Wild-type and mutant p53 differentially regulate transcription of the insulin-like growth factor I receptor gene.

The insulin-like growth factor I receptor (IGF-I-R) plays a critical role in transformation events. It is highly overexpressed in most malignant tissues where it functions as an anti-apoptotic agent by enhancing cell survival. Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. p53 is the most frequently mutated gene in human cancer. Cotransfection of Saos-2 (os-teosarcoma-derived cells) and RD (rhabdomyosarcoma-derived cells) cells with IGF-I-R promoter constructs driving luciferase reporter genes and with wild-type p53 expression vectors suppressed promoter activity in a dose-dependent manner. This effect of p53 is mediated at the level of transcription and it involves interaction with TBP, the TATA box-binding component of TFIID. On the other hand, three tumor-derived mutant forms of p53 (mut 143, mut 248, and mut 273) stimulated the activity of the IGF-I-R promoter and increased the levels of IGF-I-R/luciferase fusion mRNA. These results suggest that wild-type p53 has the potential to suppress the IGF-I-R promoter in the postmitotic, fully differentiated cell, thus resulting in low levels of receptor gene expression in adult tissues. Mutant versions of p53 protein, usually associated with malignant states, can derepress the IGF-I-R promoter, with ensuing mitogenic activation by locally produced or circulating IGFs.

Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells in vivo.

Prostate carcinoma is the second leading cause of death from malignancy in men in the United States. Prostate cancer cells express type I insulin-like growth factor receptor (IGF-IR) and prostate cancer selectively metastazises to bone, which is an environment rich in insulin-like growth factors (IGFs), thereby supporting a paracrine action for cancer cell proliferation. We asked whether the IGF-IR is coupled to tumorigenicity and invasion of prostate cancer. When rat prostate adenocarcinoma cells (PA-III) were stably transfected with an antisense IGF-IR expression construct containing the ZnSO4-inducible metallothionein-1 transcriptional promoter, the transfectants expressed high levels of IGF-IR antisense RNA after induction with ZnSO4, which resulted in dramatically reduced levels of endogenous IGF-IR mRNA. A significant reduction in expression both of tissue-type plasminogen activator and of urokinase-type plasminogen activator occurred in PA-III cells accompanying inhibition of IGF-IR. Subcutaneous injection of either nontransfected PA-III or PA-III cells transfected with vector minus the IGF-IR insert into nude mice resulted in large tumors after 4 weeks. However, mice injected with IGF-IR antisense-transfected PA-III cells either developed tumors 90% smaller than controls or remained tumor-free after 60 days of observation. When control-transfected PA-III cells were inoculated over the abraded calvaria of nude mice, large tumors formed with invasion of tumor cells into the brain parenchyma. In contrast, IGF-IR antisense transfectants formed significantly smaller tumors with no infiltration into brain. These results indicate an important role for the IGF/IGF-IR pathway in metastasis and provide a basis for targeting IGF-IR as a potential treatment for prostate cancer.

Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin.

The alpha-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, alpha-gustducin, is described here to be also expressed in the stomach and intestine. The alpha-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.

Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity.

To explore the possible involvement of STAT factors ("signal transducers and activators of transcription") in the interleukin 2 receptor (IL-2R) signaling cascade, murine HT-2 cells expressing chimeric receptors composed of the extracellular domain of the erythropoietin receptor fused to the cytoplasmic domains of the IL-2R beta or -gamma c chains were prepared. Erythropoietin or IL-2 activation of these cells resulted in rapid nuclear expression of a DNA-binding activity that reacted with select STAT response elements. Based on reactivity with specific anti-STAT antibodies, this DNA-binding activity was identified as a murine homologue of STAT-5. Induction of nuclear expression of this STAT-5-like factor was blocked by the addition of herbimycin A, a tyrosine kinase inhibitor, but not by rapamycin, an immunophilin-binding antagonist of IL-2-induced proliferation. The IL-2R beta chain appeared critical for IL-2-induced activation of STAT-5, since a mutant beta chain lacking all cytoplasmic tyrosine residues was incapable of inducing this DNA binding. In contrast, a gamma c mutant lacking all of its cytoplasmic tyrosine residues proved fully competent for the induction of STAT-5. Physical binding of STAT-5 to functionally important tyrosine residues within IL-2R beta was supported by the finding that phosphorylated, but not nonphosphorylated, peptides corresponding to sequences spanning Y392 and Y510 of the IL-2R beta tail specifically inhibited STAT-5 DNA binding.

Atrial-like phenotype is associated with embryonic ventricular failure in retinoid X receptor alpha -/- mice.

