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Ligand-independent Dimerization and Activation of the Oncogenic Xmrk Receptor by Two Mutations in the Extracellular Domain

2001; Elsevier BV; Volume: 276; Issue: 5 Linguagem: Inglês

10.1074/jbc.m006574200

1083-351X

Ana Gómez, Claudia Wellbrock, Heidrun Gutbrod, Nicola Dimitrijevic, Manfred Schartl,

Invertebrate Immune Response Mechanisms

Overexpression of the oncogenic receptor tyrosine kinase ONC-Xmrk is the first step in the development of hereditary malignant melanoma in the fish Xiphophorus. However, overexpression of its proto-oncogene counterpart (INV-Xmrk) is not sufficient for the oncogenic function of the receptor. Compared with INV-Xmrk, the ONC-Xmrk receptor displays 14 amino acid changes, suggesting the presence of activating mutations. To identify such activating mutations, a series of chimeric and mutant receptors were studied. None of the mutations present in the intracellular domain was found to be involved in receptor activation. In the extracellular domain, we found two mutations responsible for activation of the receptor. One is the substitution of a conserved cysteine (C578S) involved in intramolecular disulfide bonding. The other is a glycine to arginine exchange (G359R) in subdomain III. Either mutation leads to constitutive dimer formation and thereby to activation of the ONC-Xmrk receptor. Besides, the presence of these mutations slows down the processing of the Xmrk receptor in the endoplasmic reticulum, which is apparent as an incomplete glycosylation. Overexpression of the oncogenic receptor tyrosine kinase ONC-Xmrk is the first step in the development of hereditary malignant melanoma in the fish Xiphophorus. However, overexpression of its proto-oncogene counterpart (INV-Xmrk) is not sufficient for the oncogenic function of the receptor. Compared with INV-Xmrk, the ONC-Xmrk receptor displays 14 amino acid changes, suggesting the presence of activating mutations. To identify such activating mutations, a series of chimeric and mutant receptors were studied. None of the mutations present in the intracellular domain was found to be involved in receptor activation. In the extracellular domain, we found two mutations responsible for activation of the receptor. One is the substitution of a conserved cysteine (C578S) involved in intramolecular disulfide bonding. The other is a glycine to arginine exchange (G359R) in subdomain III. Either mutation leads to constitutive dimer formation and thereby to activation of the ONC-Xmrk receptor. Besides, the presence of these mutations slows down the processing of the Xmrk receptor in the endoplasmic reticulum, which is apparent as an incomplete glycosylation. receptor tyrosine kinase epidermal growth factor receptor polyacrylamide gel electrophoresis endoglycosidase H endoplasmic reticulum bovine serum albumin phosphate-buffered saline 4′,6-diamidino-2-phenylindole oncogenic version of Xmrk Xmrk proto-oncogene product Receptor tyrosine kinases (RTKs)1 are important components of the signaling network that controls cell growth and differentiation. Their enzymatic activity is tightly regulated in normal cells. After ligand binding and dimerization, they become activated and a cascade of phosphorylations is initiated inside the cells (1Ullrich A. Schessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4605) Google Scholar). Diverse mechanisms have been reported that can lead to the constitutive activation of these enzymes. These comprise overexpression, amplification, point mutations, truncations, and autocrine stimulation. The inappropriate constitutive activation of the RTKs results in an altered signaling inside the cell and is a widely documented process implicated in tumor formation (2Aaronson S.A. Science. 1991; 254: 1146-1153Crossref PubMed Scopus (1158) Google Scholar, 3Rodrigues G.A. Park M. Curr. Opin. Genet. Dev. 1994; 4: 15-24Crossref PubMed Scopus (119) Google Scholar). The hereditary melanoma of Xiphophorus fish is a well established genetic model system for tumor development in which the overexpression of the RTK gene Xmrk( X iphophorus melanomareceptor kinase) leads to melanoma formation (for review, see Ref. 4Schartl M. Trends Genet. 