Cell adaptation to activated FGFR3 includes Sprouty4 up regulation to inhibit the receptor-mediated ERKs activation from the endoplasmic reticulum

The kinase activity of the thanatophoric dysplasia type II-fibroblast growth factor receptor 3 mutant (TDII-FGFR3) hampers its maturation. As a consequence, the immature receptor activates extracellular regulated kinases (ERKs) from the endoplasmic reticulum (ER), which leads to apoptosis. On the other hand, in stable TDII-FGFR3 cells receptor biosynthesis is restored and ERKs are activated from the cell surface. To identify potential mediators of cell adaptation to the activated receptor we investigated gene products that are differently regulated in TDII and wild-type FGFR3 cells. cDNA representational difference analysis reveals Sprouty4 up regulation in the TDII-FGFR3 cells. Interestingly, Sprouty4 inhibits the TDII-FGFR3-mediated ERKs activation from the ER, but fails to suppress ERKs activation from cell surface. We conclude that cell adaptation to activated FGFR3 include Sprouty4 activity, which silences the premature receptor signaling and suppress apoptosis.     

Lectins and protein traffic early in the secretory pathway

Lectins of the early secretory pathway are involved in selective transport of newly synthesized glycoproteins from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). The most prominent cycling lectin is the mannose-binding type I membrane protein ERGIC-53 (ERGIC protein of 53 kDa), a marker for the ERGIC, which functions as a cargo receptor to facilitate export of an increasing number of glycoproteins with different characteristics from the ER. Two ERGIC-53-related proteins, VIP36 (vesicular integral membrane protein 36) and a novel ERGIC-53-like protein, ERGL, are also found in the early secretory pathway. ERGL may act as a regulator of ERGIC-53. Studies of ERGIC-53 continue to provide new insights into the organization and dynamics of the early secretory pathway. Analysis of the cycling of ERGIC-53 uncovered a complex interplay of trafficking signals and revealed novel cytoplasmic ER-export motifs that interact with COP-II coat proteins. These motifs are common to type I and polytopic membrane proteins including presenilin 1 and presenilin 2. The results support the notion that protein export from the ER is selective.     

K644E/M FGFR3 mutants activate Erk1/2 from the endoplasmic reticulum through FRS2 alpha and PLC gamma-independent pathways

Fibroblast growth factor receptors 3 (FGFR3) with K644M/E substitutions are associated to the severe skeletal dysplasias: severe achondroplasia with developmental delay and achanthosis nigricans(SADDAN) and thanatophoric dysplasia(TDII). The high levels of kinase activity of the FGFR3-mutants cause uncompleted biosynthesis that results in the accumulation of the immature/mannose-rich, phosphorylated receptors in the endoplasmic reticulum (ER) and STATs activation. Here we report that FGFR3 mutants activate Erk1/2 from the ER through an FRS2-independent pathway: instead, a multimeric complex by directly recruiting PLCgamma, Pyk2 and JAK1 is formed. The Erk1/2 activation from the ER however, is PLCgamma-independent, since preventing the PLCgamma/FGFR3 interaction by the Y754F substitution does not inhibit Erks. Furthermore, Erk1/2 activation is abrogated upon treatment with the Src inhibitor PP2, suggesting a role played by a Src family member in the pathway from the ER. Finally we show that the intrinsic kinase activity by mutant receptors is required to allow signaling from the ER. Overall these results highlight how activated FGFR3 exhibits signaling activity in the early phase of its biosynthesis and how segregation in a sub-cellular compartment can affect the FGFR3 multi-faceted capacity to recruit specific substrates.     

The kinase activity of fibroblast growth factor receptor 3 with activation loop mutations affects receptor trafficking and signaling

