The function of the epidermal growth factor receptor (EGFR) family member HER4 remains unclear because its activating ligand, heregulin, results in either proliferation or differentiation. This variable response may stem from the range of signals generated by HER4 homodimers versus heterodimeric complexes with other EGFR family members. The ratio of homo-and heterodimeric complexes may be influenced both by a cell's EGFR family member expression profile and by the ligand or even ligand isoform used. To define the role of HER4 in mediating antiproliferative and differentiation responses, human breast cancer cell lines were screened for responses to heregulin. Only cells that expressed HER4 exhibited heregulin-dependent antiproliferative responses. In-depth studies of one line, SUM44, demonstrated that the antiproliferative and differentiation responses correlated with HER4 activation and were abolished by stable expression of a kinase-inactive HER4. HB-EGF, a HER4-specific ligand in this EGFR-negative cell line, also induced an antiproliferative response. Moreover, introduction and stable expression of HER4 in HER4-negative SUM102 cells resulted in the acquisition of a heregulin-dependent antiproliferative response, associated with increases in markers of differentiation. The role of HER2 in these heregulin-dependent responses was examined through elimination of cell surface HER2 signaling by stable expression of a single-chain anti-HER2 antibody that sequestered HER2 in the endoplasmic reticulum. In the cell lines with either endogenously (SUM44) or exogenously (SUM102) expressed HER4, elimination of HER2 did not alter HER4-dependent decreases in cell growth. These results suggest that HER4 is both necessary and sufficient to trigger an antiproliferative response in human breast cancer cells.The epidermal growth factor receptor (EGFR) family has been implicated in breast cancer pathogenesis and progression (reviewed in references 13 and 39). Aberrant expression of at least two of the family members, EGFR and HER2, has been associated with poor prognosis and differential response to therapy (21,28,31,44). Recently, treatment targeted against HER2 has demonstrated clinical efficacy, emphasizing the importance of members of this receptor family in breast cancer prognosis and therapy (10).The EGFR family consists of four known members: EGFR (HER1, erbB-1), HER2 (erbB-2), HER3 (erbB-3), and HER4 (erbB-4) (reviewed in references 13, 34, and 39). The four receptors form homodimers or heterodimers upon activation by two sets of ligands, the EGF and heregulin/neuregulin families. There are several possible hetero-and homodimeric receptor combinations, which theoretically result in differential activation of multiple downstream signal transduction pathways. Additional heterogeneity results from varying phenotypic responses, depending on cell type and the duration or intensity of downstream signaling, determined in part by differences in ligand affinity, recycling, and intracellular environment, as well as other factors th...

Proteins are directed to the nucleus by their nuclear localization sequences (NLSs) in a multistep process. The first step, which is to dock the NLS-containing protein to the nuclear pore, is carried out in part by a recently identified NLS receptor named Srp1/importin-␣. Using the high mobility group (HMG) DNA binding domain of human lymphoid enhancer factor-1 (hLEF-1) as bait in a yeast two-hybrid screen, we have identified two different mouse Srp1 proteins (pendulin/importin-␣ and mSrp1) that each bind to a 9-amino acid sequence in hLEF-1 called the B box. We show that the B box of hLEF-1, a region essential for high affinity DNA binding, is also necessary and sufficient for nuclear localization, lending support to the model that NLSs can function both in nuclear transport and DNA binding. Pendulin and mSrp1 are the mouse homologues of hRch1/hSrp1␣/ importin-␣ and hSrp1/karyopherin ␣/NPI-1, respectively, and show considerable sequence divergence from each other. We find a surprising and significant difference in the expression pattern of pendulin and mSrp1 mRNA, suggesting that these two Srp1 proteins are distinguishable in function as well as sequence.Analysis of the nuclear localization of transcription regulatory factors has largely focused on defining the amino acid sequences within transcription factor proteins that direct nuclear entry. A universal consensus sequence that signals nuclear localization (NLS) 1 has not been found; nevertheless short regions, rich in basic residues are a feature common to