Role of N-glycans in growth factor signaling

Secreted proteins and membrane proteins are frequently post-translationally modified by oligosaccharides. Therefore, many glycoproteins are involved in signal transduction. One example is growth factor receptors, which are membrane proteins that often contain oligosaccharides. The oligosaccharides in those growth factor receptors play crucial roles in receptor functions. An analysis of glycosyltransferase-transfectants revealed that the branching structures of oligosaccharide also serve as important determinants. For example, N-glycans of epidermal growth factor receptor (EGFR) are involved in receptor sorting, ligand binding and dimerization. The addition of a bisecting GlcNAc to N-glycans increases the endocytosis of EGFR. N-glycans of Trk, a high affinity nerve growth factor receptor, also affect its function. Thus, oligosaccharides play an important role in growth factor signaling. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Role of fibroblast growth factor receptor signaling in kidney development

Fibroblast growth factor receptors (Fgfrs) consist of four signaling family members and one nonsignaling decoy receptor, Fgfr-like 1 (Fgfrl1), all of which are expressed in the developing kidney. Several studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB) in cultured tissues. Transgenic and conditional knockout approaches in whole animals have shown that Fgfr1 and Fgfr2 (predominantly the IIIc isoform) in kidney mesenchyme are critical for early MM and UB formation. Conditional deletion of the ligand, Fgf8, in nephron precursors or global deletion of Fgfrl1 interrupts nephron formation. Fgfr2 (likely the IIIb isoform signaling downstream of Fgf7 and Fgf10) is critical for ureteric morphogenesis. Moreover, Fgfr2 appears to act independently of Frs2α (the major signaling adapter for Fgfrs) in regulating UB branching. Loss of Fgfr2 in the MM leads to many kidney and urinary tract anomalies, including vesicoureteral reflux. Thus Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.     

Fibroblast growth factor signaling in tumorigenesis

Fibroblast growth factors and their signaling receptors have been associated with multiple biological activities, including proliferation, differentiation and motility. Consequently, they have evoked interest as candidate oncogenes with the potential to initiate and/or promote tumorigenesis. This has resulted in a large literature describing the presence of these growth factors and their receptors in cancer cell lines and primary tumors of diverse origin. However, it is only recently that compelling evidence has emerged to implicate the fibroblast growth factors (Fgfs) and their receptors in the genesis of human cancers. Here, we outline the model systems that demonstrate the potential oncogenic nature of Fgf signaling and summarise recent evidence that implicates aberrant Fgf signaling as important in the natural history of some common human cancers.     

