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

Abstract

POB1 has been identi®ed 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 puri®ed a protein which binds to the EH domain of POB1 from bovine brain cytosol and identi®ed 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 aected neither insulinbinding 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.

The Brain Exocyst Complex Interacts with RalA in a GTP-dependent Manner

“…The mammalian equivalent of seven of the eight yeast exocyst proteins have been cloned (23)(24)(25). Our results are the first to demonstrate that the mammalian exocyst also interacts with small GTPases and suggests that, in addition to an established role in endocytosis (14,26) activated RalA may play a central role in directing sites of exocytosis.…”

The association of epsin with ubiquitinated cargo along the endocytic pathway is negatively regulated by its interaction with clathrin

“…Epsin contains a multiplicity of binding sites, including binding sites for phosphoinositides (39), a transcription factor (40), Ub (6,14), clathrin (41,42), the clathrin adaptor AP-2 (5), and other signaling (43) and endocytic proteins (5,44,45), which implies the occurrence of regulatory mechanisms to control these interactions. As we have shown previously, some of the interactions of epsin are regulated by its phosphorylation and reversible ubiquitination (15,46).…”

Interaction of POB1, a Downstream Molecule of Small G Protein Ral, with PAG2, a Paxillin-binding Protein, Is Involved in Cell Migration

“…The entire PCR products were sequenced, and the structures of all plasmids were confirmed by restriction analyses. pGEX-2T/POB1-(1-125), pGEX-2T/POB1-(126 -227), pGEX-2T/POB1-(228 -406), pGEX-2T/ POB1-(322-521), pGEX-2T/RalBP1-(364 -647), pMAL-c2/RalBP1-(364 -647), pCGN/POB1, pGEX-2T/paxillin ␣, and pBJ-Myc/RalBP1 were constructed as described (2,19,(33)(34)(35)(36).…”

“…EGF stimulates tyrosine phosphorylation of POB1 and induces the complex formation between EGF receptor and POB1 (2). The EH domain of POB1 associates with Eps15 and Epsin (18,19). Epsin also regulates endocytosis by directly binding to phospholipids (20), ␣-adaptin (21), and clathrin (22,23).…”