most identified NLSs (1, 2). It is also common for the NLS of transcription factors to be in regions that overlap or are near the DNA binding domain, prompting speculation that (a) nuclear occupancy by a protein is due to both nuclear import and retention by DNA binding, (b) NLS sequences contribute to DNA binding, or (c) there is coordinate regulation of the two functions due to their proximity (3). It is known that the NLS directs transport through nuclear pores via a receptor-mediated, multistep process (4 -6). Recently, a cytosolic NLS receptor protein in Xenopus named importin-␣ was found to carry out the first step along with the Xenopus importin-␤ subunit (also known as p97 or karyopherin ␤) by docking NLS-containing proteins at the nuclear pore (7). Xenopus importin-␣ is homologous to yeast Srp1p, an essential gene first identified as a suppressor of RNA polymerase I mutations in Saccharomyces cerevisiae (8). Immunofluorescence studies localized ySrp1p mainly to the nuclear pore, and genetic and biochemical evidence established a tight association of ySrp1p with known protein components of the pore (8, 9). More recently, work with human Srp1␣ (hSrp1␣; also known as hRch1) and its mouse homolog pendulin/importin-␣, and hSrp1/karyopherin ␣/NPI-1, a different human Srp1 protein, has confirmed in vivo and in vitro that Srp1 proteins and p97/karyopherin ␤ function as cytosolic NLS receptors that usher NLS-containing proteins to the cytoplasmic side of the nuclear pore, where additional GTP-requiring fac...

The transcription factor lymphoid enhancer factor 1 (LEF-1) is directed to the nucleus by a nine-amino-acid nuclear localization signal (NLS; KKKKRKREK) located in the high-mobility-group DNA binding domain. This NLS is recognized by two armadillo repeat proteins (pendulin/Rch1/␣-P1/hSrp1␣ and Srp1/karyopherin-␣/␣-S1/NPI-1) which function in nuclear transport as the importin-␣ subunit of NLS receptors. T-cell factor 1 (TCF-1), a related transcription factor, contains a similar sequence (KKKRRSREK) in the identical position within its HMG DNA binding domain. We show that this sequence functions as an NLS in vivo but is not recognized by these two importin-␣ subtypes in a yeast two-hybrid assay and only weakly recognized in an in vitro binding assay. Transfer of the LEF-1 NLS to TCF-1 can confer pendulin/Rch1 binding, demonstrating that the NLS is the primary determinant for recognition. We have constructed a set of deletion mutations in pendulin/Rch1 to examine the differential NLS recognition more closely. We find that the entire armadillo repeat array of pendulin/Rch1 is necessary to maintain high affinity and specificity for the LEF-1 NLS versus the TCF-1 NLS. Importin-␤, the second subunit of the NLS receptor complex, does not influence in vitro NLS binding affinity or specificity. To test whether this differential recognition is indicative of distinct mechanisms of nuclear transport, the subcellular localization of LEF-1 and TCF-1 fused to green fluorescent protein (GFP)) was examined in an in vitro nuclear transport assay. GFP-LEF-1 readily localizes to the nucleus, whereas GFP-TCF-1 remains in the cytoplasm. Thus, LEF-1 and TCF-1 differ in several aspects of nuclear localization.A recent survey of DNA and RNA binding proteins with delimited nuclear localization signals (NLSs) found that these signals are often contained within or are near the domain involved in DNA or RNA binding (27). NLSs serve to target the protein to the nucleus through a direct binding of 60-kDa NLS receptors known collectively as importins or karyopherins (2,15,32,35). These receptors are composed primarily of reiterated hydrophobic repeat motifs called armadillo repeats (named after Drosophila armadillo) (37). Although the armadillo repeat regions are known to be involved in NLS binding, the precise domain responsible for NLS recognition has not been defined. Understanding how NLS receptors bind and direct their ligands to the nucleus is important because in the case of RNA and DNA binding proteins, many are shuttled through nuclear pores by an NLS receptor that remains tightly bound to the nucleic acid binding domain for an undetermined length of time.The DNA binding domain of the transcription factor lymphoid enhancer factor 1 (LEF-1) binds and bends specific DNA sequences within the promoters or enhancers of genes (11,50,54). LEF-1 regulates gene expression by engaging in protein-protein contact with enhancer and promoter binding proteins on these bent templates (4,5,12,30,45). The DNA binding and bending activities are carried o...