We have recently characterized a cardiac model of ventricular chamber defects in retinoid X receptor alpha (RXR alpha) homozygous mutant (-/-) gene-targeted mice. These mice display generalized edema, ventricular chamber hypoplasia, and muscular septal defects, and they die at embryonic day 15. To substantiate our hypothesis that the embryos are dying of cardiac pump failure, we have used digital bright-field and fluorescent video microscopy and in vivo microinjection of fluorescein-labeled albumin to analyze cardiac function. The affected embryos showed depressed ventricular function (average left ventricular area ejection fraction, 14%), ventricular septal defects, and various degrees of atrioventricular block not seen in the RXR alpha wild-type (+/+) and heterozygous (+/-) littermates (average left ventricular area ejection fraction, 50%). The molecular mechanisms involved in these ventricular defects were studied by evaluating expression of cardiac-specific genes known to be developmentally regulated. By in situ hybridization, aberrant, persistent expression of the atrial isoform of myosin light chain 2 was identified in the ventricles. We hypothesize that retinoic acid provides a critical signal mediated through the RXR alpha pathway that is required to allow progression of development of the ventricular region of the heart from its early atrial-like form to the thick-walled adult ventricle. The conduction system disturbances found in the RXR alpha -/- embryos may reflect a requirement of the developing conduction system for the RXR alpha signaling pathway, or it may be secondary to the failure of septal development.

Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.

Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor α knockout mouse uterus

Estrogen is known to increase progesterone receptor (PR) levels in the wild-type mouse uterus, and this estrogen induction was thought to be important for progesterone action through the PR. The estrogen receptor α knockout (ERKO) mouse uterus was observed to express PR mRNA that cannot be induced by estrogen. Progesterone action was characterized to determine whether it was diminished in ERKO mice. The PR protein is present in the ERKO uterus at 60% of the level measured in a wild-type uterus. The PR-A and PR-B isoforms are both detected on Western blot, and the ratio of isoforms is the same in both genotypes. Although the level of PR is reduced in the ERKO uterus, the receptor level is sufficient to induce genomic responses, since both calcitonin and amphiregulin mRNAs were increased after progesterone treatment. Finally, the ERKO uterus can be induced to undergo a progesterone-dependent decidual response. Surprisingly, the decidual response is estrogen independent in the ERKO, although it remains estrogen dependent in a wild type. These results indicate that estrogen receptor α modulation of PR levels is not necessary for expression of the PR or genomic and physiologic responses to progesterone in the ERKO uterus.

Molecular characteristics of fibroblast growth factor–fibroblast growth factor receptor–heparin-like glycosaminoglycan complex

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF–FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF–FGFR interaction mediated by the ‘conserved’ primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the ‘variable’ secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1β receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF–FGFR interactions. In the FGF–FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.

Crystal structure of the HLA-Cw3 allotype-specific killer cell inhibitory receptor KIR2DL2

Killer cell inhibitory receptors (KIR) protect class I HLAs expressing target cells from natural killer (NK) cell-mediated lysis. To understand the molecular basis of this receptor-ligand recognition, we have crystallized the extracellular ligand-binding domains of KIR2DL2, a member of the Ig superfamily receptors that recognize HLA-Cw1, 3, 7, and 8 allotypes. The structure was determined in two different crystal forms, an orthorhombic P212121 and a trigonal P3221 space group, to resolutions of 3.0 and 2.9 Å, respectively. The overall fold of this structure, like KIR2DL1, exhibits K-type Ig topology with cis-proline residues in both domains that define β-strand switching, which sets KIR apart from the C2-type hematopoietic growth hormone receptor fold. The hinge angle of KIR2DL2 is approximately 80°, 14° larger than that observed in KIR2DL1 despite the existence of conserved hydrophobic residues near the hinge region. There is also a 5° difference in the observed hinge angles in two crystal forms of 2DL2, suggesting that the interdomain hinge angle is not fixed. The putative ligand-binding site is formed by residues from several variable loops with charge distribution apparently complementary to that of HLA-C. The packing of the receptors in the orthorhombic crystal form offers an intriguing model for receptor aggregation on the cell surface.

Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4

The receptor 2B4 belongs to the Ig superfamily and is found on the surface of all murine natural killer (NK) cells as well as T cells displaying non-MHC-restricted cytotoxicity. Previous studies have suggested that 2B4 is an activating molecule because cross-linking of this receptor results in increased cytotoxicity and γ-interferon secretion as well as granule exocytosis. However, it was recently shown that the gene for 2B4 encodes two different products that arise by alternative splicing. These gene products differ solely in their cytoplasmic domains. One form has a cytoplasmic tail of 150 amino acids (2B4L) and the other has a tail of 93 amino acids (2B4S). To determine the function of each receptor, cDNAs for 2B4S and 2B4L were transfected into the rat NK cell line RNK-16. Interestingly, the two forms of 2B4 had opposing functions. 2B4S was able to mediate redirected lysis of P815 tumor targets, suggesting that this form represents an activating receptor. However, 2B4L expression led to an inhibition of redirected lysis of P815 targets when the mAb 3.2.3 (specific for rat NKRP1) was used. In addition, 2B4L constitutively inhibits lysis of YAC-1 tumor targets. 2B4L is a tyrosine phosphoprotein, and removal of domains containing these residues abrogates its inhibitory function. Like other inhibitory receptors, 2B4L associates with the tyrosine phosphatase SHP-2. Thus, 2B4L is an inhibitory receptor belonging to the Ig superfamily.

