The endophilin–CIN85–Cbl complex mediates ligand-dependent downregulation of c-Met

Abstract

Ligand-dependent downregulation of tyrosine kinase receptors is a critical step for modulating their activity. Upon ligand binding, hepatocyte growth factor (HGF) receptor (Met) is polyubiquitinated1 and degraded2; however, the mechanisms underlying HGF receptor endocytosis are not yet known. Here we demonstrate that a complex involving endophilins, CIN85 and Cbl controls this process. Endophilins3 are regulatory components of clathrin-coated vesicle formation. Through their acyl-transferase activity they are thought to modify the membrane phospholipids and induce negative curvature and invagination of the plasma membrane during the early steps of endocytosis4. Furthermore, by means of their Src-homology 3 domains, endophilins are able to bind CIN85, a recently identified protein that interacts with the Cbl proto-oncogene5. Cbl, in turn, binds and ubiquitinates activated HGF receptor, and by recruiting the endophilin–CIN85 complex, it regulates receptor internalization. Inhibition of complex formation is sufficient to block HGF receptor internalization and to enhance HGF-induced signal transduction and biological responses. These data provide further evidence of a relationship between receptor-mediated signalling and endocytosis, and disclose a novel functional role for Cbl in HGF receptor signalling.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Endophilins interact with CIN85 and Cbl.
Figure 2: Cbl ubiquitinates Met and recruits the CIN85-endophilin complex.
Figure 3: Expression of dominant interfering mutants of Cbl, CIN85 and endophilin impairs HGF receptor internalization and downregulation.
Figure 4: Impairment of Met downregulation enhances signal transduction and biological responses.

References

  1. 1

    Jeffers, M., Taylor, G. A., Weidner, K. M., Omura, S. & Vande Woude, G. F. Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway. Mol. Cell. Biol. 17, 799–808 (1997).

    CAS  Article  Google Scholar 

  2. 2

    Hammond, D. E., Urbé, S., Vande Woude, G. F. & Clague, M. J. Down-regulation of MET, the receptor for hepatocyte growth factor. Oncogene 20, 2761–2770 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Giachino, C. et al. A novel SH-3-containing human gene family preferentially expressed in the central nervous system. Genomics 41, 427–434 (1997).

    CAS  Article  Google Scholar 

  4. 4

    Schmidt, A. et al. Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature 401, 133–141 (1999).

    ADS  CAS  Article  Google Scholar 

  5. 5

    Take, H. et al. Cloning and characterization of a novel adaptor protein, CIN85, that interacts with c-Cbl. Biochem. Biophys. Res. Commun. 268, 321–328 (2000).

    CAS  Article  Google Scholar 

  6. 6

    Hunter, T. Signaling—2000 and beyond. Cell 100, 113–127 (2000).

    CAS  Article  Google Scholar 

  7. 7

    Waterman, H. & Yarden, Y. Molecular mechanisms underlying endocytosis and sorting of ErbB receptor tyrosine kinases. FEBS Lett. 490, 142–152 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Di Fiore, P. P. & De Camilli, P. Endocytosis and signaling: an inseparable partnership. Cell 106, 1–4 (2001).

    CAS  Article  Google Scholar 

  9. 9

    Ringstad, N., Nemoto, Y. & De Camilli, P. The SH3p4/SH3p8/SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain. Proc. Natl Acad. Sci. USA 94, 8569–8574 (1997).

    ADS  CAS  Article  Google Scholar 

  10. 10

    Kirsch, K. H., Georgescu, M. M., Ishimura, S. & Hanafusa, H. CMS: an adapter molecule involved in cytoskeletal rearrangements. Proc. Natl Acad. Sci. USA 99, 6211–6216 (1999).

    ADS  Article  Google Scholar 

  11. 11

    Dustin, M. L. et al. A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94, 667–677 (1998).

    CAS  Article  Google Scholar 

  12. 12

    Cestra, G. et al. The SH3 domains of Endophilin and Amphiphysin bind to the proline rich region of Synaptojanin 1 at distinct sites that display an unconventional binding specificity. J. Biol. Chem. 274, 32001–32007 (1999).

