Abstract
Numb is an endocytic adaptor protein that regulates the endocytosis and trafficking of transmembrane receptors including Notch, E-cadherin, and integrins. Vertebrate Numb is alternatively spliced at exons 3 and 9 to give rise to four protein isoforms. Expression of these isoforms varies at different developmental stages, and although the function of Numb isoforms containing exon 3 has been studied, the role of exon 9 inclusion has not been shown. Here we use affinity purification and tandem mass spectrometry to identify Numb associated proteins, including novel interactions with REPS1, BMP2K, and BCR. In vitro binding measurements indicated exon 9-independent Numb interaction with REPS1 and Eps15 EH domains. Selected reaction monitoring mass spectrometry was used to quantitatively compare the proteins associated with the p72 and p66 Numb isoforms, which differ by the exon 9 region. This showed that significantly more EPS15 and three AP-2 subunit proteins bound Numb isoforms containing exon 9. The EPS15 preference for exon 9-containing Numb was confirmed in intact cells by using a proximity ligation assay. Finally, we used multiplexed selected reaction monitoring mass spectrometry to assess the dynamic regulation of Numb association with endocytic proteins. Numb hyper-phosphorylation resulted in disassociation of Numb endocytic complexes, while inhibition of endocytosis did not alter Numb association with the AP-2 complex but altered recruitment of EPS15, REPS1, and BMP2K. Hence, quantitative mass spectrometric analysis of Numb protein-protein interactions has provided new insights into the assembly and regulation of protein complexes important in development and cancer.
Highlights
From the ‡Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 2M9, Canada; §The Arthur and Sonia Labatt Brain Tumour Research Center and Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; ¶Program in Molecular Structure and Function, The Hospital For Sick Children, Toronto, Ontario M5G 1X8, Canada; ʈDivision of Neurosurgery, Toronto Western Hospital, University of Toronto, Toronto M5A 2N4, Canada; **Department of Molecular Genetics, and Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5S 1A8, Canada
We used tandem mass spectrometry to identify the endocytic protein network associated with Numb and selected reaction monitoring (SRM)-MS to quantify specific interacting proteins associated with individual Numb isoforms
We identified an endocytic complex associated with Numb that includes several known Numb interacting proteins such as ␣-adaptin, its associated AP-2 subunits, and EPS15
Summary
Numb appears to regulate post-endocytic trafficking events through its association with the EHD family of proteins and ubiquitin ligases including ITCH [17, 26]. Developmental regulation of Numb alternative splicing and its association with human tumors suggests Numb protein isoforms may confer distinct functional properties. To address the hypothesis that such properties arise through the molecular interactions of Numb, we took a proteomic approach involving mass spectrometry (MS) to identify protein complexes associated with individual isoforms of Numb. We identified both known components of the endocytic complex and several proteins not previously identified as being associated with Numb. A targeted proteomic approach involving selected reaction monitoring (SRM)-MS1 was used to quantify the interacting proteins associated with Numb isoforms, and monitor the dynamics of Numb endocytic complex assembly
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