Interaction between the insulin receptor and Grb14: a dynamic study in living cells using BRET.
Grb14 is a molecular adaptor that binds to the activated insulin receptor (IR) and negatively regulates insulin signaling. We have studied the dynamics of interaction of the IR with Grb14, in real time, in living HEK cells, using bioluminescence resonance energy transfer (BRET). Insulin rapidly and dose-dependently stimulated this interaction. Removing insulin from the incubation medium only resulted in a modest decrease in BRET signal, indicating that the interaction between the IR and Grb14 can remain long after insulin stimulus has disappeared. BRET saturation experiments indicated that insulin markedly increases the affinity between IR and Grb14, resulting in recruitment of the adaptor to the activated IR. In addition, using both BRET and co-immunoprecipitation experiments, we demonstrated that insulin induced the dimerization of Grb14, most likely as a result of simultaneous binding of two Grb14 molecules on the activated IR. We also investigated the relationships between IR, Grb14 and the protein tyrosine phosphatase PTP1B. We observed that insulin-induced BRET between the IR and PTP1B was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the insulin receptor, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the ERK pathway. Our work suggests that Grb14 may regulate signalling through the insulin receptor by controlling its tyrosine-dephosphorylation in a site-specific manner.
Nouaille
Sébastien
S
Institut Cochin, Département de Biologie Cellulaire, Paris F-75014, France.
Blanquart
Christophe
C
Zilberfarb
Vladimir
V
Boute
Nicolas
N
Perdereau
Dominique
D
Burnol
Anne-Françoise
AF
Issad
Tarik
T
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007668:Kidney / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D048868:Adaptor Proteins, Signal Transducing; C099655:GRB14 protein, human; D007328:Insulin; D008164:Luminescent Proteins; D011972:Receptor, Insulin; D020928:Mitogen-Activated Protein Kinases; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases