scholarly journals Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle: studies on the role of small-molecular-mass GTP-binding proteins

1994 ◽  
Vol 301 (1) ◽  
pp. 177-182 ◽  
Author(s):  
I Uphues ◽  
T Kolter ◽  
B Goud ◽  
J Eckel
Keyword(s):  
Cardiac Muscle ◽  
Molecular Mass ◽  
G Proteins ◽  
Binding Proteins ◽  
Glucose Transporter ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Microsomal Membranes ◽  
Glucose Transporter Glut4

Subcellular fractions obtained from rat cardiac ventricular tissue were used to elucidate a possible functional relationship between small-molecular-mass G-proteins and the insulin-responsive glucose transporter GLUT4. Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Incubation with [alpha-32P]GTP revealed the presence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsomal membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriched membrane vesicles. This material was found to contain a much decreased amount of small G-proteins, with the exclusive presence of the 24 kDa species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decreased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enriched vesicles from insulin-treated animals. Western-blot analysis of microsomal membranes with a panel of antisera against rab GTP-binding proteins indicated the presence of rab4A, with a molecular mass of 24 kDa, whereas rab1A, rab2 and rab6 were not observed. rab4A was barely detectable in GLUT4-enriched vesicles; however, insulin produced an extensive shift of rab4A from the cytosol and the microsomal fraction to the plasma membrane with a parallel increase in GLUT4. These data show that a small GTP-binding protein is co-localized with GLUT4 in an insulin-responsive intracellular compartment, and strongly suggest that this protein is involved in the exocytosis of GLUT4 in cardiac muscle. Furthermore, the observed translocation of rab4A is compatible with insulin-induced endosome recycling processes, possibly including the glucose transporters.

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.

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 Characterization of GTP-binding proteins in Golgi-associated membrane vesicles from rat adipocytes

1992 ◽  
Vol 283 (3) ◽  
pp. 795-801 ◽  
Author(s):  
A Schürmann ◽  
W Rosenthal ◽  
G Schultz ◽  
H G Joost
Keyword(s):  
G Proteins ◽  
Glucose Transport ◽  
Subcellular Distribution ◽  
Sucrose Gradient ◽  
Binding Proteins ◽  
Glucose Transporters ◽  
Beta Subunits ◽  
Transport Activity ◽  
Gtp Binding Proteins ◽  
Gtp Binding

We have previously reported that guanine nucleotides inhibit glucose transport activity reconstituted from adipocyte membrane fractions. In order to further investigate the hypothetical involvement of guanine-nucleotide-binding proteins (GTP-binding proteins) in the regulation of insulin-sensitive glucose transport activity, we studied their subcellular distribution in adipocytes treated or not with insulin. Adipocytes were homogenized and fractionated to yield plasma membranes (PM) and a Golgi-enriched fraction of intracellular membranes (low-density microsomes, LDM). In these membrane fractions, total guanosine 5′-[gamma-[35S]thio]triphosphate ([35S]GTP[S]) binding, alpha- and beta-subunits of heterotrimeric G-proteins, proto-oncogenes Ha-ras and K-ras, and 23-28 kDa GTP-binding proteins were assayed. The levels of alpha s and alpha i (the alpha-subunits of Gs and Gi) were approx. 8-fold lower in LDM than in PM; beta-subunits, Ha-ras and K-ras were not detectable in LDM. Total GTP[S]-binding sites and 23-28 kDa GTP-binding proteins were present in LDM in approximately the same concentrations as in PM. Insulin gave rise to the characteristic translocation of glucose transporters, but failed to alter the subcellular distribution of any of the GTP-binding proteins. Fractionation of the LDM on a discontinuous sucrose gradient revealed that alpha s and alpha i, as detected with antiserum against a common peptide sequence (alpha common), and the bulk of the 23-28 kDa G-proteins sedimented at different sucrose densities. None of the GTP-binding proteins co-sedimented with glucose transporters. Furthermore, the inhibitory effect of GTP[S] on the reconstituted transport activity was lost in the peak fractions of glucose transporters partially purified on the sucrose gradient. These data indicate that LDM from adipocytes contain several GTP-binding proteins in discrete vesicle populations. However, the intracellular GTP-binding proteins are not tightly associated with the vesicles containing the glucose transporter.

scholarly journals Roles of insulin, guanosine 5′-[γ-thio]triphosphate and phorbol 12-myristate 13-acetate in signalling pathways of GLUT4 translocation

