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.

Evidence for Differential Roles of the Rho Subfamily of GTP-Binding Proteins in Glucose- and Calcium-Induced Insulin Secretion from Pancreatic β Cells

1997 ◽  
Vol 54 (10) ◽  
pp. 1097-1108 ◽  
Author(s):  
Anjaneyulu Kowluru ◽  
Guodong Li ◽  
Mary E Rabaglia ◽  
Venkatesh B Segu ◽  
Fred Hofmann ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Binding Proteins ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Gtp Binding Proteins ◽  
Gtp Binding

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).

scholarly journals Redistribution of ADP-ribosylation factor during stimulation of permeabilized cells with GTP analogues

1991 ◽  
Vol 275 (3) ◽  
pp. 639-644 ◽  
Author(s):  
R Regazzi ◽  
S Ullrich ◽  
R A Kahn ◽  
C B Wollheim
Keyword(s):  
Insulin Secretion ◽  
Molecular Mass ◽  
Binding Proteins ◽  
Secretory Granules ◽  
Rinm5f Cells ◽  
Permeabilized Cells ◽  
Adp Ribosylation ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Adp Ribosylation Factor

Low-molecular-mass GTP-binding proteins of the ras family were analysed by [32P]GTP binding after PAGE and transfer to nitrocellulose membranes. By this technique, several GTP-binding proteins in the 20-30 kDa range were detected in both cytosolic and microsomal fractions of RINm5F cells. One of these, displaying an apparent molecular mass of about 20 kDa and a pI of 6.7, was mainly cytosolic and was shown to be the ADP-ribosylation factor (ARF) by using specific antibodies. When permeabilized RINm5F cells were incubated with the stable GTP analogues guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) the amount of ARF increased in a fraction containing both Golgi and plasma-membrane markers, but not in the fraction containing secretory granules, mitochondria and lysosomes. GTP, GDP and its beta-thio analogue did not enhance ARF binding to membranes, smg25/rab3 and rho, as well as all the other small GTP-binding proteins detected by the [32P]GTP binding, did not redistribute under these conditions. As GTP[S] stimulates insulin secretion in these cells, we also examined the relationship between ARF translocation and insulin secretion. Both phenomena were elicited by GTP[S] with an EC50 (median effective concentration) of about 10 microM. p[NH]ppG was equipotent with GTP[S] in inducing insulin secretion (EC50 about 10 microM), but higher concentrations (about 500 microns) were required to achieve the same maximal ARF redistribution. These results suggest that: (1) ARF is subject to cycling between a membrane-associated and a free/loosely attached form, determined by the species of bound guanine nucleotide; (2) ARF alone does not seem to regulate exocytosis in insulin-secreting cells.

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

1999 ◽  
Vol 82 (09) ◽  
pp. 1177-1181 ◽  
Author(s):  
Hubert de Leeuw ◽  
Pauline Wijers-Koster ◽  
Jan van Mourik ◽  
Jan Voorberg
Keyword(s):  
Endothelial Cells ◽  
Binding Proteins ◽  
Binding Protein ◽  
Control Release ◽  
Small Gtpase ◽  
Gtp Binding Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

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