Role of complex N-glycans in plant stress tolerance

In plant cells, glycans attached to asparagine (N) residues of proteins undergo various modifications in the endoplasmic reticulum and the Golgi apparatus. The N-glycan modifications in the Golgi apparatus result in complex N-glycans attached to membrane proteins, secreted proteins and vacuolar proteins. Recently, we have investigated the role of complex N-glycans in plants using a series of Arabidopsis thaliana mutants affected in complex N-glycan biosynthesis.¹ Several mutant plants including complex glycan 1 (cgl1) displayed a salt-sensitive phenotype during their root growth, which was associated with radial swelling and loss of apical dominance. Among the proteins whose N-glycans are affected by the cgl1 mutation is a membrane anchored β1,4-endoglucanase, KORRIGAN1/RADIALLY SWOLLEN 2 (KOR1/RSW2) involved in cellulose biosynthesis. The cgl1 mutation strongly enhanced the phenotype of a temperature sensitive allele of KOR1/RSW2 (rsw2-1) even at the permissive temperature. This establishes that plant complex N-glycan modification is important for the in vivo function of KOR1/RSW2. Furthermore, rsw2-1 as well as another cellulose biosynthesis mutant rsw1-1 exhibited also a salt-sensitive phenotype at the permissive temperature. Based on these findings, we propose that one of the mechanisms that cause salt-induced root growth arrest is dysfunction of cell wall biosynthesis that induces mitotic arrest in the root apical meristem.Key words: Arabidopsis, salt stress, complex N-glycans, β1,4-endoglucanase, cell wallIn eukaryotic cells, both soluble and membrane proteins that enter the endoplasmic reticulum (ER) system may undergo post-translational modifications called N-glycosylation. N-glycosylation occurs in two phases, namely, core glycosylation in the ER and glycan maturation in the Golgi apparatus.^2,3 The process and roles of core glycosylation in the ER are well established and ubiquitous for eukaryotes. In the ER, pre-assembled core oligosaccharides (Glc₃Man₉GlcNac₂) are transferred to asparagine residues of the Asn-X-Ser/Thr motives in nascent polypeptides by the function of an oligosaccharyltransferase complex (OST). Terminal glucose residues are recognition sites for ER chaperones calnexin and calreticulin, and thus core N-glycans in the ER function in correct folding of newly synthesized proteins.^2,3Greater diversity exists in the N-glycan maturation steps in the Golgi apparatus and conspicuous roles for the resulting complex N-glycans.^2,4 In general, mature N-glycan structures are classified as oligomannosidic type, hybrid or complex type. Glycoprotein precursors that are exported from the ER carry high-mannose type N-glycan intermediates. Numerous enzymes are involved in the conversion of high-mannose type N-glycans to mature complex N-glycans. The functions of N-glycan modifications in the Golgi apparatus are well established in humans, because lack of N-glycan maturation results in Type II Congenital Disorders of Glycosylation.⁵ In Drosophila melanogaster, the Golgi pathway is necessary for development and function of the central nervous system,⁶ whereas in Candida albicans, it is necessary for cell wall integrity and virulence.⁷The first Arabidopsis thaliana mutant lacking complex N-glycans was reported in 1993.⁸ Since then, several mutants and transgenic plants altered in N-glycan maturation in the Golgi apparatus have been reported.^9–12 Plants with altered N-glycan modification pathways that are devoid of potentially immunogenic complex N-glycans are used for the production of pharmaceutical proteins^12,13 and could serve as potential food crops with reduced allergenicity. Until recently, however, plant complex N-glycans have not been associated with essential biological functions in their host plants due to lack of obvious phenotypes of mutant plants defective in complex N-glycan biosynthesis. We recently reported that mutants defective in complex N-glycans show enhanced salt sensitivity, establishing that complex N-glycans are indispensable for certain biological functions.¹Our previous study using an OST subunit mutant stt3a indicated that protein glycosylation could affect salt tolerance and root growth of A. thaliana.¹⁴ Since OST functions upstream of protein folding processes in the ER, stt3a caused an unfolded protein response (UPR), which is a general ER stress response to protein folding defects, as well as accumulation of under-glycosylated proteins. In our recent study, we tried to address whether the salt stress response of the mutant is caused by an activation of UPR, or by a shortage of functional glycoproteins produced by the cells.