Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers
The discoidin domain receptor ( DDR ) subfamily of receptor tyrosine kinases ( RTKs ) comprises two members , DDR1 and DDR2 . The DDRs regulate cell adhesion , cell migration and differentiation in a number of mammalian tissues ( Leitinger , 2014 ) . Both DDRs play key roles in embryo development: DDR1 , for instance , is essential for mammary gland development ( Vogel et al . , 2001 ) , while DDR2 mediates bone growth ( Ali et al . , 2010; Bargal et al . , 2009; Labrador et al . , 2001 ) . The DDRs also play key roles in disease progression in a wide range of disorders including organ fibrosis , inflammation , osteoarthritis , atherosclerosis and many different types of cancer ( Borza and Pozzi , 2014; Leitinger , 2014 ) . Both DDRs are well-recognised drug targets but how ligand binding translates to DDR kinase activation has been poorly defined . Uniquely among RTKs , the DDRs bind to key structural proteins found in all types of extracellular matrices , namely different types of collagen . Fibrillar collagens are ligands for both DDR1 and DDR2 , while non-fibrillar collagens have different DDR preferences , with collagen IV exclusively binding to DDR1 and collagen X preferring DDR2 over DDR1 ( Leitinger , 2003; Leitinger and Kwan , 2006; Shrivastava et al . , 1997; Vogel et al . , 1997 ) . The interactions of the DDRs with fibrillar collagens are well understood: receptor binding sites have been mapped to specific amino acid motifs with the use of collagen-mimetic triple-helical peptides ( Konitsiotis et al . , 2008; Xu et al . , 2011 ) , and structural studies have revealed the details of the interactions ( Carafoli et al . , 2009; Ichikawa et al . , 2007 ) . In contrast , the nature of DDR binding sites on non-fibrillar collagens is currently not known . Like all RTKs , the DDRs are composed of a ligand-binding extracellular region , a transmembrane domain and a cytoplasmic region that contains the catalytic kinase domain . DDR1 and DDR2 share a high degree of homology , in particular in their globular domains . The extracellular DDR region contains two globular domains: an N-terminal , ligand-binding discoidin domain that is tightly linked to a discoidin-like domain ( Leitinger , 2014 ) . These globular domains are followed by a highly flexible extracellular juxtamembrane region rich in glycine and proline residues ( Xu et al . , 2014 ) . The intracellular DDR regions contain unusually large juxtamembrane regions ( up to 171 amino acids in DDR1 , depending on the isoform , and 142 amino acids in DDR2 ) that are followed by a catalytic kinase domain lacking a C-terminal tail ( Leitinger , 2014 ) . The collagen-binding site of DDRs is entirely contained in the discoidin domain ( Ichikawa et al . , 2007; Leitinger , 2003 ) , which forms an 8-stranded β-barrel structure ( Carafoli et al . , 2009 , 2012; Ichikawa et al . , 2007 ) . A conserved trench at the top of the discoidin domain accommodates the collagen triple helix . The structures of the collagen-bound DDR2 discoidin domain ( Carafoli et al . , 2009 ) and that of the DDR1 discoidin domain in the absence of ligand ( Carafoli et al . , 2012 ) are very similar and do not provide any information on how ligand binding results in intracellular kinase activation . This is in sharp contrast to the situation of collagen-binding integrins where large conformational changes in the ligand-binding domain are transmitted to the rest of the receptor to drive transmembrane signalling ( Luo et al . , 2007 ) . A crystal structure of the inactive DDR1 kinase revealed a typical bilobal architecture , with kinase autoinhibition through interactions of the activation loop with the active site ( Canning et al . , 2014 ) . The DDR cytoplasmic regions contain several sites of potential tyrosine phosphorylation , both in the juxtamembrane regions and in the kinase domains ( Leitinger , 2014; Lemeer et al . , 2012 ) . While ligand-induced activation loop phosphorylation of the kinase domain has been demonstrated experimentally ( Iwai et al . , 2013; Lemeer et al . , 2012; Xu et al . , 2014 ) , only a few juxtamembrane tyrosines have been shown to be phosphorylated in response to collagen binding to cells ( Iwai et al . , 2013; Lemeer et al . , 2012 ) . Moreover , little is known about signalling pathways that are initiated by the phosphorylation of particular DDR tyrosine residues ( Leitinger , 2014 ) . The DDRs differ from typical RTKs by several unusual features , the mechanistic bases of which are poorly understood . One of their puzzling characteristics is the slow kinetics of collagen-induced autophosphorylation , which in some cell types can take hours to reach a maximum and then remains detectable for more than a day ( Shrivastava et al . , 1997; Vogel et al . , 1997 ) . Another distinguishing feature is that the DDRs form stable , non-covalent dimers in the absence of ligand ( Mihai et al . , 2009; Noordeen et al . , 2006; Xu et al . , 2014 ) . A key dimerisation interface is located in the DDR transmembrane domain via a leucine-based sequence motif; however , multiple receptor-receptor contacts in the extracellular and cytosolic regions also seem to contribute to DDR dimerisation ( Noordeen et al . , 2006 ) . The isolated DDR1 and DDR2 transmembrane helices interact very strongly , as detected in a bacterial TOXCAT reporter assay ( Noordeen et al . , 2006 ) . In fact , a systematic study that compared the self-interaction potential of all RTK transmembrane domains found that the DDR1 and DDR2 transmembrane domains gave the strongest signal of all RTKs in this assay ( Finger et al . , 2009 ) . In previous work , we performed cysteine-scanning mutagenesis of the 10 extracellular juxtamembrane residues closest to the DDR1 transmembrane domain and found that most of the mutant constructs were crosslinked with nearly 100% efficiency , which led us to conclude that DDR1 dimerisation is constitutive , rather than in a dynamic equilibrium with monomers ( Xu et al . , 2014 ) . We further concluded that dimerisation occurs during biosynthesis ( Noordeen et al . , 2006; Xu et al . , 2014 ) . While ligand-independent non-covalent dimerisation has been observed for other RTKs , including the EGF receptor family , these RTKs exist in a dynamic equilibrium between monomers and inactive dimers ( discussed in [Maruyama , 2014] ) . Because the DDRs are constitutively dimerised in the absence of ligand , the paradigm of ligand-induced RTK dimerisation ( Lemmon and Schlessinger , 2010 ) cannot apply to them . The insulin receptor is a covalently-linked dimer that is activated by ligand-induced conformational changes within the dimer ( Kavran et al . , 2014; Ward et al . , 2013 ) . However , structural studies found no evidence for ligand-induced conformational changes in the DDR ectodomains ( see above; [Carafoli et al . , 2009 , 2012; Ichikawa et al . , 2007] ) , and biochemical experiments led to the conclusion that transmembrane signalling of DDR1 occurs without coupling through the flexible juxtamembrane region ( Xu et al . , 2014 ) . Together , these studies ruled out a mechanism of signalling within DDR1 dimers and led to the hypothesis that DDR1 activation may be associated with ligand-induced clustering ( lateral association ) of receptors in the cell membrane ( Xu et al . , 2014 ) . Here we show evidence for a mechanism that is compatible with ligand-induced clustering of DDR1 dimers . Co-expression studies using a range of signalling-incompetent DDR1 mutants with different forms of signalling-competent DDR1 and DDR2 showed that collagen-induced DDR1 activation involves phosphorylation in trans between neighbouring DDR1 dimers . Phosphorylation between DDR1 dimers occurs both on the juxtamembrane region and the kinase activation loop and can be elicited by different types of ligand . Moreover , phosphorylation in trans requires specific contacts between the transmembrane domains , but not between the ectodomains . These findings define a unique mechanism of transmembrane signalling by DDR1 .