Amino acid substitutions at the Lys-650 codon within the activation loop kinase domain of fibroblast growth factor receptor 3 (FGFR3) result in graded constitutive phosphorylation of the receptor. Accordingly, the Lys-650 mutants are associated with dwarfisms with graded clinical severity. To assess the importance of the phosphorylation level on FGFR3 maturation along the secretory pathway, hemagglutinin A-tagged derivatives were studied. The highly activated SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) mutant accumulates in its immature and phosphorylated form in the endoplasmic reticulum (ER), which fails to be degraded. Furthermore, the Janus kinase (Jak)/STAT pathway is activated from the ER by direct recruitment of Jak1. Abolishing the autocatalytic property of the mutated FGFR3 by replacing the critical Tyr-718 reestablishes the receptor full maturation and inhibits signaling. Differently, the low activated hypochondroplasia mutant is present as a mature phosphorylated form on the plasma membrane, although with a delayed transition in the ER, and is completely processed. Signaling does not occur in the presence of brefeldin A; instead, STAT1 is activated when protein secretion is blocked with monensin, suggesting that the hypochondroplasia receptor signals at the exit from the ER. Our results suggest that kinase activity affects FGFR3 trafficking and determines the spatial segregation of signaling pathways. Consequently, the defect in down-regulation of the highly activated receptors results in the increased signaling capacity from the intracellular compartments, and this may determine the severity of the diseases.     

Cargo selectivity of the ERGIC-53/MCFD2 transport receptor complex

Exit of soluble secretory proteins from the endoplasmic reticulum (ER) can occur by receptor-mediated export as exemplified by blood coagulation factors V and VIII. Their efficient secretion requires the membrane lectin ER Golgi intermediate compartment protein-53 (ERGIC-53) and its soluble luminal interaction partner multiple coagulation factor deficiency protein 2 (MCFD2), which form a cargo receptor complex in the early secretory pathway. ERGIC-53 also interacts with the two lysosomal glycoproteins cathepsin Z and cathepsin C. Here, we tested the subunit interdependence and cargo selectivity of ERGIC-53 and MCFD2 by short interference RNA-based knockdown. In the absence of ERGIC-53, MCFD2 was secreted, whereas knocking down MCFD2 had no effect on the localization of ERGIC-53. Cargo binding properties of the ERGIC-53/MCFD2 complex were analyzed in vivo using yellow fluorescent protein fragment complementation. We found that MCFD2 is dispensable for the binding of cathepsin Z and cathepsin C to ERGIC-53. The results indicate that ERGIC-53 can bind cargo glycoproteins in an MCFD2-independent fashion and suggest that MCFD2 is a recruitment factor for blood coagulation factors V and VIII.     

The lectin ERGIC-53 is a cargo transport receptor for glycoproteins

Soluble secretory proteins are transported from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) in vesicles coated with COP-II coat proteins. The sorting of secretory cargo into these vesicles is thought to involve transmembrane cargo-receptor proteins. Here we show that a cathepsin-Z-related glycoprotein binds to the recycling, mannose-specific membrane lectin ERGIC-53. Binding occurs in the ER, is carbohydrate- and calcium-ion-dependent and is affected by untrimmed glucose residues. Binding does not, however, require oligomerization of ERGIC-53, although oligomerization is required for exit of ERGIC-53 from the ER. Dissociation of ERGIC-53 occurs in the ERGIC and is delayed if ERGIC-53 is mislocalized to the ER. These results strongly indicate that ERGIC-53 may function as a receptor facilitating ER-to-ERGIC transport of soluble glycoprotein cargo.     

Identification of ERGIC-53 as an intracellular transport receptor of alpha1-antitrypsin

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein-based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify alpha1-antitrypsin (alpha1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of alpha1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of alpha1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.     

pH-induced conversion of the transport lectin ERGIC-53 triggers glycoprotein release

The recycling mannose lectin ERGIC-53 operates as a transport receptor by mediating efficient endoplasmic reticulum (ER) export of some secretory glycoproteins. Binding of cargo to ERGIC-53 in the ER requires Ca2+. Cargo release occurs in the ERGIC, but the molecular mechanism is unknown. Here we report efficient binding of purified ERGIC-53 to immobilized mannose at pH 7.4, the pH of the ER, but not at slightly lower pH. pH sensitivity of the lectin was more prominent when Ca2+ concentrations were low. A conserved histidine in the center of the carbohydrate recognition domain was required for lectin activity suggesting it may serve as a molecular pH/Ca2+ sensor. Acidification of cells inhibited the association of ERGIC-53 with the known cargo cathepsin Z-related protein and dissociation of this glycoprotein in the ERGIC was impaired by organelle neutralization that did not impair the transport of a control protein. The results elucidate the molecular mechanism underlying reversible lectin/cargo interaction and establish the ERGIC as the earliest low pH site of the secretory pathway.     

