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Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a. [ABSTRACT FROM AUTHOR]

Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues (Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006) Genomics 88, 731-736). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans. [ABSTRACT FROM AUTHOR]

Endocytosis is regulated by several GTPases including Rab5 and one or more heterotrimeric G proteins. We show here that Rab5, in the GTP gamma S (guanosine 5'-O-(thiotriphosphate))-bound form, fully supports in vitro endosome fusion, indicating that GTP hydrolysis is not required, whereas Rab5:S34N and Rab5:N133I, mutants unable to bind GTP, are potent inhibitors of endosome fusion. Double mutants (Rab5:S34N/delta C4 and Rab5:N133I/delta C4) lacking the C-terminal prenylation site were inactive, indicating that prenylation is required. Endosomes became resistant to the inhibitory effects of Rab5:S34N by preincubating the vesicles with cytosol prior to the addition of the inhibitor. The acquisition of resistance to Rab5:S34N was more rapid than to N-ethylmaleimide, indicating that Rab5 mutants are early acting. G beta gamma subunits of heterotrimeric G proteins block endosome fusion. However the effect of G beta gamma was abrogated by Rab5-GTP gamma S, indicating that a heterotrimeric G protein may operate upstream of Rab5.

Rab5 is a Ras-like small GTPase that regulates early events of endocytosis. Previous work indicates that two GTP-binding defective Rab5 mutants (Rab5:S34N and Rab5:N133I) are dominant inhibitors of endocytosis. In this report, we have initiated experiments to address the structural features necessary for the inhibitory activity of these two Rab5 mutants. Second-site mutations were introduced into Rab5:S34N and Rab5:N133I, respectively, and the resulting double mutants were expressed in cultured BHK-21 cells via a Sindbis virus expression vector. Endocytic activity of the cells was monitored by following the uptake of a fluid-phase endocytic marker (horseradish peroxidase). The effects of the Rab5 mutants on endosome fusion in vitro were also examined. Truncation of the C-terminal isoprenylation motif CCSN abolished the inhibitory activity of both Rab5:S34N and Rab5:N133I. The same held true when the secondary mutation was a substitution mutation (F57S) in the effector domain. Another substitution mutation in this region (I53A) had no effect on the inhibitory activity of either Rab5:S34N or Rab5:N133I. The final mutation (R81A) was created immediately downstream of the second GTP binding motif (WDTAGQER), i.e. in the loop 4 region based on the structural model of Ras. This mutation greatly decreased the isoprenylation of Rab5:N133I and its inhibitory activity on endocytosis. It is believed that Rab5 function requires protein-protein interactions with Rab5-specific regulators and effectors. Some of these interactions are disrupted by Rab5:S34N and Rab5:N133I. By analogy to Ras, both Rab5:S34N and Rab5:N133I are likely to sequester a Rab5-specific guanine nucleotide exchange factor. This interaction requires the effector domain Phe57 residue and C-terminal isoprenylation of Rab5.