In this study we explored the mechanism of ligand-induced phosphorylation of DDR1 by co-expression of signalling-defective DDR1 mutants with signalling-competent DDR1 . Receptor phosphorylation was visualised by Western blotting with a phospho-specific antibody ( Ab ) against a key tyrosine phosphorylation site in the intracellular juxtamembrane region , tyrosine-513 ( Xu et al . , 2014 ) . DDR1 has two main isoforms , DDR1a and DDR1b , with DDR1b containing an additional 37 amino acids in its cytosolic juxtamembrane region ( Leitinger , 2014 ) . Tyrosine-513 within this alternatively spliced insert is one of the few experimentally verified ligand-induced tyrosine phosphorylation sites in the juxtamembrane region ( Lemeer et al . , 2012 ) . The signalling-defective DDR1 mutants had mutations in the ectodomains but intact cytosolic juxtamembrane regions and kinase domains . As detailed below , co-expression with signalling-competent DDR1 resulted in ligand-induced phosphorylation of the signalling-defective mutants , which on their own could not be phosphorylated in response to collagen . Hence , in our co-expression experiments , we use the terms ‘donor kinase’ for the signalling-competent DDR1 and ‘receiver kinase’ for the signalling-defective mutants , respectively . In order to distinguish the cytoplasmic domains of receiver and donor DDR1 we replaced tyrosine-513 in the donor receptor with a phenylalanine , thereby creating DDR1b-Y513F , which cannot be phosphorylated at this residue ( Figure 1A ) . This allowed us to monitor exclusively the phosphorylation of receiver DDR1 by Western blotting with the Ab against phosphorylated tyrosine-513 . 10 . 7554/eLife . 25716 . 003Figure 1 . Co-expression of DDR1 donor kinase with signalling-incompetent receiver DDR1 mutants leads to collagen-induced phosphorylation of receiver DDR1 . ( A ) Schematic diagrams of wild-type and mutant DDR1b . The extracellular region consists of the ligand-binding discoidin ( DS ) domain , the discoidin-like ( DS-like ) domain and a flexible juxtamembrane ( JM ) region . The transmembrane ( TM ) domain contains a dimerising motif . The cytoplasmic region contains a large unstructured JM region , followed by the catalytic kinase domain . Collagen binding induces phosphorylation of DDR1b on cytoplasmic tyrosine residues; Y513 in the JM region and the three activation loop tyrosines are shown in phosphorylated form for WT DDR1b as yellow circles . DDR1b-Y513F is phosphorylated on the activation loop but cannot be phosphorylated on Y513 . Ligand-binding defective DDR1b-W53A and signalling-defective DDR1b-R32E or DDR1b-L152E are DDR1 mutants that are not phosphorylated upon collagen incubation . ( B–D ) Wild-type or mutant DDR1b constructs were transiently expressed in HEK293 cells , either alone , or co-expressed as indicated . Cells were stimulated with collagen I or collagen IV for 90 min at 37°C . Aliquots of cell lysates were analysed by reducing SDS-PAGE and Western blotting . The blots were probed with phospho-specific Abs , as indicated , and re-probed with anti-DDR1 . * , non-specific band . ( C , D ) Co-expression was performed with different amounts of DDR1b-Y513F expression vector , with the higher amount denoted by ( 2 ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 003 We first confirmed expression and collagen-induced activation of DDR1b-Y513F in transiently transfected HEK293 cells . Collagen-induced autophosphorylation of the activation loop ( anti-pY792 ) was detected at similar levels as in wild-type DDR1b , whereas , as expected , no signal was detectable for DDR1b-Y513F with the Ab against phosphorylated tyrosine-513 ( anti-pY513; Figure 1B ) . Mutation of a key ligand-binding residue in DDR2 , tryptophan-52 ( conserved in DDR1 where it corresponds to tryptophan-53 ) , severely reduces collagen binding affinity ( Carafoli et al . , 2009; Ichikawa et al . , 2007 ) and renders the full-length receptor unable to become activated by collagen ( Carafoli et al . , 2009 ) . We created DDR1b-W53A as a ligand binding-defective DDR1 construct , which , on its own , does not respond to collagen with increased autophosphorylation . Co-expression with DDR1b-Y513F , however , led to collagen I-induced phosphorylation of tyrosine-513 in DDR1-W53A ( Figure 1C ) . In a previous study we identified a conserved patch on the surface of DDR1 that is required for transmembrane signalling seemingly without participating in ligand binding ( Carafoli et al . , 2012 ) . Co-expression of the signal-patch mutants , DDR1b-R32E or DDR1b-L152E , with DDR1b-Y513F restored collagen-induced phosphorylation of tyrosine-513 in the signal-patch mutants ( Figure 1C ) . Rescue of DDR1b-W53A phosphorylation through co-expression with DDR1b-Y513F was not restricted to activation by fibrillar collagen I , but could also be induced by binding to the network-forming collagen IV ( Figure 1D ) . Phosphorylation of signalling-defective receiver DDR1 mutants in the co-expression system could arise through transphosphorylation within heterodimers of receiver and donor receptors and/or phosphorylation between dimers in trans . To investigate whether the receiver mutants were phosphorylated as a dimer , we created covalently-linked homodimers of the DDR1 receiver mutants . Cysteine replacement of threonine-416 , a residue in the extracellular DDR1 juxtamembrane region close to the transmembrane domain , results in covalently cross-linked DDR1 dimers via a disulphide bond at residue 416 , without affecting collagen-induced receptor activation ( Xu et al . , 2014 ) . Non-reducing SDS-PAGE followed by Western blotting of cell lysates from cells expressing DDR1b-W53A/T416C , DDR1b-R32E/T416C or DDR1b-L152E/T416C ( referred to as DDR1b-W53A-Cys , DDR1b-R32E-Cys and DDR1b-L152E-Cys from here on ) , showed that the mature forms of the double mutants were efficiently dimerised , as expected ( Figure 2A and B , anti-DDR1 blot , lanes 7 and 8 ) . Co-expression of any of the dimerised signalling-defective receiver mutants with DDR1b-Y513F resulted in collagen-induced phosphorylation at the position of the disulphide-linked dimers ( Figure 2 , see also Figure 2—figure supplement 1 ) , demonstrating conclusively that collagen induces phosphorylation in trans between DDR1 dimers . 10 . 7554/eLife . 25716 . 004Figure 2 . Co-expression with DDR1 donor kinase results in ligand-induced phosphorylation of receiver DDR1 dimers . ( A , B ) Ligand-binding defective ( W53A or W53A-Cys ) or signalling-defective ( R32E or R32E-Cys ) DDR1b constructs were transiently expressed in HEK293 cells , either alone or co-expressed with DDR1b-Y513F , as indicated . Cells were stimulated with collagen I ( Coll ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blots were probed with phospho-specific anti-pY513 ( upper blots ) , and re-probed with anti-DDR1 ( lower blots ) . Co-expression was performed with different amounts of DDR1b-Y513F expression vector , with the higher amount denoted by ( 2 ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric DDR1 and of mature monomeric DDR1 are indicated on the right . ( C ) Schematic diagram showing that co-expression of dimeric receiver DDR1b ( W53A-Cys or R32E-Cys ) with donor DDR1b-Y513F leads to collagen-induced Y513 phosphorylation of receiver dimers . The blue shape denotes anti-pY513 used for Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 00410 . 7554/eLife . 25716 . 005Figure 2—figure supplement 1 . Co-expression with DDR1 donor kinase results in ligand-induced phosphorylation of DDR1-L152E dimers . ( A , B ) Signalling defective ( L152E or L152E-Cys ) DDR1b was transiently expressed in HEK293 cells , either alone or co-expressed with DDR1b-Y513F , as indicated . The cells were stimulated with collagen I ( Coll ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blot was probed with phospho-specific anti-pY513 ( upper blot ) , and re-probed with anti-DDR1 ( lower blot ) . Co-expression was performed with different amounts of DDR1b-Y513F expression vector , with the higher amount denoted by ( 2 ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric DDR1 and of mature monomeric DDR1 are indicated on the right . ( C ) Schematic diagram showing that co-expression of dimeric receiver DDR1b-L152E-Cys with donor DDR1b-Y513F leads to collagen-induced Y513 phosphorylation of DDR1b-L152E-Cys dimers . The blue shape denotes anti-pY513 used for Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 005 We next asked whether extracellular contacts were required for signalling between DDR1 dimers . Deletion of the entire DDR1 ectodomain ( except for the seven amino acids closest to the transmembrane domain ) did not impair the ability of DDR1b-Y513F to promote collagen-induced phosphorylation of the ectodomain deletion mutant ( Figure 3A , left hand side ) . Cysteine replacement of T416 ( full-length DDR1b numbering ) in the deletion mutant resulted in a construct that was predominantly dimeric , but in contrast to the full-length dimerised constructs , a proportion of monomeric protein was also detectable ( Figure 3A , bottom blot , lanes 7 and 8 ) . Interestingly , co-expression with DDR1b-Y513F led to collagen-induced phosphorylation only of the truncated dimer , with very little phosphorylated monomer detectable ( Figure 3A , top right blot ) . 