1996 ◽  
Vol 315 (3) ◽  
pp. 875-882 ◽  
Author(s):  
Mikio TODAKA ◽  
Hideki HAYASHI ◽  
Takanobu IMANAKA ◽  
Yasumasa MITANI ◽  
Seika KAMOHARA ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Glucose Transporter ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Cell System ◽  
Signalling Pathways ◽  
Dominant Negative Mutant ◽  
Glut4 Translocation ◽  
Gtp Binding Proteins ◽  
Gtp Binding

Insulin, guanosine 5´-[γ-thio]triphospate (GTP[S]) and phorbol 12-myristate 13-acetate (PMA) trigger the translocation of GLUT4 (type 4 glucose transporter; insulin-sensitive glucose transporter) from an intracellular pool to the cell surface. We have developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact 3T3-L1 adipocytes and Chinese hamster ovary (CHO) cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We examined the roles of insulin, GTP[S] and PMA in the signalling pathways of GLUT4 translocation in the CHO cell system. Among small molecular GTP-binding proteins, ras, rab3D, rad and rho seem to be candidates as signal transmitters of insulin-stimulated GLUT4 translocation. Overexpression of wild-type H-ras and the dominant negative mutant H-rasS17N in our cell system respectively enhanced and blocked insulin-stimulated activation of mitogen-activated protein kinase, but did not affect insulin-stimulated GLUT4 translocation. Overexpression of rab3D or rad in the cells did not affect GLUT4 translocation triggered by insulin, GTP[S] or PMA. Treatment with Botulinum C3 exoenzyme, a specific inhibitor of rho, had no effect on GLUT4 translocation induced by insulin, GTP[S] or PMA. Therefore these small molecular GTP-binding proteins are not likely to be involved in GLUT4 translocation. In addition, insulin, GTP[S] and PMA apparently stimulate GLUT4 translocation through independent pathways.

scholarly journals Failure of insulin-regulated recruitment of the glucose transporter GLUT4 in cardiac muscle of obese Zucker rats is associated with alterations of small-molecular-mass GTP-binding proteins

1995 ◽  
Vol 311 (1) ◽  
pp. 161-166 ◽  
Author(s):  
I Uphues ◽  
T Kolter ◽  
B Goud ◽  
J Eckel
Keyword(s):  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Glucose Transporter ◽  
Zucker Rats ◽  
Gtp Binding Protein ◽  
Insulin Resistant ◽  
Gtp Binding ◽  
Obese Rats ◽  
Obese Zucker Rats ◽  
Glucose Transporter Glut4

Cardiac ventricular tissue of lean and genetically obese (fa/fa) Zucker rats was used to study the expression, subcellular distribution and insulin-induced recruitment of the glucose transporter GLUT4 and to elucidate possible molecular alterations of the translocation process. Hearts were removed from basal and insulin-treated (20 min) lean and obese Zucker rats, and processed for subcellular fractionation and Western blotting of proteins. In obese rats, the total GLUT4 content in a crude membrane fraction was reduced to 75 +/- 8% (P = 0.019) of lean controls. In contrast, GLUT4 abundance in plasma membranes was not significantly different between lean and obese rats with a concomitant decrease (47 +/- 3%) in the microsomal fraction of obese animals. In plasma membranes of lean animals insulin was found to increase the GLUT4 abundance to 294 +/- 43% of control with a significantly (P = 0.009) reduced effect in the obese group (139 +/- 10% of control). In these animals insulin failed to recruit GLUT4 from the microsomal fraction, whereas the hormone induced a significant decrease (41 +/- 4%) of microsomal GLUT4 in lean controls. In GLUT4-enriched membrane vesicles, obtained from cardiac microsomes of lean rats, a 24 kDa GTP-binding protein could be detected, whereas no significant labelling of this species was observed in GLUT4 vesicles prepared from obese animals. In addition to the translocation of GLUT4, insulin was found to promote the movement of the small GTP-binding protein rab4A from the cytosol (decrease to 61 +/- 13% of control) to the plasma membrane (increase to 177 +/- 19% of control) in lean rats with no effect of the hormone on rab4A redistribution in the obese group. In conclusion, cardiac glucose uptake of insulin-resistant obese Zucker rats is subject to multiple cellular abnormalities involving a reduced expression, altered redistribution and defective recruitment of GLUT4. We show here an association of the latter defect with alterations at the level of small GTP-binding proteins possibly leading to an impaired trafficking of GLUT4 in the insulin-resistant state.

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