1995; 11: 185-189Abstract Full Text PDF PubMed Scopus (89) Google Scholar). Xmrk belongs to the epidermal growth factor receptor (EGFR) family, but it is an additional member, clearly distinct from the four receptors (HER1–4) already described in mammals (5Wittbrodt J. Adam D. Malitschek B. Mäueler W. Raulf F. Telling A. Robertson S.M. Schartl M. Nature. 1989; 341: 415-421Crossref PubMed Scopus (260) Google Scholar). 2A. Gómez, unpublished data.2A. Gómez, unpublished data. Two copies of the Xmrk gene have been found. One of them, INV-Xmrk, is a gene invariably present in all fish (6Dimitrijevic N. Winkler C. Wellbrock C. Gómez A. Duschl J. Altschmied J. Schartl M. Oncogene. 1998; 16: 1681-1690Crossref PubMed Scopus (29) Google Scholar). 3A. Gómez, M. Schartl, C. Winkler, and Y. Hong, unpublished data.3A. Gómez, M. Schartl, C. Winkler, and Y. Hong, unpublished data. It is ubiquitously expressed at low levels, and, although its physiological role is still unknown, it appears not to be involved in melanoma formation. It represents the proto-oncogenic form of Xmrk. The second oncogenic copy, ONC-Xmrk, is only present in some species of Xiphophorus. It originated by an ancient gene duplication event from INV-Xmrk and it is under a different transcriptional control than the proto-oncogene. Only basal levels of expression are observed if the regulatory locus R is also present in the genome. This is the situation found in nonhybrid wild fish, which are generally tumor-free. In hybrids, due to crossing conditioned elimination of the R-containing chromosome (7Ahuja M.R. Anders F. Prog. Exp. Tumor Res. 1976; 20: 380-397Crossref PubMed Google Scholar), the system is deregulated and ONC-Xmrk is overexpressed (8Adam D. Mäueler W. Schartl M. Oncogene. 1991; 6: 73-80PubMed Google Scholar). This leads to neoplastic transformation of pigment cells. A cell line (PSM) derived from Xiphophorus melanoma provides an in vitro system where ONC-Xmrk is also overexpressed. Here, as in melanoma in situ, the Xmrk receptor is highly activated, which is apparent as strong tyrosine autophosphorylation (9Malitschek B. Wittbrodt J. Fischer P. Lammers R. Ullrich A. Schartl M. J. Biol. Chem. 1994; 269: 10423-10430Abstract Full Text PDF PubMed Google Scholar,10Wellbrock C. Geissinger E. Gómez A. Fischer P. Friedrich K. Schartl M. Oncogene. 1998; 16: 3047-3056Crossref PubMed Scopus (34) Google Scholar). The fact that the highly expressed ONC-Xmrk is constitutively autophosphorylated in melanoma cells pointed to overexpression, and thus high concentration of receptors, as one mechanism for activation. However, the ectopic overexpression of INV- and ONC-Xmrk in embryos of transgenic fish showed that exclusively those fish expressing ONC-Xmrk were developing tumors with high incidence, short latency periods, and a specific pattern of affected tissues, whereas only a basal rate of tumor induction appeared in the case of INV-Xmrk-expressing fish, comparable to the rate obtained with the expression of another, nonactivated receptor (6Dimitrijevic N. Winkler C. Wellbrock C. Gómez A. Duschl J. Altschmied J. Schartl M. Oncogene. 1998; 16: 1681-1690Crossref PubMed Scopus (29) Google Scholar, 11Winkler C. Wittbrodt J. Lammers R. Ullrich A. Schartl M. Oncogene. 1994; 9: 1517-1525PubMed Google Scholar). Besides that, INV-Xmrk was shown to be not phosphorylated when transiently expressed in human cells (HEK293), in contrast to the strong autophosphorylation of ONC-Xmrk. The different behavior of INV- and ONC-Xmrk clearly indicates that a mechanism additional to overexpression is instrumental in Xmrk activation (6Dimitrijevic N. Winkler C. Wellbrock C. Gómez A. Duschl J. Altschmied J. Schartl M. Oncogene. 1998; 16: 1681-1690Crossref PubMed Scopus (29) Google Scholar). Comparison of the amino acid sequences of the two versions of Xmrk revealed that the oncogene differs from the proto-oncogene in 14 residues, including some that are highly conserved in the EGFR family of RTKs and that are present in INV-Xmrk but substituted in ONC-Xmrk (6Dimitrijevic N. Winkler C. Wellbrock C. Gómez A. Duschl J. Altschmied J. Schartl M. Oncogene. 