10 . 7554/eLife . 25716 . 006Figure 3 . Signalling between DDR1 dimers is independent of ectodomain contacts . ( A ) Ectodomain-deletion constructs ( DDR1b-ΔECD or DDR1b-ΔECD-Cys ) were transiently expressed in HEK293 cells , either alone or co-expressed with DDR1b-Y513F , as indicated . The cells were stimulated with collagen I ( Coll ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blots were probed with phospho-specific anti-pY513 ( upper blots ) , and re-probed with anti-DDR1 ( lower blots ) . Co-expression was performed with different amounts of DDR1b-Y513F expression vector , with the higher amount denoted by ( 2 ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric DDR1b-ΔECD-Cys , DDR1b-Y513F and mature monomeric DDR1b-ΔECD are indicated on the right . ( B ) Schematic diagram showing that co-expression of ectodomain-deleted dimeric DDR1b with donor DDR1b-Y513F leads to collagen-induced Y513 phosphorylation of ectodomain-deletion dimers . The blue shape denotes anti-pY513 used for Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 00610 . 7554/eLife . 25716 . 007Figure 3—figure supplement 1 . Cell surface and total protein DDR1 expression in DDR1-overexpressing HEK293 cells , primary keratinocytes and cell lines . ( A ) . HEK293 cells transiently transfected with the indicated amounts of DDR1b cDNA ( in total volume of 400 μl ) , primary dermal keratinocytes ( KER ) , MCF-7 and T47D cells were stained on ice with mouse anti-DDR1 mAb 7A9 , followed by anti-mouse FITC conjugated secondary Ab and flow cytometry . Filled gray histograms , secondary Ab only; open histograms , anti-DDR1 . ( B ) Quantification of mean fluorescence intensity ( MFI ) , expressed relative to mock secondary Ab only ( n = 3 ) . ( C ) HEK293 cells transiently transfected with the indicated amounts of DDR1b cDNA ( in μg ) , primary keratinocytes ( KER ) , MCF-7 and T47D cells were lysed and aliquots of cell lysates were analysed by reducing SDS-PAGE followed by Western blotting . The blot was probed with anti-DDR1 ( upper blot ) and anti-tubulin ( lower blot ) Abs . The positions of molecular mass markers are indicated on the left ( in KDa ) . ( D ) Quantitation of DDR1 signals , normalised to respective tubulin signals , expressed as relative band intensity with respect to the lowest signals on the blots ( HEK293- Mock; n = 3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 00710 . 7554/eLife . 25716 . 008Figure 3—figure supplement 2 . Co-expression with DDR1 donor kinase results in ligand-induced phosphorylation of receiver DDR1 dimers even at low expression levels . ( A , B ) . Signalling-defective ( R32E-Cys ) or ectodomain-deletion ( ΔECD-Cys ) DDR1b constructs were transiently co-expressed in HEK293 cells with DDR1b-Y513F . Co-expression was performed with different amounts of R32E-Cys and DDR1b-Y513F expression vectors , as indicated . Numbers refer to μg of DNA used ( 1:1 ratio of expression plasmids ) . Note that cells were transfected in total volume of 100 μl . Cells were stimulated with collagen I ( Coll ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blots were probed with phospho-specific anti-pY513 ( upper blots and middle blot in A ) , and the signals were detected with Amersham ECL Select Western blotting detection reagent and X-ray film . The middle blot in A shows a longer exposure . The blots were re-probed with anti-DDR1 ( lower blots ) , followed by detection on a Typhoon FLA 9500 imager . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric DDR1 and of mature monomeric DDR1 are indicated on the right . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 008 Because our experimental approach relies on co-expression of mutant DDR1 constructs , our experiments can only be done under transfection conditions . A potential concern is that over-expression may lead to artificial DDR1 aggregates , which are responsible for phosphorylation between dimers . Flow cytometry of DDR1 showed that under standard transfection conditions used for co-expression experiments , mean DDR1 fluorescence levels were similar to those on the breast cancer cell line T47D but approximately three times higher than those on primary keratinocytes ( Figure 3—figure supplement 1; HEK 5 μg DNA vs T47D ) . To exclude that phosphorylation between dimers is dependent on high DDR1 expression levels , we performed co-expression experiments with lower DDR1 expression levels and used the most sensitive chemiluminescence substrate for detection of tyrosine-513 phosphorylation . Ligand-induced dimer phosphorylation was detected for both DDR1-R32-Cys and DDR1-ΔECD-Cys at a range of expression levels including those that would lead to surface levels comparable to those on keratinocytes ( Figure 3—figure supplement 2 ) . In a previous study we identified a leucine-based sequence motif in the DDR1 transmembrane domain that is required for strong transmembrane helix association , as well as for collagen-induced receptor activation ( Noordeen et al . , 2006 ) . In order to analyse whether transmembrane helix interactions play a role in signalling between DDR1 dimers , we introduced the inactivating transmembrane mutation ( L430G/L431P , termed TM1 ) into the ectodomain deletion constructs . Surface immunostaining and flow cytometry confirmed that the TM1 mutants were present on the cell surface at comparable levels with the respective constructs with wild-type transmembrane regions ( Figure 4—figure supplement 1 ) , demonstrating that the transmembrane mutations did not affect protein trafficking . However , these constructs could no longer be phosphorylated through co-expression with DDR1b-Y513F , despite the presence of cysteine-linked dimers in the ΔECD-Cys-TM1 construct ( Figure 4 ) . These results indicate that specific interactions through the DDR1 transmembrane domain are required for phosphorylation between dimers . 10 . 7554/eLife . 25716 . 009Figure 4 . Signalling between DDR1 dimers is dependent on transmembrane domain contacts . ( A ) Ectodomain-deletion constructs ( DDR1b-ΔECD , DDR1b-ΔECD-TM1 , DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1 ) were transiently expressed in HEK293 cells , either alone or co-expressed with DDR1b-Y513F , as indicated . The cells were stimulated with collagen I ( Coll ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blots were probed with phospho-specific anti-pY513 ( upper blots ) , and re-probed with anti-DDR1 ( lower blots ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric DDR1b-ΔECD-Cys ( D ) , of DDR1b-Y513F ( Y ) and of mature monomeric DDR1b-ΔECD ( M ) are indicated on the right . ( B ) Quantitation of receiver DDR1 pY513 signals ( co-expressed with Y513F ) , normalised to respective DDR1 signals , expressed as relative band intensity with respect to the lowest signals on the blots ( unstimulated DDR1b-ΔECD-TM1 or unstimulated DDR1b-ΔECD-Cys-TM1 ) . US , unstimulated; Coll , stimulation with collagen I . *p<0 . 05; n = 4 . ( C ) Schematic diagram showing that co-expression of ectodomain-deleted dimeric DDR1b with a transmembrane domain mutation and donor DDR1b-Y513F does not result in collagen-induced Y513 phosphorylation of the deletion construct . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 00910 . 7554/eLife . 25716 . 010Figure 4—figure supplement 1 . Cell surface expression of ectodomain-deletion constructs . ( A ) N-terminally Flag-tagged ectodomain-deletion constructs ( DDR1b-ΔECD , DDR1b-ΔECD-TM1 , DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1 ) were transiently expressed in Cos-7 cells . Cells were incubated on ice with mouse anti-Flag Ab , before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab . Cells were imaged using a widefield microscope . Scale bars , 20 µm . ( B ) N-terminally Flag-tagged ectodomain-deletion constructs ( DDR1b-ΔECD , DDR1b-ΔECD-TM1 , DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1 ) were transiently expressed in HEK293 cells . Cells were stained on ice with rabbit anti-Flag Ab , followed by anti-rabbit Alexa-Fluor-488 conjugated secondary Ab and flow cytometry . Filled gray histograms , secondary Ab only; open histograms , anti-Flag . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 010 We next explored whether signalling between dimers required the co-expressed DDR1 constructs to have identical juxtamembrane or kinase domains . Co-expression of receiver DDR1b constructs with DDR1a ( which lacks the 37 amino acid segment that contains tyrosine-513 ) resulted in efficient collagen-induced tyrosine-513 phosphorylation of the signalling-incompetent receiver dimers ( Figure 5 ) . A more drastic test was to replace the donor kinase with the homologous DDR2 . The kinase domains of the DDRs are highly conserved ( ~67% identity ) , but the intracellular juxtamembrane regions are much less conserved ( ~30% identity between DDR1b and DDR2 ) . Co-expression with DDR2 as the donor kinase similarly led to phosphorylation of the DDR1b receiver mutants ( Figure 5 , see also Figure 5—figure supplement 1 ) . Therefore , phosphorylation between DDR dimers in trans does not have a strict requirement for a particular juxtamembrane sequence . 