Identification of novel RasGRF1 interacting partners by large-scale proteomic analysis

Paola Lavagni, Marzia Indrigo, Graziano Colombo, Enzo Martegani, Kobi Rosenblum, Nerina Gnesutta, Renata Zippel

Research output: Contribution to journalArticlepeer-review


The brain-specific Ras guanine nucleotide exchange factor RasGRF1 is a protein harbouring a complex array of structural motifs. It contains a pleckstrin homology (PH1) domain, a coiled coil region (CC) and an ilimaquinone (IQ) one in addition to the catalytic Ras and Rac exchange factor domains. In this study, we used the recombinant N-terminal PH1, CC and IQ region (PHCCIQ) fused to the chitin-binding domain to isolate interacting proteins from mouse brain extracts. The use of an advanced software tool, the Pep-Miner, allowed clustering similar spectra from multiple mass spectrometry analysis, simplifying and improving the analysis of the complex peptide mixture. The most representative classes of RasGRF1-interacting proteins were ribosomal and other RNA-binding proteins, cytoskeletal proteins and proteins involved in vesicular trafficking. We confirmed the interaction of some of the identified proteins using different experimental approaches. We also demonstrated an RNA-dependent association of the PHCCIQ moiety of RasGRF1 with ribosomal protein S6 and Ras-GTPase-activating protein SH3-domain binding protein 2. In addition, we found that purified total RNA binds to the PHCCIQ fusion protein and the recombinant protein associates with poly(A)-sepharose. These data indicate that RasGRF1 can interact with different protein categories and exhibits a potential RNA-binding property.

Original languageEnglish
Pages (from-to)212-224
Number of pages13
JournalJournal of Molecular Neuroscience
Issue number3
StatePublished - Mar 2009

Bibliographical note

Funding Information:
Acknowledgements We thank E. Sturani, for critical comments and useful discussion. This work was supported by grants from MIUR to N. Gnesutta (PRIN 2005) and FIRB-Neuroscienze (PRO-NEURO) to E. Martegani.


  • Adaptor protein complex AP-2
  • Chitin-binding domain
  • Mass spectrometry
  • PH domain
  • Pull-down assay
  • RasGRF2

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience


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