ERGIC-53 KKAA signal mediates endoplasmic reticulum retrieval in yeast

Studies on the ERGIC-53 KKAA signal have revealed a new mechanism for static retention of mammalian proteins in the endoplasmic reticulum (Andersson, H., Kappeler, F., Hauri, H. P. (1999): Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval. J. Biol. Chem. 274,15080 - 15084). To test if this mechanism was conserved in yeast, the ERGIC-53 KKAA signal was transferred on two different yeast reporter proteins. Making use of a genetic assay, we demonstrate that this signal induces COPI-dependent ER retrieval. ER retention of KKAA-tagged proteins was impaired in yeast mutants affected in COPI subunits. Furthermore, biochemical analysis of post-ER carbohydrate modifications detected on reporter proteins indicated that KKAA-tagged proteins recycle continuously within early compartments of the secretory pathway. Therefore in yeast, the KKAA signal might only function as a classical dilysine ER retrieval signal.     

Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum

The cytosolic coat-protein complex COP-I interacts with cytoplasmic 'retrieval' signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.     

Carbohydrate- and conformation-dependent cargo capture for ER-exit

Some secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture mechanism, but the signals required for the cargo-receptor interaction are largely unknown. Here, we describe a novel targeting motif that is composed of a high-mannose type oligosaccharide intimately associated with a surface-exposed peptide beta-hairpin loop. The motif accounts for lectin ERGIC-53-assisted ER-export of the lyososomal enzyme procathepsin Z. The second oligosaccharide chain of procathepsin Z exhibits no binding activity for ERGIC-53, illustrating the selective lectin properties of ERGIC-53. Our data suggest that the conformation-based motif is only present in fully folded procathepsin Z and that its recognition by ERGIC-53 reflects a quality control mechanism that acts complementary to the primary folding machinery in the ER. A similar oligosaccharide/beta-hairpin loop structure is present in cathepsin C, another cargo of ERGIC-53, suggesting the general nature of this ER-exit signal. To our knowledge this is the first documentation of an ER-exit signal in soluble cargo in conjunction with its decoding by a transport receptor.     

Lectins and traffic in the secretory pathway

Evidence is accumulating that intracellular animal lectins play important roles in quality control and glycoprotein sorting along the secretory pathway. Calnexin and calreticulin in conjunction with associated chaperones promote correct folding and oligomerization of many glycoproteins in the endoplasmic reticulum (ER). The mannose lectin ERGIC-53 operates as a cargo receptor in transport of glycoproteins from ER to Golgi and the homologous lectin VIP36 may operate in quality control of glycosylation in the Golgi. Exit from the Golgi of lysosomal hydrolases to endosomes requires mannose 6-phosphate receptors and exit to the apical plasma membrane may also involve traffic lectins. Here we discuss the features of these lectins and their role in glycoprotein traffic in the secretory pathway.     

Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II.

Thanatophoric dysplasia type II (TDII) is a neonatal lethal skeletal dysplasia caused by a recurrent Lys-650-->Glu mutation within the highly conserved activation loop of the kinase domain of fibroblast growth factor receptor 3 (FGFR3). We demonstrate here that this mutation results in profound constitutive activation of the FGFR3 tyrosine kinase, approximately 100-fold above that of wild-type FGFR3. The mechanism of FGFR3 activation in TDII was probed by constructing various point mutations in the activation loop. Substitutions at position 650 indicated that not only Glu but also Asp and, to a lesser extent, Gln and Leu result in pronounced constitutive activation of FGFR3. Additional mutagenesis within the beta10-beta11 loop region (amino acids Tyr-647 to Leu-656) demonstrated that amino acid 650 is the only residue which can activate the receptor when changed to a Glu, indicating a specificity of position as well as charge for mutations which can give rise to kinase activation. Furthermore, when predicted sites of autophosphorylation at Tyr-647 and Tyr-648 were mutated to Phe, either singly or in combination, constitutive kinase activity was still observed in response to the Lys-650-->Glu mutation, although the effect of these mutations on downstream signalling was not investigated. Our data suggest that the molecular effect of the TDII activation loop mutation is to mimic the conformational changes that activate the tyrosine kinase domain, which are normally initiated by ligand binding and autophosphorylation. These results have broad implications for understanding the molecular basis of other human developmental syndromes that involve mutations in members of the FGFR family. Moreover, these findings are relevant to the study of kinase regulation and the design of activating mutations in related tyrosine kinases.     