1998; 16: 1681-1690Crossref PubMed Scopus (29) Google Scholar). This fact suggested that mutational alteration could be involved in the activation of Xmrk. Moreover, the high phosphorylation level shown by an ONC-INV chimeric receptor containing the extracellular domain of ONC-Xmrk and the intracellular domain of INV-Xmrk pointed to one or more of the mutations in the extracellular domain as implicated in the activation (6Dimitrijevic N. Winkler C. Wellbrock C. Gómez A. Duschl J. Altschmied J. Schartl M. Oncogene. 1998; 16: 1681-1690Crossref PubMed Scopus (29) Google Scholar). However, this result did neither address a role of the intracellular mutations in the activation of Xmrk nor could it identify the extracellular oncogenic amino acid change. To understand the mechanism of activation of ONC-Xmrk, we have analyzed the effect of different mutations. We show that two mutations in the extracellular region of the ONC-Xmrk receptor are responsible for activation. Both of them independently lead to the constitutive dimerization of the receptor by aberrant intermolecular disulfide bonding formation. Additionally, the presence of these mutations slows down the processing of Xmrk receptor in the endoplasmic reticulum (ER), which is apparent as an incomplete glycosylation. 5E.2 (anti-Tyr(P)) is a mouse monoclonal antibody directed against phosphotyrosines (12Fendly B.M. Winget M. Hudziak R.M. Lipari M.T. Napier M.A. Ullrich A. Cancer Res. 1990; 50: 1550-1558PubMed Google Scholar). Anti-mrk is a polyclonal antibody raised against ONC-Xmrk (9Malitschek B. Wittbrodt J. Fischer P. Lammers R. Ullrich A. Schartl M. J. Biol. Chem. 1994; 269: 10423-10430Abstract Full Text PDF PubMed Google Scholar). The INV-ONC chimera was generated by replacing an Eco RI fragment from pRK5 ONC (pRK5 Xmrk; Ref. 13Wittbrodt J. Lammers R. Malitschek B. Ullrich A. Schartl M. EMBO J. 1992; 11: 4239-4246Crossref PubMed Scopus (59) Google Scholar) containing the extracellular, transmembrane, and juxtamembrane domains from the ONC-Xmrk receptor with an INV-Xmrk fragment corresponding to the same domains (6Dimitrijevic N. Winkler C. Wellbrock C. Gómez A. Duschl J. Altschmied J. Schartl M. Oncogene. 1998; 16: 1681-1690Crossref PubMed Scopus (29) Google Scholar). AnEco RI-Eco 47III and anEco 47III-Sal I fragment from ONC-Xmrk were used to replace the corresponding fragments in pRK5 INV and thus generate the Xmrk(G359R,P388T) and Xmrk(P470L,S476N,C578S,M595I) chimeras, respectively. The different point mutations were created using a Muta-Gene phagemid in vitro mutagenesis kit (version 2, Bio-Rad). Different fragments of INV- or ONC-Xmrk cloned in pBlueScript were used as templates. The following primers were used to generate the mutations: 5′-GAAGGATCCGATGTTGGTTG-3′ for the Xmrk(P388T) mutant, 5′-GGACCTGATGTTGGTCGAGTTG-3′ for the INV(G359R) mutant, and 5′-TGCACACTTCGAGCAGTTGG-3′ for the Xmrk (P470L,S476N,M595I) mutant. The INV(C578S) mutant was generated by replacing a Cpo I-Nsi I fragment from INV-Xmrk by the corresponding one from ONC-Xmrk containing the C578S mutation. All constructs containing point mutations were sequenced to ensure that the desired mutation was present. Three clones from a X. maculatus cosmid library were used as templates for sequencing the mutations. Cosmid L11 091 contains the X-ONC-Xmrk allele, M08 036 contains the Y-ONC-Xmrk allele, and G01 008 contains INV-Xmrk (14Nanda I. Volff J.-N. Weis S. Körting C. Froschauer A. Schmid M. Schartl M. Chromosoma. 2000; 109: 173-180Crossref PubMed Scopus (65) Google Scholar). Oligonucleotides designed from introns 4, 8, 9, 11, and 14 were used as sequencing primers. Cycle sequencing was performed using a Cycle Sequencing Kit (Amersham Pharmacia Biotech). The sequence of these cosmid clones revealed the existence in INV- and ONC-Xmrk of an additional codon in positions 1744 and 1573, respectively. This leads to the insertion of a glycine between Trp-512 and Pro-513. Therefore, the amino acid positions higher than 512 are increased by 1 as compared with the numbers published by Dimitrijevicet al. (6Dimitrijevic N. Winkler C. Wellbrock C. Gómez A. Duschl J. Altschmied J. Schartl M. Oncogene. 