Transportin Regulates Nuclear Import of CD44

Abstract

CD44 is a facultative cell surface proteoglycan that serves as the principal cell surface receptor for hyaluronan (HA). Studies have shown that in addition to participating in numerous signaling pathways, CD44 becomes internalized upon engagement by ligand and that a portion of its intracellular domain can translocate to the nucleus where it is believed to play a functional role in cell proliferation and survival. However, the mechanisms whereby fragments of CD44 enter the nucleus have not been elucidated. Here we show that CD44 interacts with two import receptors of the importin ␤ superfamily, importin ␤ itself and transportin. Inhibition of importin ␤-dependent transport failed to block CD44 accumulation in the nucleus. By contrast, inhibition of the transportin-dependent pathway abrogated CD44 import. Mutagenesis of the intracellular domain of CD44 revealed that the 20 membrane-proximal residues contain sequences required for transportin-mediated nuclear transport. Our observations provide evidence that CD44 interacts with importin family members and identify the transportin-dependent pathway as the mechanism whereby full-length CD44 enters the nucleus.Proteins cross the double membrane of the nuclear envelope through nuclear pores. To traverse this barrier, proteins larger than 40 kDa must bind to soluble transport factors or carrier molecules that shuttle between the cytoplasm and the nucleus (1). Most of the nuclear transport factors belong to the ␤-karyopherin family and are classified, according to their function, as importins or exportins (2). In the cytoplasm, cargo proteins containing a nuclear localization signal (NLS) 2 are usually bound by the adaptor protein importin ␣, followed by the carrier importin ␤ (2), although in some cases, importin ␣ binding is not required (3). Importin-bound cargo is then translocated through the nuclear pore complex and is released in the nucleus following binding of importin ␤ to RanGTP protein (2). The importin-Ran complex is exported to the cytoplasm where it dissociates upon RanGTP conversion to RanGDP. An analogous mechanism is used by mRNA-binding proteins, where cargo is recognized by the transportin1 carrier (2). Interestingly, it has also been shown that some proteins, including ␤-catenin, SMAD, and ERK2, can be imported into the nucleus without involvement of importins and RanGTP (4).Nuclear export is analogous to the import mechanism. There is a single karyopherin protein, Crm1, that upon recognition of a nuclear export sequence binds the target protein in the nucleus and upon stimulation by Ran protein releases the cargo into the cytoplasm (2). The mechanisms described above are common for cytoplasmic proteins, but there is also some evidence that the same pathways might be used for nuclear translocation of the full-length transmembrane receptors (5, 6).Growth factor receptor signaling proceeds as a cascade of intracellular events. Receptor engagement by ligand on the cell surface typically results in receptor clustering and, depending on the receptor type...

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