10 . 7554/eLife . 25716 . 011Figure 5 . Signalling between dimers does not require identical cytoplasmic regions . ( A , B ) Signalling-defective ( DDR1b-R32E-Cys ) or ectodomain-deletion ( DDR1b-ΔECD-Cys ) constructs were transiently expressed in HEK293 cells , either alone or co-expressed with DDR1a , DDR1b-Y513F or DDR2 , as indicated . The cells were stimulated with collagen I ( Coll ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blots were probed with phospho-specific anti-pY513 ( upper blots ) , and re-probed with anti-DDR1 ( lower blots ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric ( D ) or mature monomeric ( M ) DDR1 receiver constructs , as well as of DDR1a or DDR1b-Y513F donor DDR1 are indicated on the right . ( C ) Schematic diagram showing that co-expression of signalling-incompetent dimeric receiver DDR1 constructs with DDR1a ( which lacks the region encompassing Y513 ) or DDR2 as the donor kinases results in collagen-induced Y513 phosphorylation of the receiver dimers . The blue shape denotes anti-pY513 used for Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 01110 . 7554/eLife . 25716 . 012Figure 5—figure supplement 1 . Co-expression of signalling-incompetent receiver DDR1 mutants with DDR2 leads to collagen-induced phosphorylation of receiver mutants . Ligand-binding defective ( DDR1b-W53A ) or signalling-defective ( DDR1b-R32E , DDR1b-L152E ) mutant DDR1b constructs were transiently expressed in HEK293 cells , either alone , or co-expressed with DDR2 , as indicated . The cells were stimulated with collagen I ( Coll ) for 90 min at 37°C , or left untreated . Aliquots of cell lysates were analysed by reducing SDS-PAGE and Western blotting . The blots were probed with phospho-specific anti-pY513 ( upper blots ) , and re-probed with anti-DDR1 ( lower blots ) . Co-expression was performed with different amounts of DDR2 expression vector , with the higher amount denoted by ( 2 ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 012 So far we monitored activation of DDR1 mutant receiver dimers by tyrosine-513 phosphorylation . Blotting with phospho-specific Abs against the activation loop tyrosines , tyrosine-792 or tyrosine-796 , showed that DDR1 receiver dimers were also phosphorylated on the activation loop ( Figure 6A , B , see also Figure 6—figure supplement 1 ) , indicating that co-expression with functional DDR1 donor kinase results in enhanced kinase activity of the receiver kinase . We therefore analysed whether autocatalytic kinase activity of the receiver kinase was required for tyrosine-513 or activation loop phosphorylation when co-expressed with donor DDR1 . Mutation of lysine-655 ( DDR1b numbering ) in the catalytic domain of DDR1 abolishes collagen-induced DDR1 phosphorylation ( Vogel et al . , 2000 ) . We co-expressed DDR1b-K655A ( hereafter referred to as DDR1b-KD ) , with or without the T416C mutation , with functional donor DDR1 and observed efficient ligand-induced tyrosine-513 phosphorylation of both kinase-dead receiver constructs ( Figure 6C ) . Furthermore , collagen-induced activation loop phosphorylation of dimeric DDR1-Cys-KD was also observed when co-expressed with functional donor DDR1 ( Figure 6—figure supplement 2 ) , demonstrating receiver activation loop phosphorylation in the absence of receiver kinase activity . These data show that phosphorylation between dimers can occur independently of catalytic activity of the receiver kinase and suggest that the receiver kinase dimer functions as a substrate of the functional donor receptor ( DDR1a , DDR1-Y513F or DDR2 ) , which acts as an enzyme kinase in our co-expression experiments . 10 . 7554/eLife . 25716 . 013Figure 6 . Signalling between dimers leads to activation loop phosphorylation of the receiver kinase but does not require receiver kinase activity . ( A , B ) Signalling-defective ( DDR1b-R32E-Cys ) or ectodomain-deletion ( DDR1b-ΔECD-Cys ) constructs were transiently expressed in HEK293 cells , either alone or in combination with DDR1a or DDR1b-Y513F , as indicated . ( C ) Kinase-inactive DDR1 constructs ( DDR1b-KD or DDR1b-Cys-KD ) were expressed in HEK293 cells , either alone or in combination with DDR1b-Y513F , as indicated . The cells were stimulated with collagen I ( Coll ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blots were probed with phospho-specific Abs against activation loop tyrosine-792 or tyrosine-796 ( A , B ) or juxtamembrane tyrosine-513 ( C ) , as indicated ( upper blots ) , and re-probed with anti-DDR1 ( lower blots ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric ( D ) DDR1 receiver constructs , as well as DDR1a or DDR1b-Y513F donor DDR1 are indicated on the right . Note that in ( A ) , the latter position includes heterodimers between DDR1b-R32E-Cys and DDR1a/DDR1-Y513F . ( D ) Schematic diagram showing collagen-induced activation loop ( pY792 ) phosphorylation of DDR1b-R32E-Cys or DDR1b-ΔECD-Cys receiver constructs when co-expressed with donor DDR1 , as well as Y513 phosphorylation of DDR1b-Cys-KD when co-expressed with DDR1b-Y513F . The blue shape denotes anti-pY792 or anti-pY513 used for Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 01310 . 7554/eLife . 25716 . 014Figure 6—figure supplement 1 . Signalling between dimers leads to activation loop phosphorylation of the receiver kinase . Signalling-defective DDR1b-R32E-Cys was transiently expressed in HEK293 cells , either alone or in combination with DDR1a or DDR1b-Y513F , as indicated . The cells were stimulated with collagen I ( Coll ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blot was probed with a phospho-specific Ab against activation loop tyrosine-796 ( upper blot ) , and re-probed with anti-DDR1 ( lower blot ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric ( D ) DDR1 receiver constructs , as well as DDR1a or DDR1b-Y513F donor DDR1 are indicated on the right . Note that the latter position includes heterodimers between DDR1b-R32E-Cys and DDR1a/DDR1-Y513F . The schematic diagram shows collagen-induced activation loop ( pY796 ) phosphorylation of DDR1b-R32E-Cys when co-expressed with DDR1a or DDR1b-Y513F . The blue shape denotes anti-pY796 used for Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 01410 . 7554/eLife . 25716 . 015Figure 6—figure supplement 2 . Signalling between dimers leads to activation loop phosphorylation of the receiver kinase independently of receiver kinase activity . ( A ) Kinase-inactive DDR1 ( DDR1b-Cys-KD ) was transiently expressed in HEK293 cells , either alone or co-expressed with DDR1b-Y513F , as indicated . The cells were stimulated with collagen I ( Coll ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blot was probed with a phospho-specific Ab against activation loop tyrosine-792 ( upper blot ) , and re-probed with anti-DDR1 ( lower blot ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of the cysteine-linked dimeric DDR1b-Cys-KD receiver construct , as well as DDR1b-Y513F are indicated on the right . The schematic diagram shows collagen-induced activation loop ( pY792 ) phosphorylation of DDR1b-Cys-KD when co-expressed with DDR1b-Y513F . The blue shape denotes anti-pY792 used for Western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 015 The requirement for kinase activity of the donor kinase was addressed by co-expression of the ectodomain deletion dimer , DDR1b-ΔECD-Cys , as the receiver kinase , with the kinase-dead form of DDR1a , DDR1a-KD , as the donor kinase ( Figure 7 ) . Neither DDR1a-KD nor a truncated version of DDR1b , DDR1-MDN , which retains part of the DDR1b juxtamembrane region but lacks the kinase domain ( Noordeen et al . , 2006 ) , were able to act as donor kinases and induce phosphorylation of the receiver construct , demonstrating that catalytic activity of the donor kinase is necessary for phosphorylation between dimers . 10 . 7554/eLife . 25716 . 