Oligomerization and interacellular localization of the glycoprotein receptor ERGIC-53 is independent of disulfide bonds

ERGIC-53 is a type I transmembrane lectin facilitating the efficient export of a subset of secretory glycoproteins from the endoplasmic reticulum. Previous results have shown that ERGIC-53 is present as reduction-sensitive homo-oligomers, i.e. as a balanced mixture of disulfide-linked hexamers and dimers, with the two cysteine residues located close to the transmembrane domain playing a crucial role in oligomerization. Here, we demonstrate, using sucrose gradient sedimentation, cross-linking analyses, and non-denaturing gel electrophoresis, that ERGIC-53 is present exclusively as a hexameric complex in cells. However, the hexamers exist in two forms, one as a disulfide-linked, Triton X-100, perfluoro-octanic acid, and SDS-resistant complex, and the other as a non-covalent, Triton X-100, perfluoro-octanoic acid-resistant, but SDS-sensitive, complex made up of three disulfide-linked dimers that are likely to interact through the coiled-coil domains present in the luminal part of the protein. In contrast to what was previously believed, neither of the membrane-proximal cysteine residues plays an essential role in the formation, or maintenance, of the latter form of hexamers. Subcellular fractionation revealed that the double-cysteine mutant was present in the endoplasmic reticulum-Golgi-intermediate compartment, indicating that the two cysteine residues are not essential for the intracellular distribution of ERGIC-53. Based on these results, we present a model for the formation of the two hexameric forms.     

Mannose-dependent endoplasmic reticulum (ER)-Golgi intermediate compartment-53-mediated ER to Golgi trafficking of coagulation factors V and VIII.

The endoplasmic reticulum-Golgi intermediate compartment (ERGIC) is the site of segregation of secretory proteins for anterograde transport, via packaging into COPII-coated transport vesicles. ERGIC-53 is a homo-hexameric transmembrane lectin localized to the ERGIC that exhibits mannose-selective properties in vitro. Null mutations in ERGIC-53 were recently shown to be responsible for the autosomal recessive bleeding disorder, combined deficiency of coagulation factors V and VIII. We have studied the effect of defective ER to Golgi cycling by ERGIC-53 on the secretion of factors V and VIII. The secretion efficiency of factor V and factor VIII was studied in a tetracycline-inducible HeLa cell line overexpressing a wild-type ERGIC-53 or a cytosolic tail mutant of ERGIC-53 (KKAA) that is unable to exit the ER due to mutation of two COOH-terminal phenylalanine residues to alanines. The results show that efficient trafficking of factors V and VIII requires a functional ERGIC-53 cycling pathway and that this trafficking is dependent on post-translational modification of a specific cluster of asparagine (N)-linked oligosaccharides to a fully glucose-trimmed, mannose9 structure.     

Profile-based data base scanning for animal L-type lectins and characterization of VIPL,a novel VIP36-like endoplasmic reticulum protein

Consensus profiles were established to screen data bases for novel animal L-type lectins. The profiles were generated from linear sequence motifs of the human L-type lectin-like membrane proteins ERGIC-53, ERGL, and VIP36 and by optimal alignment of the entire carbohydrate recognition domain of these proteins. The search revealed numerous orthologous and homologous L-type lectin-like proteins in animals, protozoans, and yeast, as well as the sequence of a novel family member related to VIP36, named VIPL for VIP36-like. Sequence analysis suggests that VIPL is a ubiquitously expressed protein and appeared earlier in evolution than VIP36. The cDNA of VIPL was cloned and expressed in cell culture. VIPL is a high-mannose type I membrane glycoprotein with similar domain organization as VIP36. Unlike VIP36 and ERGIC-53 that are predominantly associated with postendoplasmic reticulum (ER) membranes and cycle in the early secretory pathway, VIPL is a non-cycling resident protein of the ER. Mutagenesis experiments indicate that ER retention of VIPL involves a RKR di-arginine signal. Overexpression of VIPL redistributed ERGIC-53 to the ER without affecting the cycling of the KDEL-receptor and the overall morphology of the early secretory pathway. The results suggest that VIPL may function as a regulator of ERGIC-53.     

