta-cells, and cerulenic acid, an inhibitor of protein palmitoylation, also reduced nutrient-induced secretion; an inactive analogue of AFC (which did not inhibit carboxyl methylation in islets) had no effect on secretion. In contrast with nutrients, the effects of agonists that induce secretion by directly activating distal components in signal transduction (such as a phorbol ester or mastoparan) were either unaffected or enhanced by lovastatin or AFC. These data are compatible with the hypothesis that post-translational modifications are required for one or more stimulatory GBPs to promote proximal step(s) in fuel-induced insulin secretion, whereas one or more inhibitory GBPs might reduce secretion at a more distal locus.

scholarly journals The mRNA encoding a β subunit of heterotrimeric GTP-binding proteins is localized to the animal pole of Xenopus laevis oocyte and embryos

1996 ◽  
Vol 59 (2) ◽  
pp. 141-151 ◽  
Author(s):  
Eric Devic ◽  
Laurent Paquereau ◽  
Karine Rizzoti ◽  
Armelle Monier ◽  
Bernard Knibiehler ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Binding Proteins ◽  
Xenopus Laevis Oocyte ◽  
Β Subunit ◽  
Animal Pole ◽  
Gtp Binding Proteins ◽  
Gtp Binding

scholarly journals Prolonged Depletion of Guanosine Triphosphate Induces Death of Insulin-Secreting Cells by Apoptosis*

Endocrinology ◽  
1998 ◽  
Vol 139 (9) ◽  
pp. 3752-3762 ◽  
Author(s):  
Guodong Li ◽  
Venkatesh Babu G. Segu ◽  
Mary E. Rabaglia ◽  
Rui-Hua Luo ◽  
Anjaneyulu Kowluru ◽  
...  
Keyword(s):  
Cell Death ◽  
Binding Proteins ◽  
Immunosuppressive Agents ◽  
Specific Effect ◽  
Cell Number ◽  
Immunosuppressive Drugs ◽  
Β Cells ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Hit Cells

Abstract Inhibitors of IMP dehydrogenase, such as mycophenolic acid (MPA) and mizoribine, which deplete cellular GTP, are used clinically as immunosuppressive drugs. The prolonged effect of such agents on insulin-secreting β-cells (HIT-T15 and INS-1) was investigated. Both MPA and mizoribine inhibited mitogenesis, as reflected by[ 3H]thymidine incorporation. Cell number, DNA and protein contents, and cell (metabolic) viability were decreased by about 30%, 60%, and 80% after treatment of HIT cells with clinically relevant concentrations (e.g. 1 μg/ml) of MPA for 1, 2, and 4 days, respectively. Mizoribine (48 h) similarly induced the death of HIT cells. INS-1 cells also were damaged by prolonged MPA treatment. MPA-treated HIT cells displayed a strong and localized staining with a DNA-binding dye (propidium iodide), suggesting condensation and fragmentation of DNA, which were confirmed by detection of DNA laddering in multiples of about 180 bp. DNA fragmentation was observed after 24-h MPA treatment and was dose dependent (29%, 49%, and 70% of cells were affected after 48-h exposure to 1, 3, and 10 μg/ml MPA, respectively). Examination of MPA-treated cells by electron microscopy revealed typical signs of apoptosis: condensed and marginated chromatin, apoptotic bodies, cytosolic vacuolization, and loss of microvilli. MPA-induced cell death was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating a specific effect of GTP depletion. An inhibitor of protein isoprenylation (lovastatin, 10–100μ m for 2–3 days) induced cell death and DNA degradation similar to those induced by sustained GTP depletion, suggesting a mediatory role of posttranslationally modified GTP-binding proteins. Indeed, impeding the function of G proteins of the Rho family (via glucosylation using Clostridium difficile toxin B), although not itself inducing apoptosis, potentiated cell death induced by MPA or lovastatin. These findings indicate that prolonged depletion of GTP induces β-cell death compatible with apoptosis; this probably involves a direct impairment of GTP-dependent RNA-primed DNA synthesis, but also appears to be modulated by small GTP-binding proteins. Treatment of intact adult rat islets (the β-cells of which replicate slowly) induced a modest, but definite, death by apoptosis over 1- to 3-day periods. Thus, more prolonged use of the new generation of immunosuppressive agents exemplified by MPA might have deleterious effects on the survival of islet or pancreas grafts.

Bacterial protein toxins inhibiting low-molecular-mass GTP-binding proteins

2001 ◽  
Vol 291 (4) ◽  
pp. 243-250 ◽  
Author(s):  
Ingo Just ◽  
Fred Hofmann ◽  
Harald Genth ◽  
Ralf Gerhard
Keyword(s):  
Molecular Mass ◽  
Binding Proteins ◽  
Bacterial Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Protein Toxins
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