016Figure 7 . Signalling between dimers requires kinase activity of the donor kinase . ( A ) The ectodomain-deletion construct DDR1b-ΔECD-Cys was transiently expressed in HEK293 cells , either alone or co-expressed with DDR1a , kinase-inactive DDR1a ( DDR1a-KD ) or with a truncated DDR1 construct lacking the kinase domain ( DDR1-MDN ) , as indicated . The cells were stimulated with collagen I ( Coll ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blot was probed with phospho-specific anti-pY513 ( upper blot ) , and re-probed with anti-DDR1 ( middle blot , C-terminal Ab ) . Expression of DDR1-MDN was verified by blotting with anti-DDR1 Abs against the ectodomain ( lowest blot , N-terminal Ab ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric DDR1b-ΔECD-Cys ( D ) and of mature monomeric DDR1b-ΔECD ( M ) receiver constructs , as well as of DDR1a or DDR1-MDN donor DDR1 are indicated on the right . ( B ) Quantitation of receiver DDR1 pY513 signals , normalised to respective DDR1 signals , expressed as relative band intensity with respect to the lowest signals on the blots ( unstimulated DDR1b-ΔECD-Cys ) . US , unstimulated; Coll , stimulation with collagen I . **p<0 . 01; NS , no significance; n = 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 016 In addition to stimulation by authentic collagen ligands , DDR1 phosphorylation can also be induced by an activating Ab , mAb-513 ( Figure 8A ) . This monoclonal Ab is a multimeric IgM Ab . Since mAb-513 binds to an extracellular epitope , the ectodomain deletion mutant , DDR1b-ΔECD-Cys , cannot bind this Ab and hence was not phosphorylated by incubation of cells with mAb-513 ( Figure 8B ) . Co-expression with DDR1b-Y513F , however , led to mAb-513-induced phosphorylation of DDR1b-ΔECD-Cys dimers . Similar to the situation with collagen-induced phosphorylation of the ectodomain deletion construct by DDR1-Y513F , mAb-513-induced phosphorylation was dependent on the transmembrane domain of the receiver kinase ( Figure 8B , C ) . These data show that phosphorylation between dimers is independent of the specific activation mode of the donor kinase . 10 . 7554/eLife . 25716 . 017Figure 8 . Collagen-independent activation by a multimeric anti-DDR1 Ab leads to signalling between dimers . ( A ) Wild-type DDR1b was transiently expressed in HEK293 cells , and the cells were incubated with mAb513 for 90 min at 37°C or left untreated . Cell lysates were analysed by reducing SDS-PAGE followed by Western blotting . ( B ) The ectodomain-deletion construct DDR1b-ΔECD-Cys was transiently expressed in HEK293 cells , either alone or co-expressed with DDR1b-Y513F , as indicated . The cells were stimulated with collagen I ( C ) or mAb513 ( A ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blots were probed with phospho-specific anti-pY513 ( upper blots ) , and re-probed with anti-DDR1 ( lower blots ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric DDR1b-ΔECD-Cys ( D ) and mature monomeric DDR1b-ΔECD ( M ) receiver constructs , as well as DDR1b-Y513F donor DDR1 are indicated on the right . ( C ) Quantitation of pY513 signals , normalised to respective DDR1 signals , expressed as relative band intensity with respect to the lowest signals on the blots ( unstimulated DDR1b-ΔECD-Cys ) . US , unstimulated; Coll , stimulation with collagen I; mAb , stimulation with mAb513 . **p<0 . 01; NS , no significance; n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 017 Fibrillar collagens are triple-helical proteins that assemble into fibrillar structures in tissues . In our co-expression experiments we incubated cells with soluble collagen I , but we cannot rule out that small fibrils form during the 90 min incubation times . These fibrils could provide multiple binding sites for DDR1 , thereby clustering the receptor . We previously showed that DDR1 activation could be induced by collagen-mimetic synthetic triple-helical peptides encompassing a DDR1 binding motif ( An et al . , 2016; Xu et al . , 2011 ) . Because these peptides are not able to form higher-order aggregates , we concluded that DDR phosphorylation can be induced by single collagen triple helices . Co-expression of DDR1b-ΔECD-Cys with DDR1b-Y513F showed that a collagen-mimetic DDR1 peptide ligand could also induce phosphorylation between dimers ( Figure 9 ) , demonstrating that ligand multivalency is not required for signalling between dimers . 10 . 7554/eLife . 25716 . 018Figure 9 . Ligand multivalency is not required for signalling between dimers . The ectodomain-deletion constructs DDR1b-ΔECD or DDR1b-ΔECD-Cys were transiently expressed in HEK293 cells , either alone or co-expressed with DDR1b-Y513F , as indicated . The cells were stimulated with collagen I ( C ) or a collagen-mimetic DDR-selective peptide ( P ) for 90 min at 37°C or left untreated . Cells were lysed in the presence of NEM , and aliquots of cell lysates were analysed by non-reducing SDS-PAGE followed by Western blotting . The blot was probed with phospho-specific anti-pY513 ( upper blot ) , and re-probed with anti-DDR1 ( lower blot ) . The positions of molecular mass markers are indicated on the left ( in KDa ) . The positions of cysteine-linked dimeric DDR1b-ΔECD-Cys ( D ) and mature monomeric DDR1b-ΔECD ( M ) receiver constructs , as well as DDR1b-Y513F donor DDR1 are indicated on the right . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 018 Phosphorylation between DDR1 dimers requires close apposition of DDR1 molecules , which is likely a result of ligand-induced receptor clustering . We used immunofluorescence to study collagen-induced DDR1 redistribution on the cell surface . Figure 10 demonstrates that DDR1b on the surface of unstimulated Cos-7 cells is present in a punctate distribution . Collagen binding led to redistribution of cell-surface DDR1 into a more compact structure , a phenomenon that is consistent with ligand-induced clustering . Ligand-induced clustering was prevented when cells were incubated with collagen in the presence of a function-blocking anti-DDR1 mAb . This mAb binds to an extracellular epitope and allosterically blocks collagen-induced DDR1 phosphorylation ( Carafoli et al . , 2012 ) . Hence , collagen-induced clustering is a key step in DDR1 activation . 10 . 7554/eLife . 25716 . 019Figure 10 . Function-blocking anti-DDR1 mAb inhibits collagen-induced DDR1 clustering . Wild-type DDR1b was transiently expressed in Cos-7 cells and either left unstimulated ( A ) , stimulated with 10 μg/ml collagen I ( B ) , or stimulated with 10 μg/ml collagen I in the presence of a function-blocking anti-DDR1 mAb at 10 μg/ml ( C ) , for 10 min at 37°C . Cells from all conditions were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain , before fixation and incubation with anti-mouse Alexa-Fluor-488 Ab , and mounting . Cells were imaged using a widefield microscope . White boxes in top rows indicate corresponding areas shown at higher magnification in lower rows . Scale bars , 30 μm ( top rows ) and 10 μm ( bottom rows ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 019 Eph receptors are RTKs that require clustering by ligand for receptor activation ( Nikolov et al . , 2014 ) . It was previously reported that Eph signalling clusters extend beyond the direct receptor-ligand contact area , and phosphorylated receptors were detectable over an extensive area outside the ligand-contact zone ( Wimmer-Kleikamp et al . , 2004 ) . To study the localisation of DDR1 phosphorylation with respect to ligand contact surfaces , we used collagen I-coated beads . DDR1b was expressed in Cos-7 cells , and phosphorylation was visualised with immunofluorescence . The beads efficiently recruited DDR1 , which was phosphorylated ( Figure 11A ) . However , in contrast to the situation with Eph receptors , phosphorylated DDR1 was strictly limited to the bead contact areas , even after prolonged ( 4 hr ) incubation times . 10 . 7554/eLife . 25716 . 020Figure 11 . Collagen induces local activation of DDR1 and leads to recruitment of ectodomain-deletion DDR1 into the ligand-binding contact zone . ( A ) Wild-type DDR1b was transiently expressed in Cos-7 cells and stimulated with collagen-coated latex beads ( 3 μm diameter ) for 2 or 4 hr at 37°C . Control beads were coated with BSA and incubated for 2 hr at 37°C . Cells were incubated on ice with a mouse monoclonal Ab against the DDR1 ectodomain , before fixation and permeabilisation and incubation with rabbit anti-pY513 Ab . Cells were then incubated with anti-mouse Alexa-Fluor-488 and anti-rabbit Alexa-Fluor-555 secondary Abs . DDR1 ( green ) and pY513 ( red ) staining are shown in the merge image ( right panel ) . ( B ) The ectodomain deletion construct DDR1b-ΔECD ( which contains an N-terminal Flag tag ) and DDR1b-Y513F were expressed either alone or in combination in Cos-7 cells . Cells were stimulated with collagen-coated latex beads ( 3 μm diameter ) for 2 hr at 37°C . Cells were incubated on ice with mouse anti-Flag IgG1 and mouse anti-DDR1 ectodomain IgG2b Abs , followed by fixation and permeabilisation and incubation with rabbit anti-pY513 Ab . Cells were then incubated with anti-mouse IgG1 Alexa-Fluor-488 , anti-mouse IgG2b Alexa-Fluor-555 , and anti-rabbit Alexa-Fluor-647 secondary Abs . Flag ( green ) and pY513 ( red ) staining are shown in the merge image ( right panel ) . BF , Brightfield . Scale bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 25716 . 020 DDR1 lacking its ectodomain cannot bind ligand . Expression of DDR1b-ΔECD in Cos-7 cells did not result in DDR1b-ΔECD recruitment to collagen-coated beads , as expected ( Figure 11B ) . However , co-expression of DDR1b-ΔECD with DDR1-Y513F showed efficient recruitment of DDR1b-ΔECD into the bead contact area , as well as Y513 phosphorylation that was restricted to the bead area ( Figure 11B ) . These data strongly support the concept that ligand binding leads to lateral association of DDR1 dimers .
