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MBC in Press, published online ahead of print July 12, 2006
Mol. Biol. Cell 10.1091/mbc.E06-02-0097

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Submitted on February 3, 2006
Revised on June 28, 2006
Accepted on June 30, 2006

Dynamic Regulation of Mammalian Numb by GPCR and PKC Activation: Structural Determinants of Numb Association with the Cortical Membrane

Sascha E. Dho,* Jo Ann 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 M5G 2M9, Canada; {dagger}Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365

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 PTB domain and COOH-terminal endocytic binding motifs for {alpha}-adaptin and EH 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 which colocalizes with AP2 and Eps15 at substratum plasma membrane punctae and cortical membrane-associated vesicles. 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 C-{beta} and protein kinase C 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 X100 solubility. Live-imaging analysis of mutants identified two regions within Numb which 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.

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






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