1998; 16: 1681-1690Crossref PubMed Scopus (29) Google Scholar). The INV- and ONC-Xmrk sequences in the GenBank™ data base with accession numbers U53471 for INV-Xmrk and X16891 for ONC-Xmrk have been updated accordingly. HEK293 cells (human embryonic kidney fibroblasts) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal calf serum and 1% penicillin-streptomycin. Cells were transiently transfected using a modified calcium phosphate transfection method (15Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4820) Google Scholar). For the transfections, 1 μg of plasmid DNA encoding each expression construct was used per 2.3-cm dish and 3–10 μg per 9-cm dish. Following overnight transfection the medium was changed. Cells were harvested 48 h after transfection. Ba/F3 (mouse pro-B-cell line; Ref. 16Palacios R. Steinmetz M. Cell. 1985; 41: 727-734Abstract Full Text PDF PubMed Scopus (584) Google Scholar) cell culture, transfection, and colony selection were done as described previously (10Wellbrock C. Geissinger E. Gómez A. Fischer P. Friedrich K. Schartl M. Oncogene. 1998; 16: 3047-3056Crossref PubMed Scopus (34) Google Scholar). BaF ONC and BaF INV-ONC stable clones express pRK5 ONC (pRK5 Xmrk; Ref. 13Wittbrodt J. Lammers R. Malitschek B. Ullrich A. Schartl M. EMBO J. 1992; 11: 4239-4246Crossref PubMed Scopus (59) Google Scholar) and the INV-ONC chimera, respectively. After harvesting, the cells were washed twice with cold PBS and lysed in Triton lysis buffer as described (17Wellbrock C. Fischer P. Schartl M. Int. J. Cancer. 1998; 76: 437-442Crossref PubMed Scopus (25) Google Scholar). Immunoprecipitations were done by using protein A-Sepharose and anti-Xmrk serum. Immunoprecipitates and cell lysates were electophoresed through a 6.5% or 7.5% SDS-PAGE gel or a 3–8% gradient SDS-PAGE gel. Laemmli buffer either containing or lacking 2-mercaptoethanol was added to the samples to be run under reducing or nonreducing conditions, respectively. The separated proteins were blotted onto a nitrocellulose membrane (Schleicher & Schuell) using standard protocols. Filters were blocked for 5 min in 10 mm Tris-Cl, pH 7.9, 0.5% Tween, 1.5% BSA and incubated 1 h with the first antibody. Horseradish peroxidase-coupled second antibodies were used and developed by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) according to manufacturer's instructions. When necessary, the filters were stripped in 62.5 mmTris-Cl, pH 6.7, 2% SDS, and 100 mm 2-mercaptoethanol for 20 min at 50 °C, following three washes with PBS before blocking with 1.5% BSA previous to the second probing. Equal amounts of protein extracts were denatured at 95 °C for 5 min in a digestion buffer containing 50 mm tribasic sodium citrate (pH 5.5), 0.5% SDS, 0.1 m 2-mercaptoethanol, and 0.5 mmphenylmethylsulfonyl fluoride. 5 milliunits of endoglycosidase H (Roche Molecular Biochemicals) were added to half of the extracts, and all were incubated at 37 °C for 20 h. Digestion products were analyzed on Western blots detected with an anti-Xmrk antibody. 105 BaF INV-ONC or BaF ONC cells were suspended in 500 μl of ice-cold PBS and spun onto slides at 220 × g for 5 min using a cytospin device (Hettich). After removal of the liquid, cells were consecutively fixed for 10 min at −20 °C in methanol and for 2 min at −20 °C in acetone. The fixed cells were then blocked with 1% BSA in PBS for 20 min at room temperature, washed three times with PBS, and incubated with anti-mrk serum (40 μg/ml) for 60 min. After washing, another incubation with dichlorotriazinaminofluorescein-conjugated anti-rabbit IgG (Dianova; 30 μg/ml) for 60 min followed. Cells were embedded in mounting medium containing DAPI (Vectashield, Vector Laboratories). From an earlier study (6Dimitrijevic N. Winkler C. Wellbrock C. Gómez A. Duschl J. Altschmied J. Schartl M. Oncogene. 1998; 16: 1681-1690Crossref PubMed Scopus (29) Google Scholar), it was known that the activation of ONC-Xmrk is due not only to overexpression but also to one or several mutations located in the extracellular domain of ONC-Xmrk that might contribute to the oncogenic potential of the receptor. It was shown that an ONC-INV chimera containing the extracellular part of ONC-Xmrk fused to the intracellular domains of INV-Xmrk is strongly autophosphorylated when transiently expressed in human 293 cells. However, there are five mutations in the carboxyl terminus of ONC-Xmrk whose role was still unknown. Although the carboxyl terminus is the most divergent region in this family of receptors, two of the substitutions found there correspond to highly conserved residues in all subclass I RTKs. One is the exchange of a proline for leucine (P984L), and the other is a tyrosine for asparagine (Y1038N) (6Dimitrijevic N. Winkler C. Wellbrock C. Gómez A. Duschl J. Altschmied J. Schartl M. Oncogene. 