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Identification of two novel, kinase-deficient variants of discoidin domain receptor 1: differential expression in human colon cancer cell lines¹

FRAUKE ALVES^*2, STEFFEN SAUPE^*, MARCO LEDWON^*, FRANK SCHAUB^**, WOLFGANG HIDDEMANN and WOLFGANG F. VOGEL

^* Department of Hematology and Oncology,
^** Department of Cardiology and Pneumology, Georg-August-University, 37075 Göttingen, Germany;
Department of Internal Medicine III, University Hospital Grosshadern, Ludwig-Maximilians University, 81377 München, Germany; and
Department of Extracellular Matrix Signaling, Georg-Speyer-Haus Institute for Biomedical Research, Johann Wolfgang von Goethe University, 60596 Frankfurt, Germany

²Correspondence: Department of Hematology and Oncology, Georg-August-University, Robert-Koch Str. 40, 37075 Göttingen, Germany. E-mail: falves{at}gwdg.de

SPECIFIC AIMS

The tyrosine kinase receptor DDR1 is activated by various types of collagen and consists of three isoforms—DDR1a, b, and c—as a result of alternative splicing in the cytoplasmic region. To search for the presence of additional splice variants of DDR1 within the juxtamembrane region, we applied a polymerase chain reaction (PCR) -based approach in conjunction with primers localized within the transmembrane region (exon 9) and the tyrosine kinase domain (exon 13).

PRINCIPAL FINDINGS

1. Identification of two novel kinase-deficient isoforms
All seven different human colon carcinoma cell lines analyzed express DDR1a as demonstrated by an amplification product of 513 bp. All cell lines also revealed a major amplification product of 624 bp, most likely corresponding to DDR1b mRNA, except for Colo 206F, where only a faint band was detected. The detection of additional bands with sizes of 750, 600, 420, and 270 bp suggested the presence of further isoforms (Fig. 1 ). The complete PCR product of the cell line Colo 206F was further analyzed by cloning and sequencing that confirmed the presence of DDR1a and DDR1b. Compared to DDR1a, DDR1b has an insertion of 37 amino acids in the juxtamembrane region encoded by exon 11. Furthermore, two novel sequences of shorter sizes were isolated, which we named DDR1d and DDR1e. Both are produced by alternative splicing in the juxtamembrane region using different acceptor sites. The DDR1d amplification product was 268 bp long and was most abundant in the PCR of Colo 206F and SW 480 (Fig. 1) . DDR1d not only lacks the 111 bp corresponding to exon 11, but also 245 bp corresponding to exon 12. Whereas the deletion of exon 11 retains the reading frame, the deletion of exon 12 results in a frame shift mutation. By joining exon 10 with exon 13, a new reading frame is generated. Translation of this reading frame in exon 13 resulted in the amino acid sequence GAPV, followed by a stop codon (Fig. 2 ). The translation product of the DDR1d cDNA is predicted to be a membrane-anchored receptor missing the kinase and carboxyl-terminal tail region. The protein consists of 508 amino acids with a calculated molecular mass of 56 kDa. The second novel sequence, named DDR1e, was 186 bp long but was not detected as major product of the PCR reaction (Fig. 1) . Sequence comparison showed that the first half of exon 10 (exon 10a, 82 bp) is missing in addition to exons 11 and 12 (Fig. 2) . By joining exon 9 with the second half of exon 10 (exon 10b, 84 bp), a shift in the open reading frame is generated. Joining exon 10b with exon 13 reverts the sequence back into the previous reading frame. The second half of exon 10 in DDR1e translates to the new sequence VLESHPRTRSPGLVGIRPTPLPVSPMAL, followed by the unaltered sequence coded by exon 13. Upon careful examination of the 5' intron/exon boundary sequence of exon 10, it was apparent there were two possible splice acceptor sites. The use of the distal 5' acceptor site in the sequence gcc cag GTC CTA (nucleotides 1615–1627) resulted in the deletion of exon 10a and the production of a cDNA of 84 bp (exon 10b) with disruption of the open reading frame (Fig. 2) . The deletion of exon 10a, exon 11, and exon 12 resulted in an altered sequence of 28 amino acids in the juxtamembrane region and loss of the ATP binding site GEGQFG (amino acids 617–623), producing a receptor molecule lacking intrinsic tyrosine kinase activity with 767 amino acids and a predicted molecular mass of 86 kDa.