Here we demonstrate an activation mechanism for DDR1 that is distinct from that of all other RTK families . Previous work showed that DDR1 forms constitutive dimers ( or higher oligomers ) in the absence of ligand ( Mihai et al . , 2009; Noordeen et al . , 2006; Xu et al . , 2014 ) ruling out the widely accepted RTK activation model of ligand-induced dimerisation ( Lemmon and Schlessinger , 2010 ) . Structural studies did not detect conformational differences between ligand-bound and unliganded DDR discoidin domains ( Carafoli et al . , 2009 , 2012; Ichikawa et al . , 2007 ) . Moreover , the long and flexible extracellular DDR1 juxtamembrane region does not appear to couple conformational changes to the intracellular region ( Xu et al . , 2014 ) . These observations led to the hypothesis that collagen binding may induce clustering of DDR1 in the cell membrane . In the present study we show that collagen binding results in DDR1 redistribution into a more compact structure on the cell surface . Furthermore , we demonstrate that ligand binding induces phosphorylation in trans between DDR1 dimers ( as opposed to phosphorylation exclusively within a dimer ) , which can only occur if neighbouring dimers are brought into close proximity . These data , therefore , strongly support a clustering mechanism of DDR1 activation . Our experiments with disulphide-linked constructs clearly demonstrate that inactive DDR1 mutants can be phosphorylated in a dimeric state . Phosphorylation of a key juxtamembrane tyrosine in the receiver construct ( Y513 ) requires kinase activity of the donor but not of the receiver kinase . These data indicate a mechanism where the juxtamembrane regions of DDR1 dimers act as substrates for the kinase activity of neighbouring DDR1 molecules , rather than being phosphorylated exclusively through kinase activity within the same dimer . In addition to juxtamembrane phosphorylation , the activation loop of the receiver kinase was phosphorylated in our co-expression studies , again independently of receiver kinase activity , indicating that the activation loop can also act as a substrate for neighbouring DDR1 dimers . Activation loop phosphorylation is a major regulatory mechanism that releases cis-autoinhibition of kinases , including the majority of RTKs ( Lemmon and Schlessinger , 2010 ) . The DDR1 kinase domain is auto-inhibited through activation loop interactions with the active site ( Canning et al . , 2014 ) . Our collective biochemical and microscopic data suggest that DDRs are activated by a mechanism in which collagen binding promotes lateral association of DDR1 dimers , which contributes to activation loop phosphorylation and perhaps also to the release of cis-autoinhibition of the kinase domain . Co-clustering with , and phosphorylation by , catalytically active DDR1 was independent of ectodomain contacts but required specific transmembrane domain interactions . These data reinforce a key role for the DDR1 transmembrane domain in signalling . DDR1 transmembrane helices have the highest propensity for self-association amongst all RTKs ( Finger et al . , 2009 ) , and DDR1 mutants with mutations that weaken transmembrane helix interactions are severely compromised in collagen-induced receptor activation ( Noordeen et al . , 2006 ) . The present data show that intact transmembrane associations are not only essential for DDR1 as an enzyme kinase but also as a substrate kinase . It is possible that weakened transmembrane domain contacts affect the conformation of the cytoplasmic region in such a way that it no longer functions as a substrate for donor DDR1 kinase . Transmembrane domain contacts may also be required for DDR1 clustering , which could either involve direct receptor-receptor contacts or another membrane protein . In breast cancer cells , it was recently shown that the tetraspanin TM4SF1 promotes clustering of DDR1; however , in this scenario the DDR1 ectodomain was necessary for interaction with TM4SF1 , while the transmembrane and cytoplasmic regions were dispensable ( Gao et al . , 2016 ) . DDR1 activation in our co-expression experiments was achieved with fibrillar and non-fibrillar collagens , a collagen-mimetic peptide , as well as by clustering with a multimeric anti-DDR1 mAb . The fact that a short collagen-mimetic peptide efficiently induced signalling between DDR1 dimers suggests that DDR1 clustering is receptor-mediated , rather than achieved through cross-linking via the ligand . While most RTKs are thought to signal as dimers , ligand-induced clustering is a key step in signalling of the Eph family of RTKs ( Himanen et al . , 2010; Seiradake et al . , 2010 ) . DDR1 activation shares some , but not all of the characteristics of Eph receptor activation . Similar to what we show in this report for DDR1 , ligand binding-defective Eph receptors co-cluster with functional Eph receptors ( Seiradake et al . , 2013; Wimmer-Kleikamp et al . , 2004 ) . Furthermore , kinase-dead EphB6 clusters with EphB1 , resulting in EphB6 transphosphorylation ( Freywald et al . , 2002 ) . However , unlike in DDR1 clusters , structurally defined ectodomain contacts are important for Eph receptor clusters ( Himanen et al . , 2010; Seiradake et al . , 2010 , 2013; Xu et al . , 2013 ) . Another difference is that Eph receptor signalling clusters can exceed the size of the ligand contact zone several fold ( Wimmer-Kleikamp et al . , 2004 ) , while we observed that DDR1 signalling clusters were strictly confined to the ligand contact area . Interestingly , signalling between DDR1 dimers did not require identical cytoplasmic regions and was also observed with DDR2 as the donor kinase . These findings have functional implications in that catalytically incompetent DDR1 isoforms ( Alves et al . , 2001 ) could contribute to signalling when co-expressed with catalytically competent DDR1 isoforms or DDR2 , with the signalling outcome likely dependent on the balance between catalytically active and inactive receptors , as has been suggested for Eph receptor signalling ( Truitt and Freywald , 2011 ) . Recent studies have shown that oligomerisation beyond dimerisation also plays a role in signalling of RTKs that have traditionally been assumed to signal exclusively as dimers . Thus , EGF binding to the EGF receptor can result in formation of tetramers and higher-order oligomers , which may boost autophosphorylation of C-terminal tail tyrosines ( Clayton et al . , 2005; Huang et al . , 2016; Needham et al . , 2016 ) . However , conflicting structural models have been proposed for the architecture and stoichiometry of EGF-bound EGF receptor multimers: one model involves self-association of ligand-bound dimers ( Huang et al . , 2016 ) , while another model involves association via unoccupied ligand-binding sites , resulting in oligomers that can bind two EGF molecules at most ( Needham et al . , 2016 ) . Our microscopic and biochemical data define a ligand-induced clustering mechanism and phosphorylation between neighbouring DDR1 dimers in trans; the size and structure of DDR1 signalling clusters and whether other molecules are required to co-associate with DDR1 remain to be determined .