1998; 16: 1681-1690Crossref PubMed Scopus (29) Google Scholar). To exclude a possible contribution of the intracellular domain of ONC-Xmrk in its oncogenic activation, an INV-ONC chimera containing the extracellular sequences of INV-Xmrk and the intracellular region of ONC-Xmrk was generated. After transfection of 293 cells and immunoprecipitation of the chimeric receptor, the INV-ONC chimera showed a level of phosphorylation clearly lower than the ONC-INV construct and similar to that of INV-Xmrk (Fig.1). This indicates that all activating mutation(s) should be located in the extracellular part and none of the intracellular mutations of ONC-Xmrk is involved in activation. This was confirmed by introducing the five COOH-terminal amino acid changes (P984L, N1025T, A1035T, Y1038N, and L1156Q) into INV-Xmrk. All mutant receptors did not show enhanced autophosphorylation when compared with INV-Xmrk (data not shown). For technical reasons, the cDNAs from INV-Xmrk and ONC-Xmrk genes were originally isolated from two different species ofXiphophorus. The cDNA from INV-Xmrk was isolated from the Xiphophorus xiphidium-derived A2 cell line and ONC-Xmrk gene from the PSM cell line derived fromXiphophorus maculatus melanoma (18Wakamatsu Y. Cancer Res. 1981; 41: 679-680PubMed Google Scholar). The different species origin of these two genes could account for some of the effective nucleotide differences between them. To distinguish which of the changes in the extracellular domains were due to a species-specific variation and which were potentially functional mutations of the oncogenic Xmrk, we sequenced a series of cosmid clones that contain genomic DNA from different alleles of X. maculatus INV- and ONC-Xmrk. The alignment on Fig.2 shows that none of the nucleotide polymorphisms results in an amino acid difference between X. maculatus and X. xiphidium INV-Xmrk. However, two of the amino acid changes noted earlier (P195H and S446R) probably are irrelevant as they do not appear in the ONC-Xmrk sequence obtained from X. maculatus DNA. They may be a cell line-specific characteristic as they appear exclusively in the PSM cells. They were not considered further; thus, the number of supposed effective mutations in the extracellular region of ONC-Xmrk is reduced to six. Four of these six mutations (G359R, P470L, S476N, and M595I) involve amino acids that are not conserved in other members of the EGFR family, the fifth includes the loss of a semiconserved proline (P388T), and the sixth eliminates a highly conserved cysteine (C578S) from the second cysteine-rich domain of the receptor. One mechanism described for RTKs resulting in constitutive activation is ligand-independent dimerization. Using a heterologous system (293 cells) for transient expression of the different Xmrk constructs, we had observed traces of a high molecular weight phosphorylated form of the molecule that, however, could not be resolved in the routine SDS-polyacrylamide gels. This form appeared exclusively in the constructs where the extracellular domain of ONC-Xmrk was present (see Fig. 1). To test whether this could correspond to a dimeric form of the ONC-Xmrk receptor, electrophoreses in gradient denaturing gels under reducing and nonreducing conditions were performed. After blotting and detection of the proteins with an Xmrk antibody for both INV- and ONC-Xmrk, the appearance of a 160-kDa form that corresponds to the Xmrk monomer was recorded (Fig.3 A, lanes 1 and 2). In addition, for ONC-Xmrk under nonreducing conditions, another signal with a higher molecular weight was appearing that was consistent with the size of a dimer. This signal was not present under reducing conditions. When the ONC-INV and INV-ONC chimeras were subjected to the same analysis, the signal corresponding to the dimer was only present in the case of the ONC-INV chimera under nonreducing conditions (Fig. 3 A, lanes 3 and 4). These data point to the presence of one or several mutations in the extracellular part of ONC-Xmrk allowing ligand-independent covalent dimerization. To investigate whether disulfide-linked dimers were also present in ONC-Xmrk from fish melanoma cells, we analyzed PSM cells and melanoma