Localization and modulation of ErbB receptor tyrosine kinases

The ErbB family of receptor tyrosine kinases serves as a model for understanding the propagation of growth factor signals across the plasma membrane and the interpretation of those signals into a cellular response. Recent studies point to a critical role for the accumulation of ErbBs at specific cell-surface locations in the fidelity of ErbB signaling. The past year has witnessed significant advances in our understanding of the molecular mechanisms of ErbB localization and the role of PDZ-domain-containing proteins and cell-surface glycoproteins in directly modulating signaling through ErbBs.     

ErbB2 is necessary for induction of carcinoma cell invasion by ErbB family receptor tyrosine kinases

The epidermal growth factor (EGF) family of tyrosine kinase receptors (ErbB1, -2, -3, and -4) and their ligands are involved in cell differentiation, proliferation, migration, and carcinogenesis. However, it has proven difficult to link a given ErbB receptor to a specific biological process since most cells express multiple ErbB members that heterodimerize, leading to receptor cross-activation. In this study, we utilize carcinoma cells depleted of ErbB2, but not other ErbB receptor members, to specifically examine the role of ErbB2 in carcinoma cell migration and invasion. Cells stimulated with EGF-related peptides show increased invasion of the extracellular matrix, whereas cells devoid of functional ErbB2 receptors do not. ErbB2 facilitates cell invasion through extracellular regulated kinase (ERK) activation and coupling of the adaptor proteins, p130CAS and c-CrkII, which regulate the actin-myosin cytoskeleton of migratory cells. Overexpression of ErbB2 in cells devoid of other ErbB receptor members is sufficient to promote ERK activation and CAS/Crk coupling, leading to cell migration. Thus, ErbB2 serves as a critical component that couples ErbB receptor tyrosine kinases to the migration/invasion machinery of carcinoma cells.     

Culture of mouse germ cells isolated from fetal gonads

Mouse germ cells isolated from male or female genital ridges at 121/2 days post coitum were cultured at room temperature for up to 6 days, with [3H]thymidine present in the culture medium for either the first 24 h or the last 24 h of each culture period. Germ cells were also isolated 131/2-161/2 days post coitum and cultured for 24 h in the presence of [3H]thymidine. The proportion of cells in metaphase, and the proportion of labelled interphase and metaphase nuclei, was recorded. The labelling index declined from 131/2 days onwards, after development either in vivo or in vitro. No labelled metaphase plates were seen after 24 h in the presence of [3H]thymidine, suggesting that under these culture conditions the duration of the G2 phase exceeded 24 h. The results showed that the culture system, in spite of the low temperature, allowed the germ cells to replicate their DNA and undergo mitosis for up to 6 days. Addition of db-cAMP to the culture medium proved highly toxic to male germ cells, and did not markedly increase the proliferation rate of female germ cells.     

Endothelial receptor tyrosine kinases involved in angiogenesis

The Saccharomyces cerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellular lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-terminal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which conforms with the consensus for di-lysine-based ER-localization signals. Despite the presence of this motif, Emp47p was shown to be a Golgi protein at steady-state. The di-lysine motif of Emp47p was functional when transplanted onto Ste2p, a plasma membrane protein, conferring ER localization. Nevertheless, the di-lysine motif was required for Golgi-localization of Emp47p and showed the same charge- independent, position-dependent characteristics of other di-lysine motifs. Alpha-COP has been shown to be required for ER localization of di-lysine-tagged proteins. Consistent with this finding, the Ste2p- Emp47p hybrid protein was mislocalized to the cell surface in the alpha- COP mutant, ret1-1. Surprisingly, the Golgi-localization of Emp47p was unaffected by the ret1-1 mutation. To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di- lysine-tagged proteins we developed an assay to measure this step after block of forward transport in a sec12 mutant. Under these conditions retrograde transport led to a specific redistribution of Emp47p from the Golgi to the ER. This recycling occurred from a Golgi subcompartment containing alpha 1,3 mannose-modified oligosaccharides suggesting that it originated from a medial-or later Golgi compartment. Thus Emp47p cycles between the Golgi apparatus and the ER and requires a di-lysine motif for its alpha-COP-independent, steady state localization in the Golgi.     

Roles of receptor tyrosine kinases in Drosophila development.