View larger version (43K): [in this window] [in a new window]   Figure 1. Expression of multiple DDR1 transcripts in colon carcinoma cell lines. PCR amplification products of the juxtamembrane region of DDR1 from various human colon carcinoma cell lines and reaction products with plasmids coding for DDR1a, DDR1b, and DDR1d as templates and without template (control) were analyzed by agarose gel electrophoresis. The sizes of the PCR products of DDR1b (624 bp), DDR1a (513 bp), and DDR1d (268 bp) and of the DNA marker are indicated.

View larger version (45K): [in this window] [in a new window]   Figure 2. Schematic diagram of the different isoforms of DDR1. The upper panel shows the organization of the genomic locus of DDR1 between exon 9 and 14. The arrows indicate the approximate position of the primer pair used for PCR amplification. The various splicing events that result in the previously known isoforms DDR1a, DDR1b, and DDR1c as well in the novel isoforms DDR1d and DDR1e are depicted underneath the genomic locus. In the lower panel, the predicted length, molecular weight, and overall structure of the different DDR1 isoforms are shown.

2. RNA expression of DDR1 isoforms in human colon carcinoma cell lines
Northern blot analysis using a cDNA fragment coding for the extracellular domain as a probe, which is common in all isoforms, revealed a 4.3 kb DDR1 mRNA signal in both cell lines used, Colo 206F and SW 480 (data not shown). With a cDNA probe corresponding to exon 12, no signal was seen in Colo 206F but was observed in SW 480. This correlates with the mRNA content detected by PCR amplification, where Colo 206F cells had high amounts of DDR1d but very little product for DDR1b and reduced mRNA amplification of DDR1a (Fig. 1) . In contrast, SW 480 cells showed high amounts of DDR1a and DDR1b as well as DDR1d, which are reflected by the mRNA expression signals seen with the two different probes. The presence of mRNA for both isoforms was confirmed by nucleic acid sequence-based amplification (NASBA) analysis.

3. Expression of DDR1 isoforms in human colon carcinoma cell lines
The existence of DDR1d and DDR1e in vivo is supported by Western blot analysis (Fig. 3A ). A 68 kDa protein representing the glycosylated DDR1d receptor was seen only when an antibody against the extracellular domain was used ( DDR1 N), but not when the carboxyl-terminal antibody was applied ( DDR1 C). A protein of the apparent molecular mass of 95 kDa was seen when applying DDR1 N and DDR1 C, representing most likely DDR1e (see Fig. 3A , B , e.g.). Most of the colon carcinoma cell lines tested coexpress the kinase-deficient isoform DDR1d, together with kinase active DDR1a/b, with an apparent molecular mass of 125 kDa in comparable amounts. Furthermore, we performed an immunoprecipitation with a monoclonal antibody raised against the extracellular domain of DDR1, followed by Western blot analysis. As shown in Fig. 3C , no expression of DDR1a and DDR1b could be observed in Colo 206F, whereas in Colo 678 and HCT 116, a prominent band of 125 kDa was detected with both antibodies DDR1 N and DDR1 C. The ß-subunit of the receptor could not be detected by the DDR1 C, because the cell lysates were precipitated with an amino-terminal antibody. In all cell lines, most prominently in Colo 206F cells, a band with an apparent molecular mass of 68 kDa was detected by DDR1 N (Fig. 3C , right panel) that represents the glycosylated DDR1d receptor. In Colo 206F and HCT 116, a protein of 95 kDa was detected after immunoblotting with both antibodies, most likely corresponding to DDR1e. In Colo 678, a notable larger band with 98 kDa was seen with the DDR1 N, but not with the DDR1 C, indicating there is potentially an even higher diversity of DDR1 isoforms in this cell line. The absence of DDR1e in Colo 678 cells is supported by the fact that we failed to detect DDR1e transcripts by NASBA (data not shown).