¹ The cgl1 mutant is defective in N-acetylglucosaminyltransferase in the Golgi apparatus¹⁵ and only able to produce oligomannosidic-type N-glycans but not complex-type N-glycans.⁸ cgl1 mutants exposed to salt stress exhibited root growth arrest and radial swelling similar to stt3a mutants, however, unlike stt3a, the cgl1 mutation did not cause UPR as judged by expression of an UPR marker gene, BiP_pro-GUS. This indicated that salt sensitivity of cgl1 (and likely also of stt3a) is due to lack of mature N-glycans essential for functionality of certain glycoprotein(s).We have determined that a membrane-anchored β1,4-endoglucanase, KORRIGAN1/RADIAL SWELLING2 (KOR1/RSW2), which functions in cellulose biosynthesis, is a target of CGL1 and involved in the salt stress response of A. thaliana.¹ A temperature sensitive rsw2-1 allele¹⁶ showed specific genetic interaction with both cgl1 and stt3a mutations. The corresponding double mutants exhibited spontaneous growth defects at the permissive temperature that were reminiscent of those of rsw2-1 at the restrictive temperature, of cgl1 and stt3a plants treated with salt, and of the rsw1-1 rsw2-1 double mutant that combines two cellulose deficiency mutations. This showed that cgl1 and stt3a enhance cellulose deficiency of rsw2-1, and in turn indicate that the KOR1/RSW2 protein requires complex N-glycans for its function in vivo. Further pyramiding of these mutations resulted in incremental enhancement of growth defects as well as developmental defects of the host plants (Kang et al., (2008), and Fig. 1). Importance of functional cellulose biosynthesis for salt tolerance was further supported by the novel finding of increased salt-sensitivity of rsw2-1 and rsw1-1 single mutants.¹Our previous and current data have implications that affect our view of protein N-glycosylation in plants. First, after all, plant complex N-glycans confer important in vivo functions to secreted/secretory glycoproteins, i.e., protect root growth from salt/osmotic stress. In contrast to core oligosaccharides in the ER, which globally affect protein folding, complex N-glycans appear to function at the individual protein level. Second, one of the targets of salt/osmotic stress is a component of the cellulose biosynthesis machinery, namely KOR1/RSW2 that requires complex N-glycans for its function. KOR1/RSW2 provides a link to how complex N-glycans protect plants from salt/osmotic stress. However, the mechanism by which salt stress triggers the growth arrest via KOR1/RSW2 dysfunction is not yet understood. We have previously shown that the root apical meristem of stt3a exhibits cell cycle arrest under salt stress, but cell differentiation and lateral root formation continued in the same root tip.¹⁴ This implies that plants, in response to salt stress and compromised cell-wall biosynthesis at the root apical meristem, specifically attenuate cell cycle progression at the old meristem and initiate new meristems. A signal transduction pathway that coordinates cell-wall integrity and cell proliferation is well documented in Sacchromyces cerevisiae, where Protein kinase C1 (Pkc1) and a MAP kinase cascade play essential roles.17 Interestingly, both S. cerevisiae Stt3 and Och1 (a mannosyltransferase in the Golgi apparatus) are involved in the cell-wall integrity pathway.¹⁷ In A. thaliana, mutations in the receptor kinase THESEUS1 suppressed hypocotyl elongation defects and ectopic lignification in several cellulose deficient mutants.¹⁸ However, since THE1 is expressed in elongation zones but not in cell division zones of root tips, and the1 did not suppress the kor1-1 phenotype,¹⁸ it is unlikely that THE1 is involved in the regulation of the salt stress response at the root apical meristem. This implies that dividing cells and expanding cells employ distinct mechanism to sense cellulose deficiency. Understanding how complex N-glycans regulate cell-wall biosynthesis and cell proliferation is an exciting task for the coming years.? Open in a separate windowFigure 1Scanning electron micrograph of one-week-old wild type (A and D), rsw2-1 stt3a-2 cgl1-T (B and E) and rsw2-1 rsw1-1 stt3a-2 cgl1-T (C and F) seedlings grown at 18°C. Severe growth defects in mutants are obvious. In shoot apical meristem (D–F), aberrant trichome development is seen in rsw2-1 stt3a-2 cgl1-T (E). In rsw2-1 rsw1-1 stt3a-2 cgl1-T (F), the meristem is transformed into unorganized mass of cells. Bars indicate 0.5 mm.     

