Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis

Hereditary hemochromatosis (HH) is a common autosomal recessive disease associated with loss of regulation of dietary iron absorption and excessive iron deposition in major organs of the body. Recently, a candidate gene for HH (also called HFE) was identified that encodes a novel MHC class I-like protein. Most patients with HH are homozygous for the same mutation in the HFE gene, resulting in a C282Y change in the HFE protein. Studies in cultured cells show that the C282Y mutation abrogates the binding of the recombinant HFE protein to β2-microglobulin (β2M) and disrupts its transport to the cell surface. The HFE protein was shown by immunohistochemistry to be expressed in certain epithelial cells throughout the human alimentary tract and to have a unique localization in the cryptal cells of small intestine, where signals to regulate iron absorption are received from the body. In the studies presented here, we demonstrate by immunohistochemistry that the HFE protein is expressed in human placenta in the apical plasma membrane of the syncytiotrophoblasts, where the transferrin-bound iron is normally transported to the fetus via receptor-mediated endocytosis. Western blot analyses show that the HFE protein is associated with β2M in placental membranes. Unexpectedly, the transferrin receptor was also found to be associated with the HFE protein/β2M complex. These studies place the normal HFE protein at the site of contact with the maternal circulation where its association with transferrin receptor raises the possibility that the HFE protein plays some role in determining maternal/fetal iron homeostasis. These findings also raise the question of whether mutations in the HFE gene can disrupt this association and thereby contribute to some forms of neonatal iron overload.

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse)

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

β2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced β2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating β2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

Odorants induce the phosphorylation of the cAMP response element binding protein in olfactory receptor neurons

Although odorants are known to activate olfactory receptor neurons through cAMP, the long-term effects of odorant detection are not known. Our recent findings indicate that there is also a delayed and sustained cAMP response, with kinetics sufficient to mediate long-term cellular responses. This cAMP response is mediated by cGMP through activation of adenylyl cyclase by protein kinase G (PKG). Therefore, we investigated the ability of odorants to regulate gene expression in rat olfactory epithelium. The cAMP-responsive binding protein (CREB) is a well-characterized transcription factor regulated by cAMP. We examined CREB activity in rat olfactory epithelium and olfactory receptor neurons (ORNs) after stimulation with odorants. Odorants increased levels of phosphorylated CREB in olfactory epithelium in vivo, and this increase was localized to ORNs in vitro. Incubation with 8-bromo-cGMP or sodium nitroprusside, a guanylyl cyclase activator, also increased phosphorylated CREB. In vitro, cAMP-dependent protein kinase phosphorylated CREB. In contrast, PKG failed to phosphorylate CREB directly in vitro. Our results demonstrate that the delayed odorant-induced cAMP signal activates CREB, which in turn may modulate gene expression in ORNs. In addition, cGMP indirectly affects CREB activation. This effect of cGMP on CREB activity through cAMP provides another mechanism for the modulation of CREB.

Metabotropic glutamate receptor-initiated translocation of protein kinase p90rsk to polyribosomes: A possible factor regulating synaptic protein synthesis

Maintenance of lasting synaptic efficacy changes requires protein synthesis. We report here a mechanism that might influence translation control at the level of the single synapse. Stimulation of metabotropic glutamate receptors in hippocampal slices induces a rapid protein kinase C-dependent translocation of multifunction kinase p90rsk to polyribosomes; concomitantly, there is enhanced phosphorylation of at least six polyribosome binding proteins. Among the polyribosome bound proteins are the p90rsk-activating kinase ERK-2 and a known p90rsk substrate, glycogen synthase kinase 3β, which regulates translation efficiency via eukaryotic initiation factor 2B. Thus metabotropic glutamate receptor stimulation could induce synaptic activity-dependent translation via translocation of p90rsk to ribosomes.

Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells

In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814–4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription–PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of ≈7.5 kb, ≈3.0 kb, and ≈2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.

Protein kinase C potentiation of N-methyl-d-aspartate receptor activity is not mediated by phosphorylation of N-methyl-d-aspartate receptor subunits