tissue extracts on a gradient gel. The detection with an anti-mrk serum showed, in both cases, the presence of dimers under nonreducing conditions, whereas these were not present under reducing conditions (Fig. 3 B). This suggests that in vivo the same mechanism is present as studied after overexpression of ONC-Xmrk in 293 cells. To identify the extracellular mutation(s) present in ONC-Xmrk responsible for dimerization, additional chimeric constructs were made. In these constructs portions of the ONC-Xmrk extracellular domain were used to replace the corresponding regions in INV-Xmrk, thus introducing groups of mutations in the backbone of the proto-oncogene. Two new chimeras were constructed, Xmrk(G359R,P388T), which contains the two most amino-terminal mutations in the extracellular domain, and Xmrk(P470L,S476N,C578S,M595I), which contains the four last extracellular mutations of ONC-Xmrk. When these chimeras were subjected to Western blot analysis, dimer formation under nonreducing conditions was observed in both cases (Fig. 4), suggesting that more than one activating mutation exists in ONC-Xmrk that leads to ligand-independent receptor dimerization. It has been already described for RTKs (19Santoro M. Carlomagno F. Romano A. Bottaro D.P. Dathan N.A. Grieco M. Fusco A. Vecchio G. Matoskova B. Kraus M.H. Di Fiore P.P. Science. 1995; 267: 381-383Crossref PubMed Scopus (796) Google Scholar,20Neilson K.M. Friesel R. J. Biol. Chem. 1996; 271: 25049-25057Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) that when one conserved cysteine involved in intramolecular disulfide bonding is lost the remaining cysteine of the pair is able to form an intermolecular disulphide bridge. This aberrant bonding leads to the formation of receptor dimers that can be observed under nonreducing conditions. In the case of ONC-Xmrk, a cysteine in position 578 is lost and substituted by a serine. To verify whether this change was one of the mutations involved in activation, an INV-Xmrk receptor containing the Cys-to-Ser mutation was constructed. After transient expression in 293 cells, the INV(C578S) mutant was immunoprecipitated with an anti-mrk serum and detected with an anti-phosphotyrosine antibody. This analysis showed increased tyrosine phosphorylation of the mutant receptor compared with INV-Xmrk, demonstrating that the introduction of this mutation was sufficient to activate Xmrk receptor (Fig. 5 A). When the INV(C578S) receptor was analyzed on a denaturing gradient gel under nonreducing conditions, the appearance of a band was observed consistent with the size of the dimer as it appeared also for ONC-Xmrk (Fig.5 B). However, the extent of dimerization was lower than that observed for ONC-Xmrk, suggesting additional differences between the two receptors. To further limit the mutations being involved in dimer formation, a new construct was made wherein the Xmrk(P470L,S476N,C578S,M595I) chimera S578 was reverted to the wild type cysteine. The expression of the Xmrk(P470L,S476N,M595I) variant in 293 cells and subsequent analysis of the receptor protein showed that this receptor is not able to produce dimers (Fig. 5 C). This finding confirms that P470L, S476N, and M595I do not contribute to ONC-Xmrk activation. As shown by the Xmrk(G359R,P388T) chimera, there exists a second activating mutation in ONC-Xmrk that also leads to aberrant disulfide bridging and dimerization. To find out which of these two mutations was responsible for dimer formation, the Arg in position 359 was reverted to the wild type Gly. The analysis in gradient denaturing gels of 293 extracts expressing the Xmrk(P388T) mutant showed an almost complete disappearance of the dimer band, suggesting that the Arg in position 359 was the second activating mutation (Fig.6 A). To further support this, we introduced the Arg mutation in the backbone of INV-Xmrk and studied its level of phosphorylation. Cellular extracts from 293 cells transiently transfected with the INV(G359R) mutant were immunoprecipitated with an anti-mrk serum and subsequently detected with an anti-Tyr(P) antibody. The INV(G359R) mutant appeared strongly phosphorylated (Fig. 6 B), showing as in the case of the C578S mutation that covalent dimer formation correlates with increased receptor phosphorylation. Analysis of INV- and ONC-Xmrk