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling

Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.     

Galectin-1 signaling in leukocytes requires expression of complex-type N-glycans

Dimeric galectin-1 (dGal-1) is a homodimeric lectin with multiple proposed functions. Although dGal-1 binds to diverse glycans, it is unclear whether dGal-1 preferentially binds to specific subsets of glycans on cell surfaces to transmit signals. To explore this question, we selectively inhibited major glycan biosynthetic pathways in human HL60, Molt-4, and Jurkat cells. Inhibition of N-glycan processing blocked surface binding of dGal-1 and prevented dGal-1-induced Ca(2+) mobilization and phosphatidylserine exposure. By contrast, inhibition of O-glycan or glycosphingolipid biosynthesis did not affect dGal-1 binding or dGal-1-induced Ca(2+) mobilization and phosphatidylserine exposure. These results demonstrate that dGal-1 preferentially binds to and signals through glycoproteins containing complex-type N-glycans in at least some leukocyte subsets.     

Fibroblast growth factor signaling in Caenorhabditis elegans.

Growth factor receptor tyrosine kinases (RTKs), such as the fibroblast growth factor receptor (FGFR), play a major role in how cells communicate with their environment. FGFR signaling is crucial for normal development, and its misregulation in humans has been linked to developmental abnormalities and cancer. The precise molecular mechanisms by which FGFRs transduce extracellular signals to effect specific biologic responses is an area of intense research. Genetic analyses in model organisms have played a central role in our evolving understanding of these signal transduction cascades. Genetic studies in the nematode C. elegans have contributed to our knowledge of FGFR signaling by identifying genes involved in FGFR signal transduction and linking their gene products together into signaling modules. This review will describe FGFR-mediated signal transduction in C. elegans and focus on how these studies have contributed to our understanding of how FGFRs orchestrate the assembly of intracellular signaling pathways.     

Antagonism or synergism. Role of tyrosine phosphatases SHP-1 and SHP-2 in growth factor signaling

SHP-1 and SHP-2 are two Src homology 2 domain-containing tyrosine phosphatases with major pathological implications in cell growth regulating signaling. They share significant overall sequence identity, but their biological functions are often opposite. SHP-1 is generally considered as a negative signal transducer and SHP-2 as a positive one. However, the precise role of each enzyme in shared signaling pathways is not well defined. In this study, we investigated the interaction of these two enzymes in a single cell system by knocking down their expressions with small interfering RNAs and analyzing the effects on epidermal growth factor signaling. Interestingly, knockdown of either SHP-1 or SHP-2 caused significant reduction in the activation of ERK1/2 but not Akt. Furthermore, SHP-1, SHP-2, and Gab1 formed a signaling complex, and SHP-1 and SHP-2 interact with each other. The interaction of SHP-1 with Gab1 is mediated by SHP-2 because it was abrogated by knockdown of SHP-2, and SHP-2, but not SHP-1, binds directly to tyrosine-phosphorylated Gab1. Together, the data revealed that both SHP-1 and SHP-2 have a positive role in epidermal growth factor-induced ERK1/2 activation and that they act cooperatively rather than antagonistically. The interaction of SHP-1 and SHP-2 may be responsible for previously unexpected novel regulatory mechanism of cell signaling by tyrosine phosphatases.     

Divergence of epidermal growth factor - transforming growth factor beta signaling in embryonic orofacial tissue

The epidermal growth factor (EGF) and transforming growth factor beta (TGFbeta) families of signaling molecules play a major role in growth and development of embryos. Abrogation of either signaling pathway results in defects in embryogenesis, including cleft palate. In the developing palate, both EGF and TGFbeta regulate cellular proliferation, extracellular matrix synthesis, and cellular differentiation but often in an opposing manner. Evidence from various adult cell types suggests the existence of cross talk between the EGF and TGFbeta signaling pathways, although it is unclear whether such cross talk exists in murine embryonic maxillary mesenchymal cells, from which the developing palate is derived. In this study, embryonic maxillary mesenchymal cells in culture were treated with EGF and TGFbeta, either singly or in combination, and the cells were subsequently examined for signaling interactions between these two pathways. Immunoblot analyses of nuclear extracts of embryonic maxillary mesenchymal cells revealed that TGFbeta-induced nuclear translocation of Smad 2 and Smad 3 proteins was not affected by EGF. Conversely, immunoblot analyses of whole-cell extracts of these cells indicated that EGF-induced phosphorylation of extracellular signal-regulated kinase proteins, ERK1 and ERK2, was not affected by TGFbeta. Expression of a transfected luciferase reporter gene driven by a promoter with Smad binding elements was induced by TGFbeta in these cells but was not affected by EGF. Last, TGFbeta was found to induce expression of the endogenous gelatinase B gene in embryonic maxillary mesenchymal cells; however, this effect was independent of any interaction of EGF. Collectively, data from this study suggest that the EGF and TGFbeta signal transduction pathways do not converge in murine embryonic maxillary mesenchymal cells.     