Human embryonic kidney ( HEK ) 293 cells , monkey Cos-7 fibroblast-like kidney cells , and human breast cancer MCF-7 cells were from ATCC , Manassas , VA , and were cultured in Dulbecco’s modified Eagle’s medium/F12 nutrient mixture ( DMEM/F12; Invitrogen ) supplemented with 2 mM L-glutamine , 100 units/ml penicillin , 100 µg/ml streptomycin and 10% foetal bovine serum . Human breast cancer T47D cells were obtained from the Cell Production Laboratory , Imperial Cancer Research Fund , UK , and were cultured in Roswell Park Memorial Institute medium ( Invitrogen ) supplemented with 2 mM L-glutamine , 100 units/ml penicillin , 100 µg/ml streptomycin and 10% foetal bovine serum . MCF-7 cells ( on the list of commonly misidentified cell lines established by the International Cell Line Authentication Committee ) and T47D cells were only used as example cell lines with endogenous DDR1 expression , for comparison of their DDR1 surface levels to those in our HEK-293 cell expression system; their authenticity was not confirmed . Neonatal normal human epidermal keratinocytes ( Lonza , Switzerland; passages 3–5 ) were cultured on a mitomycin C-treated feeder layer of 3T3 fibroblasts in DMEM/F12 medium supplemented with 1 . 8 mM CaCl2 , 100 units/ml penicillin , 100 µg/ml streptomycin , 10% foetal calf serum , 5 mM L-glutamine , 5 μg/ml insulin , 0 . 5 μg/ml hydrocortisone , 0 . 1 nM cholera toxin , and 10 ng/ml epidermal growth factor . All cells were grown at 37°C , 5% CO2 . Cells were tested for mycoplasma contamination every four months and were confirmed negative . Bovine serum albumin ( BSA ) was obtained from Fisher Scientific ( Loughborough , UK ) . Collagen I ( acid-soluble from rat tail; C-7661 ) and collagen IV ( acid-soluble from placenta; C-5533 ) were purchased from Sigma ( Gillingham , UK ) . The collagen-mimetic DDR-selective peptide , ( GPP ) 5-GPRGQOGVNleGFO- ( GPP ) 5GPC-NH2 , was obtained from Prof . Richard Farndale , University of Cambridge , UK . The peptide was synthesized by Fmoc ( N- ( 9-fluorenyl ) methoxycarbonyl ) chemistry as a C-terminal amide on TentaGel R RAM resin in an Applied Biosystems Pioneer automated synthesizer and purified as described ( Raynal et al . , 2006 ) . N-ethylmaleimide ( NEM ) was purchased from Sigma ( E-1271 ) . The following primary Abs were used: rabbit anti-DDR1 ( SC-532 , Santa Cruz , Dallas , TX ) ; goat anti-DDR1 extracellular domain Ab ( AF2396 , R&D Systems , Minneapolis , MN ) ; goat anti-DDR2 ( AF2538 from R&D Systems ) ; rabbit anti-phospho-DDR1 ( Tyr513 ) and rabbit anti-phospho-DDR1 ( Tyr792 ) from Cell Signalling ( Danvers , MA ) ; rabbit anti-phospho-DDR2 ( Tyr740 ) from R&D Systems; mouse anti-Flag IgG1 clone M2 ( Sigma ) ; rabbit anti-Flag ( Sigma ) ; and mouse anti-tubulin ( Sigma ) . Anti-phospho-DDR1 ( Tyr513 ) , here referred to as anti-pY513 , is a phospho-specific Ab that recognises phosphorylated tyrosine-513 in DDR1b . Anti-phospho-DDR1 ( Tyr792 ) , here referred to as anti-pY792 , recognises phosphorylated tyrosine-792 in the activation loop of DDR1a and DDR1b . Anti-phospho-DDR2 ( Tyr740 ) , here referred to as anti-pY796 , recognises phosphorylated Tyr740 in the activation loop of DDR2 and cross-reacts with the equivalent residue in DDR1 , phosphorylated tyrosine-796 . Mouse anti-DDR1 mAbs , 7A9 and IF10 , were generated in our lab ( Carafoli et al . , 2012 ) . The multimeric anti-DDR1 IgM Ab ( mAb513 ) was used in the form of ascites and was a gift from Dr Michel Faure , SUGEN Inc . Secondary Abs were as follows: goat anti-rabbit Ig-horseradish peroxidase conjugated ( P0448 , DAKO A/S , Denmark ) ; rabbit anti-goat Ig-horseradish peroxidase ( Zymed Laboratories Inc . , San Francisco , CA ) ; sheep anti-mouse Ig-horseradish peroxidase ( Amersham Biosciences , Little Chalfont , UK ) ; goat anti-mouse IgG FITC-conjugated ( F-9006 , Sigma ) ; goat anti-mouse IgG Alexa-Flour-488 ( Invitrogen ) ; goat anti-mouse IgG1 Alexa-Fluor-488 ( Invitrogen ) ; goat anti-rabbit Alexa-Fluor-488 ( Invitrogen ) , goat anti-mouse IgG2b Alexa-Fluor-555 ( Invitrogen ) ; goat anti-rabbit IgG Alexa-Fluor-555 ( Invitrogen ) ; and goat anti-rabbit IgG Alexa-Fluor-647 ( Invitrogen ) . DDR1b-R32E and DDR1b-L152E were previously generated ( Carafoli et al . , 2012 ) . DDR1b-KD ( K655A ) and DDR1a-KD ( K655A ) constructs were generated by strand overlap extension PCR as described previously ( Leitinger , 2003 ) . DDR1b-W53A and DDR1b-Y513F constructs were generated with the QuickChange method using wild type DDR1b cDNA cloned into a pSP72 vector ( Promega ) as a template . All cysteine substitution mutants have the T416C mutation and were generated by subcloning using a previously made cDNA construct encoding DDR1b-T416C . To obtain DDR1b-W53A-Cys , DDR1b-R32E-Cys and DDR1b-L152E-Cys , DDR1b-W53A , DDR1b-R32E and DDR1b-L152E constructs were cut with EcoRI and SacI and subcloned into a pGEM-3Z ( Promega ) -based vector encoding DDR1b-T416C ( Xu et al . , 2014 ) , after the corresponding wild-type EcoRI-SacI sequence was removed . The DDR1b-KD-Cys construct was generated by subcloning the XhoI-BamHI fragment of DDR1b-KD into a pRK5 ( BD PharMingen ) -based vector encoding DDR1b-T416C , after the corresponding wild-type sequence was removed . DDR1b-ΔECD ( with an N-terminal Flag tag followed by a short linker ) was generated with the FastCloning PCR method ( Li et al . , 2011 ) , eliminating residues Gly24 to Lys410 of WT-DDR1b . The mature construct after signal peptide cleavage has the following N-terminal sequence: DADMK23DYKDDDKSGSAEGSPTA417ILI ( Flag tag and SGS linker underlined; first three transmembrane domain residues in bold ) . An N-terminally Flag-tagged DDR1b cDNA , cloned into pGEM-3Z vector , was used as a template . DDR1b-ΔECD-Cys was generated by introducing the T416C mutation using the QuickChange method . DDR1b-ΔECD-TM1 and DDR1b-ΔECD-Cys-TM1 were generated by introducing the L430G/L431P mutation ( Noordeen et al . , 2006 ) using DDR1b-ΔECD or DDR1b-ΔECD-Cys constructs as templates . The cytoplasmic DDR1 deletion construct MDN ( DDR1b truncated after Arg525 ) was a gift from Dr Michel Faure , SUGEN Inc . All cDNAs encoding mutant DDR1b were cloned into the mammalian expression vector pRK5 ( BD Pharmingen ) for transient transfection in cells . Restriction and modification enzymes were purchased from New England Biolabs ( Hitchin , UK ) or Promega ( Southampton , UK ) . All PCR-derived DNA constructs were verified by DNA sequencing . PCR primers used for generating the mutant constructs can be obtained on request . HEK293 cells were grown in 24-well ( for DDR1 autophosphorylation assays ) or 6-well ( for flow cytometry and comparison of protein expression levels ) tissue culture plates and transfected with relevant DDR1 expression plasmids by calcium phosphate precipitation . Cos-7 cells were grown on coverslips in 24-well tissue culture plates and transfected with Fugene HD ( Promega , Madison , WI ) according to the manufacturer’s instructions . The assay was performed as described before ( Leitinger , 2003 ) . Briefly , 24 hr after transfection , the cells were incubated with serum-free medium for 16 hr . Cells were then stimulated with 10 µg/ml collagen I , 50 µg/ml collagen IV , 50 µg/ml collagen-mimetic DDR-selective peptide , or the multimeric anti-DDR1 Ab ( mAb513 , 1:500 dilution from ascites ) for 90 min at 37°C . Cells were lysed in 1% Nonidet P-40 , 150 mM NaCl , 50 mM Tris , pH 7 . 4 , 1 mM EDTA , 1 mM phenylmethylsulfonyl fluoride , 50 µg/ml aprotinin , 1 mM sodium orthovanadate , and 5 mM NaF . Aliquots of the lysates were analysed by reducing SDS-PAGE on 7 . 5% polyacrylamide gels . In experiments with cysteine mutants , the lysis procedure was as described ( Xu et al . , 2014 ) . Cells were lysed in lysis buffer containing 30 mM NEM to avoid potential non-native disulphide formation during lysis , and aliquots of lysates were analysed by non-reducing SDS-PAGE on 5% or 7 . 5% polyacrylamide gels . This was followed by blotting onto nitrocellulose membranes . The blots were probed first with anti-phospho-DDR1 Abs followed by horseradish peroxidase conjugated goat anti-rabbit secondary Abs , then stripped in Antibody Stripping Solution ( Alpha Diagnostic International , San Antonio , Texas ) and re-probed with anti-DDR1 Abs followed by horseradish peroxidase conjugated goat anti-rabbit secondary Abs . To detect expression of DDR2 or the truncated DDR1 construct lacking the kinase domain ( DDR1-MDN ) in co-expression experiments , blots were probed first with anti-DDR2 or anti-DDR1 against the ectodomain , respectively , followed by horseradish peroxidase conjugated anti-goat secondary Abs . Signal detection was performed using Enhanced Chemiluminescence Plus or Enhanced Chemiluminescence Select reagents ( Amersham Biosciences ) on an Ettan DIGE imager , a Typhoon FLA 9500 Imager ( GE Healthcare Biosciences ) or X-ray film . HEK293 , T47D and MCF7 cells were grown in 6-well tissue culture plates and primary keratinocytes were grown in 10 cm dishes . HEK293 cells were transfected with varying amounts of DDR1b expression plasmid . Cells were serum-starved for 16 hr and lysed as above . Equal amounts of protein were analysed by reducing SDS-PAGE on 7 . 