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Originally published as MBC in Press, 10.1091/mbc.E06-02-0097 on July 12, 2006

Vol. 17, Issue 9, 4142-4155, September 2006

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Dynamic Regulation of Mammalian Numb by G Protein-coupled Receptors and Protein Kinase C Activation: Structural Determinants of Numb Association with the Cortical MembraneFormula Formula

Sascha E. Dho*, JoAnn Trejo{dagger}, David P. Siderovski{dagger}, and C. Jane McGlade*,{ddagger}

*The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; {ddagger}Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5S 2E4, Canada; and {dagger}Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365

Submitted February 3, 2006; Revised June 28, 2006; Accepted June 30, 2006
Monitoring Editor: Ben Margolis

The cell fate determinant Numb is a membrane-associated adaptor protein involved in both development and intracellular vesicular trafficking. It has a phosphotyrosine-binding (PTB) domain and COOH-terminal endocytic-binding motifs for {alpha}-adaptin and Eps15 homology domain-containing proteins. Four isoforms of Numb are expressed in vertebrates, two of which selectively associate with the cortical membrane. In this study, we have characterized a cortical pool of Numb that colocalizes with AP2 and Eps15 at substratum plasma membrane punctae and cortical membrane-associated vesicles. Green fluorescent protein (GFP)-tagged mutants of Numb were used to identify the structural determinants required for localization. In addition to the previously described association of the PTB domain with the plasma membrane, we show that the AP2-binding motifs facilitate the association of Numb with cortical membrane punctae and vesicles. We also show that agonist stimulation of G protein-coupled receptors (GPCRs) that are linked to phospholipase Cbeta and protein kinase C (PKC) activation causes redistribution of Numb from the cortical membrane to the cytosol. This effect is correlated with Numb phosphorylation and an increase in its Triton X-100 solubility. Live-imaging analysis of mutants identified two regions within Numb that are independently responsive to GPCR-mediated lipid hydrolysis and PKC activation: the PTB domain and a region encompassing at least three putative PKC phosphorylation sites. Our data indicate that membrane localization of Numb is dynamically regulated by GPCR-activated phospholipid hydrolysis and PKC-dependent phosphorylation events.

Formula Formula The online version of this contains supplemental material at MBC Online (http://www.molbiolcell.org).

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05.02-0097) on July 12, 2006.

Address correspondence to: C. Jane McGlade (jmcglade{at}sickkids.ca)






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