Platelet-derived growth factor signaling and human cancer

Platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. The vital functions of PDGFs for angiogenesis, as well as development of kidney, brain, cardiovascular system and pulmonary alveoli during embryogenesis, have been well demonstrated by gene knock-out approaches. Clinical studies reveal that aberrant expression of PDGF and its receptor is often associated with a variety of disorders including atherosclerosis, fibroproliferative diseases of lungs, kidneys and joints, and neoplasia. PDGF contributes to cancer development and progression by both autocrine and paracrine signaling mechanisms. In this review article, important features of the PDGF isoforms and their cell surface receptor subunits are discussed, with regards to signal transduction, PDGF-isoform specific cellular responses, and involvement in angiogensis, and tumorstromal interactions.     

N-聚糖和O-聚糖在极性化细胞蛋白质分拣中的作用

极性化上皮细胞的质膜因其所含蛋白质、脂质等组分不同,可以分为细胞膜顶端和细胞膜基底侧端两个区域,而新合成的蛋白质向这两个区域的有效分拣是上皮细胞维持其自身极性及正常功能所必需的。细胞膜基底侧端蛋白质的分拣主要由位于该蛋白质胞质区的信号肽所介导,关于这方面的研究是比较深入的;而细胞膜顶端蛋白质的分拣机制目前尚未阐明,因而显得比较复杂。近年来,糖类分子作为生物体内细胞识别和调控过程的信息分子日益受到关注,人们通过干扰聚糖合成、基因突变以及构建糖基化缺陷细胞株等实验方法,逐渐地认识到糖类分子在极性化上皮细胞的蛋白质分拣调节中起重要作用。由于糖分子本身结构非常复杂,而且目前缺乏研究糖类分子的有效手段,使得糖生物学的研究远远落后于蛋白质和核酸的研究。从而导致探讨糖类分子在蛋白质分拣过程的具体机制相对来说比较困难。本综述拟简要概括糖类分子中N-聚糖和O-聚糖在极性化上皮细胞的蛋白质分拣过程中的作用,以及两种聚糖在此过程中行使分拣信号功能的可能机制。     

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.     

Role of transforming growth factor beta in cancer

Transforming growth factor beta (TGF-beta) is an effective and ubiquitous mediator of cell growth. The significance of this cytokine in cancer susceptibility, cancer development and progression has become apparent over the past few years. TGF-beta plays various roles in the process of malignant progression. It is a potent inhibitor of normal stromal, hematopoietic, and epithelial cell growth. However, at some point during cancer development the majority of transformed cells become either partly or completely resistant to TGF-beta growth inhibition. There is growing evidence that in the later stages of cancer development TGF-beta is actively secreted by tumor cells and not merely acts as a bystander but rather contributes to cell growth, invasion, and metastasis and decreases host-tumor immune responses. Subtle alteration of TGF-beta signaling may also contribute to the development of cancer. These various effects are tissue and tumor dependent. Identifying and understanding TGF-beta signaling pathway abnormalities in various malignancies is a promising avenue of study that may yield new modalities to both prevent and treat cancer. The nature, prevalence, and significance of TGF-beta signaling pathway alterations in various forms of human cancer as well as potential preventive and therapeutic interventions are discussed in this review.     

Role of platelet-derived growth factor in wound healing

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.     

Inhibition of growth factor signaling pathways by lovastatin

Human fibroblasts treated with the antihypercholesterolaemic drug, lovastatin, displayed a diminished signaling response to epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). Supplementing the culture medium with mevalonic acid restored the signaling response. Not all growth factor signaling pathways were impaired, however, as platelet-derived growth factor (PDGF-BB) and basic fibroblast growth factor (bFGF) responses were refractory to lovastatin treatment. These results suggest the involvement of product(s) of mevalonate metabolism (e.g., prenylated proteins such as p21ras or G proteins) in the signal transduction of EGF, insulin and IGF-I. The inhibition of cell growth by lovastatin may be caused by the inability of the cell to enter the S phase of the cell cycle due to obstruction of the signaling of progression factors.