View larger version (52K): [in this window] [in a new window]   Figure 3. Expression of DDR1 protein in colon carcinoma cell lines. A) Cell lysates from different human colon carcinoma cell lines were analyzed by Western blotting with an affinity-purified polyclonal antibody against the amino terminus of DDR1 ( DDR1 N). B) The blot was reprobed with an affinity-purified polyclonal antibody against the carboxyl terminus of DDR1 ( DDR1 C). Arrows indicate DDR1a/b, DDR1e and the proteolytically processed ß -subunit that is detected by DDR1 C. C) Cell lysates were immunoprecipitated with a monoclonal antibody against the extracellular domain ( DDR1). Western blotting was performed with DDR1 C (left panel) and subsequently with DDR1 N (right panel).

4. Evidence for a nondominant negative mechanism of DDR1d signal modulation
Since Colo 206F cells showed a substantial amount of DDR1d protein expression in comparison to the full-length receptors DDR1a and DDR1b, we studied whether the kinase-deficient isoform DDR1d functions as dominant negative receptor. We could show by immunoprecipitation of DDR1b from cells stimulated with rat type I collagen for 3 h that tyrosine phosphorylation of DDR1b took place in response to collagen in cells expressing high amounts of DDR1d (data not shown). To further understand the role of the DDR1d isoform during kinase activation, a full-length DDR1d cDNA was constructed by a PCR-based approach and transiently overexpressed in human embryonic kidney fibroblasts 293 cells. Compared with DDR1a-transfected cells, a prominent band of 68 kDa was detected in DDR1d-transfected cells only with the DDR1 N antibody (data not shown). To study signaling, we examined tyrosine phosphorylation in response to rat type I collagen. In cells overexpressing DDR1d, no tyrosine phosphorylation was observed in response to collagen. Cotransfection of DDR1a together with increasing amounts of DDR1d receptor showed that receptor phosphorylation in response to rat collagen was not efficiently blocked in the presence of the tyrosine kinase-deficient DDR1d (data not shown).

CONCLUSIONS

Two novel isoforms of DDR1—DDR1d and DDR1e—were identified from human colon carcinoma Colo 206F cells. Both new isoforms are predicted to be membrane anchored but kinase-deficient receptors. The alternative splicing event takes place in the juxtamembrane region, which contains sequence motives essential for the interaction with cellular substrates and regulatory proteins. Through a frameshift in exon 13, DDR1d displays the sequence motif GAPV at the carboxyl terminus. This motif can function as a potential binding site for the growing family of proteins containing one or several PDZ (PSD-95/DLG/ZO-1) domains. Based on their structure, receptors with mutated or deleted kinase domain have been proposed to act as suppressors of full-length, enzymatic active receptors by forming heterodimers and blocking signaling in a dominant negative manner. Two lines of evidence suggest that at least DDR1d behaves differently: 1) in Colo 206F, DDR1b is tyrosine phosphorylated upon collagen stimulation despite strong expression of DDR1d, and 2) in transfected 293 cells, a fourfold excess of DDR1d did not efficiently block DDR1 tyrosine phosphorylation in response to collagen. Therefore, our study argues against a role of DDR1d as dominant negative inhibitor of DDR1 signaling. If DDR1d (and presumably DDR1e as well) does not influence collagen-mediated DDR1 signaling, other roles of the truncated receptors might be postulated in analogy to the kinase-deficient TrkB and TrkC receptors such as a role in cell adhesion, having its own signaling abilities, or sequestering and presenting collagen as ligand to the DDR1 full-length receptor. The challenge for future work will be to define the mechanisms that regulate the expression pattern of the novel DDR1 isoforms—for example, during embryogenesis and tumor progression—and to analyze the function and signaling events of DDR1 isoforms by overexpression of different isoforms in different cellular environment.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0626fje ; to cite this article, use FASEB J. (March 28, 2001) 10.1096/fj.00-0626fje

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This Article Full Text (PDF) All Versions of this Article: 15/7/1321 00-0626fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ALVES, F. Articles by VOGEL, W. F. Search for Related Content PubMed PubMed Citation Articles by ALVES, F. Articles by VOGEL, W. F.