5% polyacrylamide gels . This was followed by blotting onto nitrocellulose membranes . The blots were probed with anti-DDR1 Ab followed by horseradish peroxidase conjugated goat-anti-rabbit secondary Ab , or anti-tubulin Ab followed by horseradish peroxidase conjugated sheep-anti-mouse secondary Ab . Signal detection was performed as above . Cos-7 cells transiently expressing the ectodomain deletion constructs , DDR1b-ΔECD , DDR1b-ΔECD-TM1 , DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1 , which contain N-terminal Flag tags , were washed with PBS , and incubated with mouse anti-Flag IgG1 for 1 hr on ice to label cell surface DDR1 constructs . This was followed by washing , fixing with 4% paraformaldehyde ( Sigma ) in PBS for 15 min at room temperature , blocking in 8% BSA in PBS for 1 hr , and incubation with anti-mouse IgG1 Alexa-Fluor-488 secondary Ab . Cos-7 cells transiently expressing DDR1b wildtype were stimulated with 10 µg/ml collagen I or a mixture of 10 µg/ml collagen and 10 µg/ml mouse anti-DDR1 7A9 IgG1 mAb , for 10 min at 37°C . The cells were washed with PBS , and incubated with mAb 7A9 for 1 hr on ice to label cell surface DDR1 . This was followed by fixation and incubation with secondary Ab as above . The coverslips were mounted on slides with Prolong Gold Antifade Reagent ( Invitrogen ) . The cells were examined using an Olympus BX-51 widefield microscope . For detection of cell surface expression of DDR1 ectodomain deletion mutants , HEK293 cells in 6-well tissue culture plates were transfected with N-terminally Flag-tagged DDR1 ectodomain deletion expression plasmids ( DDR1b-ΔECD , DDR1b-ΔECD-TM1 , DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1 ) and grown for 48 hr before being dissociated and resuspended in PBS containing 1% BSA . The cells were incubated for 30 min on ice with rabbit anti-Flag Ab in 100 μl of PBS/BSA . Cells were then washed three times with PBS/BSA and incubated with goat anti-rabbit Alexa-Fluor-488 Ab for 30 min on ice . After three washes as above , the cells were resuspended in 2% formaldehyde in PBS . For comparison of surface DDR1 expression , HEK293 , T47D and MCF7 cells were grown in 6-well tissue culture plates and primary keratinocytes were grown in 10 cm dishes . HEK293 cells were transfected with varying amounts of DDR1b expression plasmid . Cells dissociation , Ab staining and fixation were done as above , except that the primary Ab was anti-mouse DDR1 ( mAb 7A9 ) and the secondary Ab was FITC-conjugated goat-anti-mouse IgG . Data were collected on a BD LSRFortessa cell analyser using BD FACSDiva software 6 . 0 ( BD Biosciences ) and further analysed on FlowJo software 10 . 2 ( Tree Star , Inc . ) . 3 µm diameter latex beads ( Sigma ) were washed in distilled water , followed by washes in coating buffer ( 50 mM Tris , 100 mM NaCl , pH 8 . 5 ) and incubation with 20 µg/ml collagen I in coating buffer overnight at 4°C on a rotating wheel . Control beads were coated with 20 μg/ml BSA . The beads were then washed in PBS , blocked in 1% BSA in PBS for 1 hr , and re-suspended in serum-free medium for use on cells . Cos-7 cells transiently expressing wild-type DDR1b were grown on coverslips and incubated with serum-free medium for 16 hr . Cells were then stimulated with collagen-coated beads or control BSA-coated beads for the indicated time at 37°C . After stimulation , cells were washed with PBS , and incubated with mouse anti-DDR1 7A9 IgG1 mAb for 1 hr on ice to label surface DDR1 . This was followed by washing in PBS , fixing with 4% paraformaldehyde in PBS for 15 min at room temperature , washing in PBS , permeabilisation with 0 . 1% Triton X-100 ( Sigma ) in PBS for 5 min , blocking in 8% BSA in PBS for 1 hr , and incubation with rabbit anti-pY513 Ab . After washing , the cells were then incubated with anti-mouse IgG Alexa-Fluor-488 and anti-rabbit Alexa-Fluor-555 secondary Abs . Cos-7 cells transiently expressing the ectodomain deletion construct DDR1b-ΔECD ( which contains an N-terminal Flag tag ) , DDR1b-Y513F or both were stimulated with collagen-coated beads for 2 hr at 37°C . After stimulation , cells were washed with PBS , and incubated with mouse anti-Flag IgG1 and mouse anti-DDR1 ectodomain IF10 IgG2b Abs for 1 hr on ice to label surface DDR1b-ΔECD and DDR1b-Y513F , respectively . This was followed by washing , fixing , permeabilisation , blocking , and incubation with rabbit anti-pY513 Ab as described above . The cells were then incubated with anti-mouse IgG1 Alexa-Fluor-488 , anti-mouse IgG2b Alexa-Fluor-555 and anti-rabbit Alexa-Fluor-647 secondary Abs . The coverslips were mounted on slides with Prolong Gold Antifade Reagent ( Invitrogen ) . The cells were examined using an Olympus BX-51 widefield microscope . All experiments were performed at least three times with similar results . Blots show representative examples . Intensities of protein bands were quantitated using ImageJ software . All data are expressed as mean ± standard error of the mean ( SEM ) from at least three independent experiments . Statistical significance was tested with a Kruskal-Wallis multiple comparison test , followed by Mann-Whitney tests using GraphPad Prism 7 ( San Diego , CA ) . Statistical significance was set at a p value < 0 . 05 .
The collagen-binding receptor tyrosine kinase DDR1 ( discoidin domain receptor 1 ) is a drug target for a wide range of human diseases , but the molecular mechanism of DDR1 activation is poorly defined . Here we co-expressed different types of signalling-incompetent DDR1 mutants ( ‘receiver’ ) with functional DDR1 ( ‘donor’ ) and demonstrate phosphorylation of receiver DDR1 by donor DDR1 in response to collagen . Making use of enforced covalent DDR1 dimerisation , which does not affect receptor function , we show that receiver dimers are phosphorylated in trans by the donor; this process requires the kinase activity of the donor but not that of the receiver . The receiver ectodomain is not required , but phosphorylation in trans is abolished by mutation of the transmembrane domain . Finally , we show that mutant DDR1 that cannot bind collagen is recruited into DDR1 signalling clusters . Our results support an activation mechanism whereby collagen induces lateral association of DDR1 dimers and phosphorylation between dimers .
The membrane surrounding each living cell contains a variety of proteins that carry out different roles . For example , proteins called receptor tyrosine kinases help a cell to receive signals from its external environment . Receptor tyrosine kinases span the membrane so that one part of the protein known as the ectodomain sticks out from the surface of the cell , while another part ( called the kinase domain ) sits inside the cell . When a signalling molecule binds to the ectodomain , the kinase domain becomes active and starts to add chemical groups called phosphates to other proteins . This process , known as phosphorylation , changes the protein’s activity , which in turn influences the cell’s behaviour . In most cases , the signalling molecule causes two receptor tyrosine kinase proteins to bind to each other and form a “dimer” in which the kinase domains are able to phosphorylate , and thus activate , each other . Female mammals need a receptor tyrosine kinase called DDR1 to develop mammary glands ( the glands that produce milk ) . DDR1 is activated when a signalling molecule called collagen binds to its ectodomain . Unlike many other receptor tyrosine kinases , DDR1 exists as a dimer even before it binds to collagen , so it is not clear how collagen activates DDR1 . One possibility is that collagen causes several DDR1 dimers to form clusters on the membrane so that kinases on neighbouring dimers can phosphorylate each other . Juskaite et al . explored this idea by pairing up normal DDR1 proteins with mutant versions that are unable to bind to collagen . The experiments show that when collagen binds to the normal DDR1 molecules , DDR1 dimers do indeed form clusters . This enables the normal protein molecules in neighbouring dimers to phosphorylate each other as well as the mutant proteins . In this way , the clustered DDR1 dimers can become active even if the clusters contain one or more mutant versions that are unable to detect collagen . Further experiments show that specific contacts need to form between neighbouring dimers for this phosphorylation to occur . Abnormal DDR1 activity is associated with several diseases including cancer , inflammation and fibrosis . The findings of Juskaite et al . suggest that developing new drugs that can prevent DDR1 from forming clusters may help to treat people with these conditions . Further work is also needed to analyse the size and structure of DDR1 clusters and investigate if other proteins also associate with the clusters .