ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2004) 7 S43

Monitoring the dynamic of interaction between the insulin receptor and protein tyrosine phosphatase-1B in living cells using BRET (bioluminescence resonance energy transfer)

N Boute, S Boubekeur, D Lacasa & T Issad

Institut Cochin, Paris, France.

Objective: PTP1B is a protein tyrosine-phosphatase, predominantly localized on the endoplasmic reticulum, which plays a major role in the regulation of the tyrosine-kinase activity of the insulin receptor (IR). A better understanding of the dynamics of the interaction between the IR and PTP1B constitutes a major task for the development of compounds that will improve insulin sensitivity.

Methods: The BRET methodology represents a major advance for the study of protein-protein interactions in intact living cells. In this methodology, one of the partners is fused to a luciferase and the other partner is fused to a fluorescent protein (YFP). If the two partners interact, resonance energy transfer occurs between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, can be detected.

Results: The interaction of the IR fused to Renilla luciferase (IR-Rluc) with a substrate-trapping mutant of PTP1B fused to YFP (YFP-PTP1B-D181A) could be followed in real time for more than 30 minutes. Insulin dose-dependently stimulated this interaction, with a half-maximal effect at 5 nM. Insulin effect could be detected at very early time-points (30 seconds), indicating the tyrosine-kinase activity of the IR is tightly controlled by PTP1B. Interestingly, basal (insulin-independent) interaction of the IR with PTP1B was much lower with a soluble form than with the endoplasmic reticulum-targeted form of PTP1B. Moreover, inhibition of IR processing suggested that basal interaction occurs during IR biosynthesis in the endoplasmic reticulum.

Conclusion: The BRET methodology can be used to monitor, in living cell, the interaction of the IR with one of the main tyrosine-phosphatases that control its activity. Moreover, localisation of PTP1B to the endoplasmic reticulum may be important to prevent insulin-independent autonomous activity of the IR precursor.

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