Mer is a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family, a family whose physiological function is not well defined. We constructed a Mer chimera using the epidermal growth factor receptor (EGFR) extracellular and transmembrane domains and the Mer cytoplasmic domain. Stable transfection of the Mer chimera into interleukin 3 (IL-3)-dependent murine 32D cells resulted in ligand-activable surface receptor that tyrosine autophosphorylated, stimulated intracellular signaling, and dramatically reduced apoptosis initiated by IL-3 withdrawal. However, unlike multiple other ectopically expressed receptor tyrosine kinases including full-length EGFR or an EGFR/Axl chimera, the Mer chimera did not stimulate proliferation. Moreover, and in contrast to EGFR, Mer chimera activation induced adherence and cell flattening in the normally suspensiongrowing 32D cells. The Mer chimera signal also blocked IL-3-dependent proliferation leading to G 1 /S arrest, dephosphorylation of retinoblastoma protein, and elongation of cellular processes. Unlike other agonists that lead to a slow (4 -8 days) ligand-dependent differentiation of 32D cells, the combined Mer and IL-3 signal resulted in differentiated morphology and growth cessation in the first 24 h. Thus the Mer chimera blocks apoptosis without stimulating growth and produces cytoskeletal alterations; this outcome is clearly separable from the proliferative signal produced by most receptor tyrosine kinases.

We cloned the Xenopus laevis form of Gqa subunit to study its effects on oocyte maturation. Injection of Xenopus Gqa mRNA into stage 6 oocytes activated the phospholipase C/phosphatidylinositol pathway. The oocyte membrane became permeable to calcium ions and was able to generate transient inward currents (Ti.), due to the opening of Ca2l-dependent Cl- The induction of oocyte maturation by progesterone leads to a series of events, involving both inhibition and activation of second-messenger systems, that culminates in germinal vesicle breakdown (GVBD) and progression to metaphase of meiosis II (1). Changes in second-messenger concentrations are usually the result of agonist-receptor association and activation of second-messenger effectors, either directly or through receptor-associated G proteins. However, it is not yet clear whether the only progesterone receptor described so far in Xenopus oocytes (2) is directly responsible for maturation.Immediately after exposure to progesterone the diacylglycerol (DAG) level in the oocyte falls "30%, and after 2 min it begins to rise, reaching control levels by 15 min and rising further until GVBD (3, 4). A similar increase in DAG, but without the initial drop, has been reported (5), and a decrease in inositol 1,4,5-triphosphate (InsP3) immediately after progesterone exposure has also been reported (3 (11), and maintained in OR2 medium (12) supplemented with gentamicin at 0.1 mg/ml (GIBCO) or incubated at 17°C with either cholera toxin at 2 ptg/ml (Sigma) or pertussis toxin at 4 ,ug/ml (Sigma) 24 hr before and after mRNA injection. To induce maturation, oocytes were continuously exposed to progesterone at 1 ,ug/ ml. Maturation was monitored by the appearance of a white spot on the animal hemisphere, indicative of GVBD, and confirmed by manual dissection (1).Cloning of Xenopus Gq a Subunit (xGqa). A mouse Gqa cDNA clone (13) was used to screen a X laevis whole-ovary cDNA library (14). Two partial cDNA clones were ligated to make the full-length xGqa homolog. Sequence data are available from GenBank under accession no. L05540. described (17). After 1 hr, the oocytes were homogenized in 100 ,ul of 50 mM NaCl/0.5 mM phenylmethylsulfonyl fluoride and centrifuged; the supernatant was then precipitated with 4 vol of acetone. The protein precipitate was resuspended and subjected to SDS/PAGE.Measurement of Diacylglyceride Mass. At specified times, five oocytes were homogenized in 3 ml of chloroform/ methanol, 1:2, 1 ml of chloroform and 1.8 ml of 1 M NaCl were added, and the organic phase was dried under N2 as described (3). The DAG mass was quantitated using a DAG kinase assay (18). The resulting 32P-labeled phospholipids were spotted on TLC plates. The plates were developed in chloroform/ methanol/acetic acid, 130:30:10, and exposed to film. Spots corresponding to phosphatidic acid (DAG plus P04) were scraped, and radioactivity was counted in a scintillation counter.