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Identification of Novel RasGRF1 Interacting Partners by Large-Scale Proteomic Analysis

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Abstract

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.

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Abbreviations

PH:

Pleckstrin homology

CC:

coiled coil

IQ:

Ilimaquinone

CBD:

Chitin-binding domain

MBP:

Maltose binding protein

MF:

microsomal fraction

CF:

cytosolic fraction

G3BP-2:

Ras-GTPase-activating protein SH3-domain binding protein 2

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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.

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Authors and Affiliations

  1. Department of Biomolecular Sciences and Biotechnology, University of Milan, Via Celoria 26-20133, Milan, Italy

    Paola Lavagni, Marzia Indrigo, Graziano Colombo, Nerina Gnesutta & Renata Zippel

  2. Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy

    Enzo Martegani

  3. Department of Neurobiology & Ethology, University of Haifa, Haifa, Israel

    Kobi Rosenblum

Authors
  1. Paola Lavagni
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  2. Marzia Indrigo
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  3. Graziano Colombo
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  4. Enzo Martegani
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  6. Nerina Gnesutta
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  7. Renata Zippel
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Correspondence to Renata Zippel.

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Lavagni, P., Indrigo, M., Colombo, G. et al. Identification of Novel RasGRF1 Interacting Partners by Large-Scale Proteomic Analysis. J Mol Neurosci 37, 212–224 (2009). https://doi.org/10.1007/s12031-008-9118-y

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  • DOI: https://doi.org/10.1007/s12031-008-9118-y

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