Reticulon 3 is involved in membrane trafficking between the endoplasmic reticulum and Golgi

Reticulons (RTNs) constitute a family of endoplasmic reticulum (ER)-associated proteins with a reticular distribution. Despite the implication of their neuronal isoforms in axonal regeneration, the function of their widely expressed isoforms is largely unknown. In this study, we examined the role of the ubiquitously expressed RTN3 in membrane trafficking. Ectopically expressed RTN3 exhibited heterogeneous patterns; filamentous, reticular, and granular distributions. The ER morphology changed accordingly. In cells where RTN3 displayed a filamentous/reticular distribution, protein transport between the ER and Golgi was blocked, and Golgi proteins were dispersed. In contrast, ERGIC-53, a marker for the ER-Golgi intermediate compartment, accumulated at the perinuclear region, and remained there even after cells were treated with agents that induce redistribution of Golgi proteins to the ER, indicating an inhibition of Golgi-to-ER transport of ERGIC-53. These results suggest that RTN3 plays a role in membrane trafficking in the early secretory pathway.     

Enhanced signaling and morphological transformation by a membrane-localized derivative of the fibroblast growth factor receptor 3 kinase domain.

Fibroblast growth factor (FGF) receptors (FGFRs) are membrane-spanning tyrosine kinase receptors that mediate regulatory signals for cell proliferation and differentiation in response to FGFs. We have previously determined that the Lys650-->Glu mutation in the activation loop of the kinase domain of FGFR3, which is responsible for the lethal skeletal dysplasia thanatophoric dyplasia type II (TDII), greatly enhances the ligand-independent kinase activity of the receptor. Here, we demonstrate that expression of this construct induces a c-fos promoter construct approximately 10-fold but does not lead to proliferation or morphological transformation of NIH 3T3 cells. In contrast, the isolated kinase domain of activated FGFR3, targeted to the plasma membrane by a myristylation signal, is able to stimulate c-fos expression by 40-fold, induce proliferation of quiescent cells, and morphologically transform fibroblasts. This result suggests that the extracellular and transmembrane domains of FGFRs exert a negative regulatory influence on the activity of the kinase domain. Targeting of the activated kinase domain to either the cytoplasm or the nucleus does not significantly affect biological signaling, suggesting that signals from FGFR3 resulting in mitogenesis originate exclusively from the plasma membrane. Furthermore, our novel observation that expression of a highly activated FGFR3 kinase domain is able to morphologically transform fibroblasts suggests that dysregulation of FGFR3 has the potential to play a role in human neoplasia.     

Proteomics of endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membranes from brefeldin A-treated HepG2 cells identifies ERGIC-32, a new cycling protein that interacts with human Erv46

Cycling proteins play important roles in the organization and function of the early secretory pathway by participating in membrane traffic and selective transport of cargo between the endoplasmic reticulum (ER), the intermediate compartment (ERGIC), and the Golgi. To identify new cycling proteins, we have developed a novel procedure for the purification of ERGIC membranes from HepG2 cells treated with brefeldin A, a drug known to accumulate cycling proteins in the ERGIC. Membranes enriched 110-fold over the homogenate for ERGIC-53 were obtained and analyzed by mass spectrometry. Major proteins corresponded to established and putative cargo receptors and components mediating protein maturation and membrane traffic. Among the uncharacterized proteins, a 32-kDa protein termed ERGIC-32 is a novel cycling membrane protein with sequence homology to Erv41p and Erv46p, two proteins enriched in COPII vesicles of yeast. ERGIC-32 localizes to the ERGIC and partially colocalizes with the human homologs of Erv41p and Erv46p, which mainly localize to the cis-Golgi. ERGIC-32 interacts with human Erv46 (hErv46) as revealed by covalent cross-linking and mistargeting experiments, and silencing of ERGIC-32 by small interfering RNAs increases the turnover of hErv46. We propose that ERGIC-32 functions as a modulator of the hErv41-hErv46 complex by stabilizing hErv46. Our novel approach for the isolation of the ERGIC from BFA-treated cells may ultimately lead to the identification of all proteins rapidly cycling early in the secretory pathway.