    CAS  Article  Google Scholar 

  13. 13

    Watanabe, S. et al. Characterization of the CIN85 adaptor protein and identification of components involved in CIN85 complexes. Biochem. Biophys. Res. Commun. 278, 167–174 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Thien, C. B. F. & Langdon, W. Y. Cbl: many adaptations to regulate protein tyrosine kinases. Nature Rev. Mol. Cell Biol. 2, 294–307 (2001).

    CAS  Article  Google Scholar 

  15. 15

    Levkowitz, G. et al. Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1. Mol. Cell 4, 1029–1040 (1999).

    CAS  Article  Google Scholar 

  16. 16

    Fixman, E. D. et al. Efficient cellular transformation by the Met oncoprotein requires a functional Grb2 binding site and correlates with phosphorylation of the Grb2-associated proteins, Cbl and Gab1. J. Biol. Chem. 272, 20167–20172 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Giordano, S., Ponzetto, C., Di Renzo, M. F., Cooper, C. S. & Comoglio, P. M. Tyrosine kinase receptor indistinguishable from the c-met protein. Nature 339, 155–156 (1989).

    ADS  CAS  Article  Google Scholar 

  18. 18

    Weidner, K. M. et al. Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature 384, 173–176 (1996).

    ADS  CAS  Article  Google Scholar 

  19. 19

    Ponzetto, C. et al. A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell 77, 261–271 (1994).

    CAS  Article  Google Scholar 

  20. 20

    Garcia-Guzman, M., Larse, E. & Vuori, K. The proto-oncogene c-Cbl is a positive regulator of Met-induced MAP kinase activation: a role for the adaptor protein Crk. Oncogene 19, 4058–4065 (2000).

    CAS  Article  Google Scholar 

  21. 21

    Brodin, L., Low, P. & Shupliakov, O. Sequential steps in clathrin-mediated synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 10, 312–320 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Huttner, W. B. & Schmidt, A. Lipids, lipid modification and lipid-protein interaction in membrane budding and fission-insights from the roles of endophilin A1 and synaptophysin in synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 10, 543–551 (2000).

    CAS  Article  Google Scholar 

  23. 23

    Hill, E., van Der Kaay, J., Downes, C. P. & Smythe, E. The role of dynamin and its binding partners in coated pit invagination and scission. J. Cell Biol. 152, 309–323 (2001).

    CAS  Article  Google Scholar 

  24. 24

    Soubeyran, P., Kowanetz, K., Szymkiewicz, I., Langdon, W. Y. & Dikic, I. Cbl–CIN85–endophilin complex mediates ligand-induced downregulation of EGF receptors. Nature 416, 183–187 (2001).

    ADS  Article  Google Scholar 

  25. 25

    Prat, M., Crepaldi, T., Pennacchietti, S., Bussolino, F. & Comoglio, P. M. Agonistic monoclonal antibodies against the Met receptor dissect the biological responses to HGF. J. Cell Sci. 111, 237–247 (1998).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank E. Lantelme, C. Giachino and L. Lanzetti for production of anti-endophilin antibodies and for GST–Endo3, GST–Endo1-2-SH3 constructs; L. Tamagnone for ΔPlexin A3 cDNA; P. De Camilli for dynamin cDNA; Y. Yarden for Cbl and ubiquitin plasmids; I. Dikic for CIN85 reagents and Cbl-ΔSH2 plasmid; and W. Y. Langdon for Cbl-70Z and Cbl-480 constructs. We also thank Y. Yarden, L. Tamagnone and our colleagues for discussions; A. Cignetto for secretarial assistance; and E. Wright for editing the manuscript. The technical assistance of L. Palmas, R. Albano and E. Clemente is acknowledged. This work was partly supported by Associazione E. & E. Rulfo per la Genetica Medica to N.M., by Italian Association for Cancer Research to P.M.C. and by MURST COFIN to S.G.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Silvia Giordano.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Petrelli, A., Gilestro, G., Lanzardo, S. et al. The endophilin–CIN85–Cbl complex mediates ligand-dependent downregulation of c-Met. Nature 416, 187–190 (2002). https://doi.org/10.1038/416187a

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.