Communication between cells is a fundamental component of development and morphogenesis. Identification of the molecules mediating cell-cell communication is crucial for elucidation of the molecular basis of these processes. Receptor tyrosine kinases (RTKs) appear to play a central role in this context by transmitting into cells information dictating their fate. The functions of RTKs in Drosophila are extremely diverse, and include maternal determination of embryonic polarity (torso and torpedo), determination of neuroblast identity (faint little ball), and guidance of tracheal cell migration in the embryo (breathless). During compound eye development, RTKs affect the number of photoreceptor clusters (Ellipse) and the determination of photoreceptor R7 identity (sevenless). The phenotypes of mutations in RTK loci serve as a starting point for understanding processes dictating cell identity at the level of the whole organism. Recently, they have also begun to provide a basis for selection of second-site suppressor mutations, encoding additional elements in their signal transduction pathway. Common themes between the functions, regulation, and signal transduction pathways of Drosophila RTKs are drawn.     

Molecular mechanisms underlying endocytosis and sorting of ErbB receptor tyrosine kinases

The major process that regulates the amplitude and kinetics of signal transduction by tyrosine kinase receptors is endocytic removal of active ligand–receptor complexes from the cell surface, and their subsequent sorting to degradation or to recycling. Using the ErbB family of receptor tyrosine kinases we exemplify the diversity of the down regulation process, and concentrate on two sorting steps whose molecular details are emerging. These are the Eps15-mediated sorting to clathrin-coated regions of the plasma membrane and the c-Cbl-mediated targeting of receptors to lysosomal degradation. Like in yeast cells, sorting involves not only protein phosphorylation but also conjugation of ubiquitin molecules. The involvement of other molecules is reviewed and recent observations that challenge the negative regulatory role of endocytosis are described. Finally, we discuss the relevance of receptor down regulation to cancer therapy.     

Uncovering gene regulatory networks during mouse fetal germ cell development

The commitment of germ cells to either oogenesis or spermatogenesis occurs during fetal gonad development: germ cells enter meiosis or mitotic arrest, depending on whether they reside within an ovary or a testis, respectively. Despite the critical importance of this step for sexual reproduction, gene networks underlying germ cell development have remained only partially understood. Taking advantage of the W(v) mouse model, in which gonads lack germ cells, we conducted a microarray study to identify genes expressed in fetal germ cells. In addition to distinguishing genes expressed by germ cells from those expressed by somatic cells within the developing gonads, we were able to highlight specific groups of genes expressed only in female or male germ cells. Our results provide an important resource for deciphering the molecular pathways driving proper germ cell development and sex determination and will improve our understanding of the etiology of human germ cell tumors that arise from dysregulation of germ cell differentiation.     

Members of the plant NIMA-related kinases are involved in organ development and vascularization in poplar, Arabidopsis and rice

NIMA-related kinases (Neks) are a family of serine/threonine kinases that have been linked to cell-cycle regulation in fungi and mammals. Information regarding the function of Neks in plants is very limited. We screened the three plant species that have had their genomes sequenced in an attempt to improve our understanding of their role in plants. We retrieved seven members in Arabidopsis thaliana, nine in Populus trichocarpa and six in Oryza sativa. Phylogenetic analysis showed that plant Neks are closely related to each other and contain paralogous genes. Moreover, their chromosome distribution and their exon-intron structure revealed that the actual plant Nek family was derived from a single representative followed by large segmental duplication events. Functional expression analyses in the three species relied on RTqPCR in poplar and publicly available microarray data for Arabidopsis and rice. Although plant Neks are present in every organ analyzed, their expression profiles suggest their involvement in plant development processes. Furthermore, we showed that PNek1, a member of the poplar family, is expressed at sites of free auxin synthesis and is specifically involved during the vascularization process.     

Drug-induced ubiquitylation and degradation of ErbB receptor tyrosine kinases: implications for cancer therapy

Overexpression of ErbB-2/HER2 is associated with aggressive human malignancies, and therapeutic strategies targeting the oncoprotein are currently in different stages of clinical application. Tyrosine kinase inhibitors (TKIs) that block the nucleotide-binding site of the kinase are especially effective against tumors. Here we report an unexpected activity of TKIs: along with inhibition of tyrosine phosphorylation, they enhance ubiquitylation and accelerate endocytosis and subsequent intracellular destruction of ErbB-2 molecules. Especially potent is an irreversible TKI (CI-1033) that alkylates a cysteine specific to ErbB receptors. The degradative pathway stimulated by TKIs appears to be chaperone mediated, and is common to the heat shock protein 90 (Hsp90) antagonist geldanamycin and a stress-induced mechanism. In agreement with this conclusion, CI-1033 and geldanamycin additively inhibit tumor cell growth. Based upon a model for drug-induced degradation of ErbB-2, we propose a general strategy for selective destruction of oncoproteins by targeting their interaction with molecular chaperones.     

