Shintaro Nakashima

Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors.

The involvement of Ral and its downstream molecules in receptor‐mediated endocytosis was examined. Expression of either RalG23V or RalS28N, which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of RalG23V or RalS28N in CHO‐IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by full‐length RalBP1 which is an effector protein of Ral, but was inhibited by its C‐terminal region which binds directly to Ral and POB1. POB1 is a binding protein of RalBP1 and has the Eps15 homology (EH) domain. Deletion mutants of POB1 inhibited internalization of EGF and insulin. However, internalization of transferrin was unaffected by Ral, RalBP1, POB1 and their mutants. Epsin and Eps15 have been reported to be involved in the regulation of endocytosis of the receptors for EGF and transferrin. The EH domain of POB1 bound directly to Epsin and Eps15. Taken together with the observation that EGF and insulin activate Ral, these results suggest that Ral, RalBP1 and POB1 transmit the signal from the receptors to Epsin and Eps15, thereby regulating ligand‐dependent receptor‐mediated endocytosis.

Axin prevents Wnt-3a-induced accumulation of β-catenin

When Axin, a negative regulator of the Wnt signaling pathway, was expressed in COS cells, it coeluted with glycogen synthase kinase-3β (GSK-3β), β-catenin, and adenomatous polyposis coli protein (APC) in a high molecular weight fraction on gel filtration column chromatography. In this fraction, GSK-3β, β-catenin, and APC were co-precipitated with Axin. Although β-catenin was detected in the high molecular weight fraction in L cells on gel filtration column chromatography, addition of conditioned medium expressing Wnt-3a to the cells increased β-catenin in the low molecular weight fraction. However, Wnt-3a-dependent accumulation of β-catenin was greatly inhibited in L cells stably expressing Axin. Axin also suppressed Wnt-3a-dependent activation of Tcf-4 which binds to β-catenin and acts as a transcription factor. These results suggest that Axin forms a complex with GSK-3β, β-catenin, and APC, resulting in the stimulation of the degradation of β-catenin and that Wnt-3a induces the dissociation of β-catenin from the Axin complex and accumulates β-catenin.

Epsin binds to the EH domain of POB1 and regulates receptor-mediated endocytosis.

POB1 has been identified as a RalBP1-binding protein and has the Eps15 homology (EH) domain. The EH domain-containing proteins have been suggested to be involved in clathrin-dependent endocytosis. To clarify the function of POB1, we purified a protein which binds to the EH domain of POB1 from bovine brain cytosol and identified it as Epsin, which is known to bind to the EH domain of Eps15. Epsin has three Asn-Pro-Phe (NPF) motifs in the C-terminal region, which are known to form the core sequence for the binding to the EH domain. The EH domain of POB1 interacted directly with the region containing the NPF motifs of Epsin. Expression of Epsin in CHO-IR cells inhibited internalization of insulin although it affected neither insulin-binding nor autophosphorylation activities of the insulin receptor. Taken together with the observations that Epsin is involved in internalization of the receptors for epidermal growth factor and transferrin, these results suggest that Epsin is a binding partner of POB1 and their binding regulates receptor-mediated endocytosis.