Negative feedback regulation of Wnt signaling by Gbetagamma-mediated reduction of Dishevelled

Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gbeta2) bound to Axin and Gbeta2 inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gbeta2gamma2 mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gbeta2gamma2 into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by beta-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gbeta2gamma2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gbeta2gamma2 is required because a mutant of Gbeta2, Gbeta2 (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gbeta1 and Gbeta2. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled-->Gbetagamma-->PLC-->Ca+2/PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gbeta2gamma2 inhibits Wnt signaling by degradation of Dishevelled.

Internalization and down-regulation of the epidermal growth factor receptor in a human keratinocyte cell line HaCaT / 中华皮肤科杂志

Objective To understand the molecular mechanism underlying the epidermal growth factor receptors(EGFR)signal transduction and its feed-back regulation.Methods Two human keratinocyte cell lines,HaCaT and CHOwt,were cultured and treated with a certain concentration of different ligands,including epidermal growth factor(EGF),heparin-bounding(HB)-EGF,transforming growth factor α (TGFα)and heregulin(HER),for various durations(2,4,8,16,20 hours).After the treatment,cells were collected and protein was extracted.The amount of total and active EGFR was measured by immunoprecipitation and immunoblot assay.The internalization and down-regulation of EGFR were visualized with immunofluorescence and laser seanning confocal microscopy.Results As shown by immunoblot technique,EGF and HB-EGF continuously down-regulated the total amount of EGFRs,whereas TGFα and HER had no significant effect on the degradation of EGFRs.The activation of EGFRs was also attenuated to different extent after long-time treatment with EGF,HB-EGF and TGFα.As indirect immunofluorescenee revealed,in untreated HaCaT and CHOwt cells,EGFRs were essentially located at the plasma membrane,with a little cytosolic distribution;after ten-minute treatment with EGF,EGFRs clustered into patch-like structures which were particularly obvious in HaCaT cells,and translocated into cytoplasmic vesicles resembling endosomes (relatively apparent in CHOwt cells),while the total amount of EGFRs remained constant in these cells.The fluorescence signal from the total EGFRs decreased evidently after four-hour treatment with EGF,indicating a strong reduction in the receptors.Conclusions EGF and HB-EGF,but not TGFα or Heregulin,could down-regulate the amount of total and active EGFRs.There might be different mechanisms for the signal transduction related to EGFRs intemalization and down-regulation between HaCaT and CHOwt cells.