N-methyl-d-aspartate receptors (NMDARs) are Ca2+-permeable glutamate-gated ion channels whose physiological properties in neurons are modulated by protein kinase C (PKC). The present study was undertaken to determine the role in PKC-induced potentiation of the NR1 and NR2A C-terminal tails, which serve as targets of PKC phosphorylation [Tingley, W. G., Ehlers, M. D., Kameyama, K., Doherty, C., Ptak, J. B., Riley, C. T. & Huganir, R. L. (1997) J. Biol. Chem. 272, 5157–5166]. Serine residue 890 in the C1 cassette is a primary target of PKC phosphorylation and a critical residue in receptor clustering at the membrane. We report herein that the presence of the C1 cassette reduces PKC potentiation and that mutation of Ser-890 significantly restores PKC potentiation. Splicing out or deletion of other C-terminal cassettes singly or in combination had little or no effect on PKC potentiation. Moreover, experiments involving truncation mutants reveal the unexpected finding that NMDARs assembled from subunits lacking all known sites of PKC phosphorylation can show PKC potentiation. These results indicate that PKC-induced potentiation of NMDAR activity does not occur by direct phosphorylation of the receptor protein but rather of associated targeting, anchoring, or signaling protein(s). PKC potentiation of NMDAR function is likely to be an important mode of NMDAR regulation in vivo and may play a role in NMDA-dependent long-term potentiation.

The Epstein–Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-κB

The Epstein–Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-κB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-κB activation also appears to be a critical component of long-term outgrowth.

N-methyl-d-aspartate receptor activation and visual activity induce elongation factor-2 phosphorylation in amphibian tecta: A role for N-methyl-d-aspartate receptors in controlling protein synthesis

N-methyl-d-aspartate receptor (NMDAR) activation has been implicated in forms of synaptic plasticity involving long-term changes in neuronal structure, function, or protein expression. Transcriptional alterations have been correlated with NMDAR-mediated synaptic plasticity, but the problem of rapidly targeting new proteins to particular synapses is unsolved. One potential solution is synapse-specific protein translation, which is suggested by dendritic localization of numerous transcripts and subsynaptic polyribosomes. We report here a mechanism by which NMDAR activation at synapses may control this protein synthetic machinery. In intact tadpole tecta, NMDAR activation leads to phosphorylation of a subset of proteins, one of which we now identify as the eukaryotic translation elongation factor 2 (eEF2). Phosphorylation of eEF2 halts protein synthesis and may prepare cells to translate a new set of mRNAs. We show that NMDAR activation-induced eEF2 phosphorylation is widespread in tadpole tecta. In contrast, in adult tecta, where synaptic plasticity is reduced, this phosphorylation is restricted to short dendritic regions that process binocular information. Biochemical and anatomical evidence shows that this NMDAR activation-induced eEF2 phosphorylation is localized to subsynaptic sites. Moreover, eEF2 phosphorylation is induced by visual stimulation, and NMDAR blockade before stimulation eliminates this effect. Thus, NMDAR activation, which is known to mediate synaptic changes in the developing frog, could produce local postsynaptic alterations in protein synthesis by inducing eEF2 phosphorylation.

Basolateral Localization of the Caenorhabditis elegans Epidermal Growth Factor Receptor in Epithelial Cells by the PDZ Protein LIN-10

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.

Radiation-induced Release of Transforming Growth Factor α Activates the Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase Pathway in Carcinoma Cells, Leading to Increased Proliferation and Protection from Radiation-induced Cell Death

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0–10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90–240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor α receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor α (TGFα) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFα cleavage 120–180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFα. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFα. Neutralization of TGFα function by an anti-TGFα antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFα–EGFR–MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.

Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase

Androgen receptor (AR) belongs to the nuclear receptor superfamily and mediates the biological actions of male sex steroids. In this work, we have characterized a novel 130-kDa Ser/Thr protein kinase ANPK that interacts with the zinc finger region of AR in vivo and in vitro. The catalytic kinase domain of ANPK shares considerable sequence similarity with the minibrain gene product, a protein kinase suggested to contribute to learning defects associated with Down syndrome. However, the rest of ANPK sequence, including the AR-interacting interface, exhibits no apparent homology with other proteins. ANPK is a nuclear protein that is widely expressed in mammalian tissues. Its overexpression enhances AR-dependent transcription in various cell lines. In addition to the zinc finger region, ligand-binding domain and activation function AF1 of AR are needed, as the activity of AR mutants devoid of these domains was not influenced by ANPK. The receptor protein does not appear to be a substrate for ANPK in vitro, and overexpression of ANPK does not increase the extent of AR phosphorylation in vivo. In view of this, it is likely that ANPK-mediated activation of AR function is exerted through modification of AR-associated proteins, such as coregulatory factors, and/or through stabilization of the receptor protein against degradation.

Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways

Many of the protein–protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-β, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-β was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain–containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways.

Protein engineering of Bacillus thuringiensis δ-endotoxin: Mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae

Substitutions or deletions of domain II loop residues of Bacillus thuringiensis δ-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) ≈4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (>21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering δ-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.

Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kit gene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.