The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2-Å resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus–ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the insertion (I)-domain on the α chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

A DNA damage and stress inducible G protein-coupled receptor blocks cells in G2/M

Cell cycle progression is monitored by highly coordinated checkpoint machinery, which is activated to induce cell cycle arrest until defects like DNA damage are corrected. We have isolated an anti-proliferative cell cycle regulator named G2A (for G2 accumulation), which is predominantly expressed in immature T and B lymphocyte progenitors and is a member of the seven membrane-spanning G protein-coupled receptor family. G2A overexpression attenuates the transformation potential of BCR-ABL and other oncogenes, and leads to accumulation of cells at G2/M independently of p53 and c-Abl. G2A can be induced in lymphocytes and to a lesser extent in nonlymphocyte cell lines or tissues by multiple stimuli including different classes of DNA-damaging agents and serves as a response to damage and cellular stimulation which functions to slow cell cycle progression.

Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: A receptor for human placental infection

Malaria during the first pregnancy causes a high rate of fetal and neonatal death. The decreasing susceptibility during subsequent pregnancies correlates with acquisition of antibodies that block binding of infected red cells to chondroitin sulfate A (CSA), a receptor for parasites in the placenta. Here we identify a domain within a particular Plasmodium falciparum erythrocyte membrane protein 1 that binds CSA. We cloned a var gene expressed in CSA-binding parasitized red blood cells (PRBCs). The gene had eight receptor-like domains, each of which was expressed on the surface of Chinese hamster ovary cells and was tested for CSA binding. CSA linked to biotin used as a probe demonstrated that two Duffy-binding-like (DBL) domains (DBL3 and DBL7) bound CSA. DBL7, but not DBL3, also bound chondroitin sulfate C (CSC) linked to biotin, a negatively charged sugar that does not support PRBC adhesion. Furthermore, CSA, but not CSC, blocked the interaction with DBL3; both CSA and CSC blocked binding to DBL7. Thus, only the DBL3 domain displays the same binding specificity as PRBCs. Because protective antibodies present after pregnancy block binding to CSA of parasites from different parts of the world, DBL-3, although variant, may induce cross-reactive immunity that will protect pregnant women and their fetuses.

Targeted ablation of the vitamin D receptor: An animal model of vitamin D-dependent rickets type II with alopecia

Vitamin D, the major steroid hormone that controls mineral ion homeostasis, exerts its actions through the vitamin D receptor (VDR). The VDR is expressed in many tissues, including several tissues not thought to play a role in mineral metabolism. Studies in kindreds with VDR mutations (vitamin D-dependent rickets type II, VDDR II) have demonstrated hypocalcemia, hyperparathyroidism, rickets, and osteomalacia. Alopecia, which is not a feature of vitamin D deficiency, is seen in some kindreds. We have generated a mouse model of VDDR II by targeted ablation of the second zinc finger of the VDR DNA-binding domain. Despite known expression of the VDR in fetal life, homozygous mice are phenotypically normal at birth and demonstrate normal survival at least until 6 months. They become hypocalcemic at 21 days of age, at which time their parathyroid hormone (PTH) levels begin to rise. Hyperparathyroidism is accompanied by an increase in the size of the parathyroid gland as well as an increase in PTH mRNA levels. Rickets and osteomalacia are seen by day 35; however, as early as day 15, there is an expansion in the zone of hypertrophic chondrocytes in the growth plate. In contrast to animals made vitamin D deficient by dietary means, and like some patients with VDDR II, these mice develop progressive alopecia from the age of 4 weeks.

The macrophage/endothelial cell mannose receptor cDNA encodes a protein that binds oligosaccharides terminating with SO4-4-GalNAcβ1,4GlcNAcβ or Man at independent sites

Lutropin (LH) and other glycoproteins bearing oligosaccharides with the terminal sequence SO4-4-GalNAcβ1,4GlcNAcβ1,4Man- (S4GGnM) are rapidly removed from the circulation by an S4GGnM-specific receptor (S4GGnM-R) expressed at the surface of hepatic endothelial cells. The S4GGnM-R isolated from rat liver is closely related to the macrophage mannose-specific receptor (Man-R) isolated from rat lung both antigenically and structurally. The S4GGnM-R and Man-R isolated from these tissues nonetheless differ in their ability to bind ligands bearing terminal GalNAc-4-SO4 or Man. In this paper, we have explored the structural relationship between the Man-R and the S4GGnM-R by examining the properties of the recombinant Man-R in the form of a transmembrane protein and a soluble chimeric fusion protein in which the transmembrane and cytosolic domains have been replaced by the Fc region of human IgG1. Like the S4GGnM-R isolated from liver, the chimeric fusion protein is able to bind ligands terminating with GalNAc-4-SO4 and Man at independent sites. When expressed in CHO cells the recombinant Man-R is able to mediate the uptake of ligands bearing either terminal GalNAc-4-SO4 or terminal Man. We propose that the Man-R be renamed the Man/S4GGnM receptor on the basis of its multiple and independent specificities.