Characterization of four mammalian numb protein isoforms - Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain

Numb is a membrane-associated, phosphotyrosine binding (PTB) domain contain ing protein that functions as an intrinsic determinant of cell fate during Drosophila development. We have identified four isoforms of mammalian Numb with predicted molecular masses of 65, 66, 71, and 72 kDa that are generate d by alternative splicing of the Numb mRNA. The different isoforms result f rom the presence of two sequence inserts within the PTB domain and the cent ral region of the protein. The endogenous expression pattern of these isofo rms, examined using specific antisera, varied in different tissues and cell lines, In addition, differentiation of P19 cells with retinoic acid leads to the specific loss of expression of the 71- and 72-kDa Numb proteins, sug gesting that the expression of certain forms of Numb protein is regulated i n a cell type-specific manner. Expression of Numb proteins fused to green fluorescent protein revealed tha t the form of the PTB domain with the alternatively spliced insert constitu tively associated with the plasma membrane in polarized Madin-Darby canine kidney cells. In contrast, the isoform without the insert was cytoplasmic, suggesting that different PTB domain isoforms may regulate the subcellular localization of Numb proteins. The membrane localization may be due, in par t, to differential affinity for acidic phospholipids. The distinct expressi on and localization patterns of the different mammalian Numb isoforms sugge st that they have distinct functional properties.