Differential endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers.

Both homo- and hetero-dimers of ErbB receptor tyrosine kinases mediate signaling by a large group of epidermal growth factor (EGF)-like ligands. However, some ligands are more potent than others, although they bind to the same direct receptor. In addition, signaling by receptor heterodimers is superior to homodimers. We addressed the mechanism underlying these two features of signal tuning by using three ligands: EGF; transforming growth factor alpha (TGFalpha); and their chimera, denoted E4T, which act on cells singly expressing ErbB-1 as a weak, a strong, and a very strong agonist, respectively. Co-expression of ErbB-2, a developmentally important co-receptor whose expression is frequently elevated in human cancers, specifically potentiated EGF signaling to the level achieved by TGFalpha, an effect that was partially mimicked by ErbB-3. Analysis of the mechanism underlying this trans-potentiation implied that EGF-driven homodimers of ErbB-1 are destined for intracellular degradation, whereas the corresponding heterodimers with ErbB-2 or with ErbB-3, dissociate in the early endosome. As a consequence, in the presence of either co-receptor, ErbB-1 is recycled to the cell surface and its signaling is enhanced. This latter route is followed by TGFalpha-driven homodimers of ErbB-1, and also by E4T-bound receptors, whose signaling is further enhanced by repeated cycles of binding and dissociation from the receptors. We conclude that alternative endocytic routes of homo- and hetero-dimeric receptor complexes may contribute to tuning and diversification of signal transduction. In addition, the ability of ErbB-2 to shunt ligand-activated receptors to recycling may explain, in part, its oncogenic potential.     

Interaction of the TEK and TIE receptor tyrosine kinases during cardiovascular development.

TEK (TIE2) and TIE (TIE1) are structurally related receptor tyrosine kinases expressed in endothelial cells and their precursors. Genetic studies in the mouse have revealed essential functions of both receptors in angiogenic expansion of the vasculature during development. As previously shown, mouse embryos homozygous for a disrupted Tek allele die by day 10.5 of embryogenesis due to endocardial defects, hemorrhaging, and impaired vascular network formation. Furthermore, TIE is required cell autonomously for endothelial cell survival and extension of the vascular network during late embryogenesis. Here we have investigated possible redundancy in the TEK and TIE signalling pathways during vascular development. Vasculogenesis proceeds normally in embryos lacking both TEK and TIE, although such embryos die early in gestation of multiple cardiovascular defects. Mosaic analysis revealed an absolute requirement for TEK in the endocardium at E10.5, whereas TEK and TIE are dispensable for the initial assembly of the rest of the vasculature. In contrast, both receptors are required in the microvasculature during late organogenesis and in essentially all blood vessels of the adult. This analysis demonstrates essential functions for TEK and TIE in maintaining the integrity of the mature vasculature.     

Ultrastructure of germ cell development in the human fetal testis

Summary Electron-microscopic examination of the human fetal testis between 10 and 20 weeks gestation reveals the presence of two distinct cell types within the tubules: Sertoli cells and germ cells. The latter are distinguished by their spherical shape, smooth nuclear membranes, globular mitochondria and paucity of cytoplasmic organelles. The gonocytes, or primitive germ cells, occur as single cells in the central portions of the tubules. Their chromatin is finely granular and evenly dispersed. Nucleoli are centrally placed and of uniform electron density. Various stages in the migration of gonocytes to the tubular periphery are indicated by the extension of cytoplasmic processes toward the basal lamina. Bands of microtubules are present within the processes. Spermatogonia are arranged in pairs and groups at the tubular periphery. They lack the nucleolar and mitochondrial characteristics of adult spermatogonia. Except for slight changes in chromatin density and nucleolar structure, the fetal spermatogonia retain the ultrastructural characteristics of gonocytes. Intercellular bridges connect adjacent spermatogonia. Degeneration affecting large numbers of germ cells, but primarily gonocytes, begins with nuclear infolding and chromatin condensation and eventually involves both nuclear and cytoplasmic structures. The degenerated cells are removed by phagocytosis by adjacent Sertoli cells. Large phagosomes are present in the cytoplasm of many of the Sertoli cells.Supported by a grant from the Ford Foundation and by General Research Support Grant RR055511 from the National Institutes of Health. Technical assistance was provided by Mrs. Lucy A. Conner.     

