A novel method to monitor insulin-stimulated GTP-loading of Rab11a in cardiomyocytes
Introduction
The Rab family of small GTPases has been implicated in the regulation of intracellular vesicle transport in secretory and endocytic pathways [1], [2]. These proteins are also thought to initiate vesicle docking by interaction with the actin- and microtubule-based cytoskeleton [3] and to facilitate the formation of SNARE complexes [1], [2]. Rab GTPases are therefore considered to play a key role in the control of GLUT4 vesicle trafficking [4]. Among the large number of known Rab proteins, Rab4 [5], [6] and Rab11a [7] have been found to co-localize with GLUT4 in adipose tissue and muscle. In several studies the functional role of Rab4 in GLUT4 trafficking was assigned to intracellular sequestration of the transporter and the biogenesis of the GLUT4 storage compartment [8], [9], [10]. However, the amount of Rab4 present in GLUT4 containing vesicles decreases after insulin stimulation [5] while the presence of Rab11a was found to increase in those vesicles [7]. It was also observed, that localization of Rab11a after insulin stimulus shifted from microsomal fractions to the plasma membrane [7]. Additionally, involvement of Rab11a in trafficking between the trans-Golgi network (TGN) and the endosomal recycling compartment as well as from endosomes back to the TGN could be shown [11]. In a recent publication from our group we reported that Rab11a mediates the exo- and endocytotic movement of GLUT4 and the uptake of 2-deoxy-glucose (2-DOG) after insulin stimulation [12].
The activity of Rab GTPases is controlled by their nucleotide binding status. In this regard, a GDP-bound Rab protein represents an inactive enzyme while the GTP-bound form is an active Rab GTPase [13], [14]. Due to cycling between the active and inactive form, Rab GTPases change their intracellular localization, binding partners and catalysed functions [15], [16], [17], [18], [19]. It has therefore been of major interest to specify the nucleotide binding status of different Rab proteins related to their specific function in vesicle movement and fusion. Most experiments addressing this question have been done by adding 32P-GTP or other GTP-analogs to cell extracts or Western Blot membranes [20], [21], [22] or have been done in vitro with purified recombinant Rab proteins [23].
Based on our earlier observation of Rab11a accumulating in GLUT4 containing vesicles after insulin stimulation, the goal of the present study was to establish a method to monitor GTP-loading of Rab11a in an intact cell system in response to insulin. By introducing a biotinylated photoactive GTP-analog into the rat cardiac muscle cell line H9c2-hIR stably overexpressing the human insulin receptor, we here establish a new non-radioactive method to observe GTP-loading of HA-tagged Rab11a in living cells as a result of an extracellular stimulus. Using this approach we show that (i) Rab11a is a component of the insulin signalling cascade, (ii) that its activation is only transient and (iii) is mediated downstream of PI3-kinase and Akt.
Section snippets
Materials
Dulbecco's modified Eagles medium (DMEM), fetal calf serum (FCS), non-essential amino acids (NEAA), penicillin and streptomycin (PenStrep) were purchased from Invitrogen (Karlsruhe, Germany). G418 was a product from Calbiochem (Merck KGaA, Darmstadt, Germany). The cDNA of rat Rab11a was derived from H9c2 cells as described earlier (11) and inserted at the 3'-end of an expression cassette for HA peptide (YPYDVPDYA) into a modified pCMVβ vector (Clontech, Heidelberg, Germany) with SalI and NotI
Electroporation of H9c2-hIR cells
To introduce a GTP-analog into living H9c2-hIR cells, a transfection procedure was established using 10 μg/ml propidiumiodide as a marker for successful transfer through the plasma membrane. For optimizing the transfection procedure, the voltage as well as the number of pulses were varied at a constant capacitance of 500 μF. After electroporation, cells were resuspended in complete culture medium and incubated for 1 h before they were fixed with 2% paraformaldehyde and fluorescence was measured
Discussion
Rab GTPases are considered to play a key role in the control of GLUT4 vesicle trafficking [4]. Rab11a has been found to co-localize with GLUT4 in adipose tissue and muscle. After stimulation with insulin the presence of Rab11a was found to increase in those vesicles [7] and overexpression of different Rab11a mutants changed the abundance of GLUT4 in the plasma membrane in the basal as well as in the insulin-stimulated state [12].
As the activity of Rab small GTPases is modulated by their
Conclusion
In conclusion, the data of the present study show that Rab11a is a target of insulin action. It is short-time activated by insulin involving a PI3-kinase-dependent pathway. Future work will be needed to identify the direct upstream regulator of Rab11a that mediates insulin action. However, our data suggest that this regulator is downstream of Akt.
Acknowledgements
This work was supported by the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Bundesministerium für Gesundheit and European Union COST Action B17.
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