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GTPase activating protein activity for Rab4 is enriched in the plasma membrane of 3T3-L1 adipocytes. Possible involvement in the regulation of Rab4 subcellular localization

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Summary

The small guanosine 5′-triphosphate (GTP)ase Rab4 has been suggested to play a role in insulin-induced GLUT4 translocation. Under insulin stimulation, GLUT4 translocates to the plasma membranes, while Rab4 leaves the GLUT4-containing vesicles and becomes cytosolic. Rab proteins cycle between a GTP-bound active form and a guanosine 5′-diphosphate (GDP)-bound inactive form. The intrinsic GTPase activity of Rab proteins is low and the interconversion between the two forms is dependent on accessory factors. In the present work, we searched for a GTPase activating protein (GAP) for Rab4 in 3T3-L1 adipocytes. We used a glutathione-S-transferase (GST)-Rab4 protein which possesses the properties of a small GTPase (ability to bind GDP and GTP and to hydrolyse GTP) and can be isolated in a rapid and efficient way. This GAP activity was observed in 3T3-L1 adipocyte lysates, and was able to accelerate the hydrolysis of the [α-32P]GTP bound to GST-Rab4 into [α-32P]GDP. This activity, tentatively called Rab4-GAP, was also present in 3T3-L1 fibroblasts. The Rab4-GAP activity was present in total membrane fractions and nearly undetectable in cytosol. Following subcellular fractionation, Rab4-GAP was found to be enriched in plasma membranes when compared to internal microsomes. Insulin treatment of the cells had no effect on the total Rab4-GAP activity or on its subcellular localization. Taking our results together with the accepted model of Rab cycling in intracellular traffic, we propose that Rab4-GAP activity plays a role in the cycling between the GTP- and GDP-bound forms of Rab4, and thus possibly in the traffic of GLUT4-containing vesicles.

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Abbreviations

GAP:

GTPase activating protein

GDI:

guanosine dissociation inhibitor

GDS:

guanosine dissociation stimulator

GDF:

GDI dissociation factor

GEF:

GDP exchange factor

GST:

glutathione-S-transferase

p44mapk :

MAP-kinase isoform with an Mr 44000

PM:

plasma membranes

HLDM:

high and low density microsomes

DMEM:

Dulbecco's modified Eagle's medium

BSA:

bovine serum albumin

PVDF:

polyvinylidene difluoride

KLH:

Keyhole limpet haemocyanin

CHAPS:

3-[(3-cholamidopropyl)dimethylammonic]-1-propane sulphonate

AS:

α subunit of Gi1,2

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Authors and Affiliations

  1. Faculté de Médecine, INSERM U145, Avenue de Vallombrose, F-06107, Nice Cedex 02, France

    M. -N. Bortoluzzi,  M. Cormont, N. Gautier, E. Van Obberghen & Y. Le Marchand-Brustel

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  1. M. -N. Bortoluzzi
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  2. M. Cormont
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  3. N. Gautier
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  4. E. Van Obberghen
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  5. Y. Le Marchand-Brustel
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Bortoluzzi, M.N., Cormont, M., Gautier, N. et al. GTPase activating protein activity for Rab4 is enriched in the plasma membrane of 3T3-L1 adipocytes. Possible involvement in the regulation of Rab4 subcellular localization. Diabetologia 39, 899–906 (1996). https://doi.org/10.1007/BF00403908

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  • DOI: https://doi.org/10.1007/BF00403908

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