with the same antibody on Western blots revealed that both receptors had slightly different electrophoretic mobilities in SDS denaturing gels. ONC-Xmrk always migrated as a smaller molecular weight form than INV-Xmrk (Fig.7 A). This different extent of migration was obviously associated with the activating mutations. The ONC-INV chimera showed the same mobility as ONC-Xmrk (data not shown). However, when the chimeras Xmrk(P470L,S476N,C578S,M595I) and Xmrk(G359R,P388T) containing only one of the activating mutations each were analyzed in Western blots, a mixture of the two forms characteristic of ONC- and INV-Xmrk appeared (Fig. 7 B). When both activating mutations were reverted to the proto-oncogenic residues, the resulting Xmrk(P388T) and Xmrk(P470L, S476N,M595I) proteins migrated in the SDS denaturing gels like the proto-oncogenic form of the receptor. As all these proteins contain the same number of amino acids, this could reflect a different post-translational modification. The main post-translational modification in the extracellular domain of a receptor tyrosine kinase from the family of the EGFR is the glycosylation of certain asparagine residues. However, none of the mutations affects a canonical glycosylation sequence. It has been reported that proteins that expose reactive thiol groups can be retained in the ER (21Isidoro C. Maggioni C. Demoz M. Pizzagalli A. Fra A.M. Sitia R. J. Biol. Chem. 1996; 42: 26138-26142Abstract Full Text Full Text PDF Scopus (37) Google Scholar). Because of incorrect folding, they are kept there by some kind of quality control mechanism and they are not processed further to the Golgi (22Roussel M.F. Downing J.R. Rettenmier C.W. Sherr C.J. Cell. 1988; 55: 979-988Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 23Gethring M.-J. McCammon K. Sambrook J. Cell. 1986; 46: 939-950Abstract Full Text PDF PubMed Scopus (474) Google Scholar). Glycoproteins stopped in this cellular compartment contain high mannose type oligosaccharides and are therefore sensitive to endoglycosidase H (endo H) digestion. In contrast, fully N-glycosylated receptors anchored in the membrane are endo H-resistant. In the case of ONC-Xmrk, we have demonstrated that the activating mutations lead to the formation of receptor dimers by intermolecular disulfide bridging between unpaired cysteines. To test whether these proteins containing activating mutations and thereby leaving active thiol groups were endo H-sensitive, we performed digestions on extracts corresponding to transient transfections of INV- and ONC-Xmrk in 293 cells. The ONC-Xmrk form of approximately 155 kDa was completely digested, and a band of about 135 kDa appeared (Fig. 7 A), which is consistent with the size of the unglycosylated form of ONC-Xmrk as already described (9Malitschek B. Wittbrodt J. Fischer P. Lammers R. Ullrich A. Schartl M. J. Biol. Chem. 1994; 269: 10423-10430Abstract Full Text PDF PubMed Google Scholar). However, the INV-Xmrk form of 160 kDa is barely affected by the action of endo H (Fig. 7 A). Endo H digestion of the Xmrk(G359R,P388T) and Xmrk(P470L,S476N,C578S,M595I) chimeras also showed the existence of the ONC-Xmrk endo H-sensitive form that was not present when the mutations were reverted to the wild type amino acids as shown in the Xmrk(P388T) and Xmrk(P470L,S476N,M595I) chimeras (Fig. 7 B). These results suggest that ONC-Xmrk containing both mutations is retained in the ER and is not properly transported and located in the outer cellular membrane. To elucidate the cellular localization of the different Xmrk receptors, we performed immunohistochemical analysis of stable Ba/F3 clones expressing the ONC or INV-ONC receptors. The BaF INV-ONC cells showed stronger membrane and weaker cytoplasmic anti-mrk staining, indicating a correct transport of the receptor to the plasma membrane (Fig.8). However, in BaF ONC cells, the anti-mrk signals were preferentially detected directly around the nuclear membrane, suggesting a localization in the ER and confirming that the processing of the Xmrk receptor is affected when the extracellular mutations are present. ONC-Xmrk activation results from at least two mechanisms: overexpression and mutational alteration. In the present study, we have identified the activating mutations and characterized their mode of action.