EphB receptor tyrosine kinases control morphological development of the ventral midbrain

EphB receptor tyrosine kinases and ephrin-B ligands regulate several types of cell-cell interactions during brain development, generally by modulating the cytoskeleton. EphB/ephrinB genes are expressed in the developing neural tube of early mouse embryos with distinct overlapping expression in the ventral midbrain. To test EphB function in midbrain development, mouse embryos compound homozygous for mutations in the EphB2 and EphB3 receptor genes were examined for early brain phenotypes. These mutants displayed a morphological defect in the ventral midbrain, specifically an expanded ventral midline evident by embryonic day E9.5-10.5, which formed an abnormal protrusion into the cephalic flexure. The affected area was comprised of cells that normally express EphB2 and ephrin-B3. A truncated EphB2 receptor caused a more severe phenotype than a null mutation, implying a dominant negative effect through interference with EphB forward (intracellular) signaling. In mutant embryos, the overall number, size, and identity of the ventral midbrain cells were unaltered. Therefore, the defect in ventral midline morphology in the EphB2;EphB3 compound mutant embryos appears to be caused by cellular changes that thin the tissue, forcing a protrusion of the ventral midline into the cephalic space. Our data suggests a role for EphB signaling in morphological organization of specific regions of the developing neural tube.     

Regulation of primordial germ cell development in the mouse

Primordial germ cells (PGCs) are the founders of the gametes. They arise at the earliest stages of embryonic development and migrate to the gonadal ridges, where they differentiate into oogonia/oocytes in the ovary, and prospermatogonia in the testis. The present article is a review of the main studies undertaken by the author with the aim of clarifying the mechanisms underlying the development of primordial germ cells. Methods for the isolation and purification of migratory and post-migratory mouse PGCs devised in the author's laboratory are first briefly reviewed. Such methods, together with the primary culture of PGCs onto suitable cell feeder layers, have allowed the analysis of important aspects of the control of their development, concerning in particular survival, proliferation and migration of mouse PGCs. Compounds and growth factors affecting PGC numbers in culture have been identified. These include survival anti-apoptotic factors (SCF, LIF) and positive regulators of proliferation (cAMP, PACAPs, RA). Evidence has been provided that the motility of migrating PGCs relies on integrated signals from extracellular matrix molecules and the surrounding somatic cells. Moreover, homotypic PGC-PGC interaction has been evidenced that might play a role in PGC migration and in regulating their development. Several molecules (i.e. integrins, specific types of oligosaccharides, E-cadherin, the tyrosine kinase receptor c-kit) have been found to be expressed on the surface of PGCs and to mediate adhesive interactions of PGCs with the extracellular matrix, somatic cells and neighbouring PGCs.     

Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads

Fluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCA_I) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEA_I) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only). Distinct developmental-related changes were observed when female germ cells were labeled with fluorescein-conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled. I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.     

The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function.     

Inhibition of vascular endothelial growth factor receptor tyrosine kinases impairs adipose tissue development in mouse models of obesity

We have studied the effect of PTK787 (Vatalanib), an inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, on adipose tissue development. Oral administration of PTK787 for 4 weeks (2 mg/g high fat diet, HFD) to C57Bl/6 mice resulted in a significant reduction in total body weight and of subcutaneous (SC) and gonadal (GON) adipose tissue mass, as compared to control animals fed HFD only (all p<0.0005). In the GON adipose tissue adipocytes were hypertrophic after PTK787 treatment. Blood vessel size and density were not significantly affected by PTK787 treatment. Expression of Flk-1 (VEGFR-2) mRNA was significantly reduced in SC and GON adipose tissues of PTK787 treated mice. De novo fat pad formation following injection of preadipocytes in NUDE mice was significantly (p<0.005) impaired by PTK787 administration (2 mg/g HFD for 4 weeks), without associated effect on blood vessel size or density. Thus, in nutritionally induced murine obesity models, oral administration of the VEGFR tyrosine kinases inhibitor PTK787 resulted in reduced adipose tissue development.