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Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in multiple cellular processes during development. The two PDGFRs, PDGFRα and PDGFRβ, dimerize to form homodimers and/or heterodimers. Here, we overcome previous limitations in studying PDGFR dimer-specific dynamics by generating cell lines stably expressing C-terminal fusions of each PDGFR with bimolecular fluorescence complementation (BiFC) fragments corresponding to the N-terminal or C-terminal regions of the Venus fluorescent protein. We find that PDGFRβ receptors homodimerize more quickly than PDGFRα receptors in response to PDGF ligand, with increased levels of autophosphorylation. Furthermore, we demonstrate that PDGFRα homodimers are trafficked and degraded more quickly, whereas PDGFRβ homodimers are more likely to be recycled back to the cell membrane. We show that PDGFRβ homodimer activation results in a greater amplitude of phospho-ERK1/2 and phospho-AKT signaling, as well as increased proliferation and migration. Finally, we demonstrate that inhibition of clathrin-mediated endocytosis leads to changes in cellular trafficking and downstream signaling, particularly for PDGFRα homodimers. Collectively, our findings provide significant insight into how biological specificity is introduced to generate unique responses downstream of PDGFR engagement. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The α6β1-integrin is a major laminin receptor, and formation of a laminin-rich basement membrane is a key feature in tumour blood vessel stabilisation and pericyte recruitment, processes that are important in the growth and maturation of tumour blood vessels. However, the role of pericyte α6β1-integrin in angiogenesis is largely unknown. We developed mice where the α6-integrin subunit is deleted in pericytes and examined tumour angiogenesis and growth. These mice had: (1) reduced pericyte coverage of tumour blood vessels; (2) reduced tumour blood vessel stability; (3) increased blood vessel diameter; (4) enhanced blood vessel leakiness, and (5) abnormal blood vessel basement membrane architecture. Surprisingly, tumour growth, blood vessel density and metastasis were not altered. Analysis of retinas revealed that deletion of pericyte α6-integrin did not affect physiological angiogenesis. At the molecular level, we provide evidence that pericyte α6-integrin controls PDGFRβ expression and AKT-mTOR signalling. Taken together, we show that pericyte α6β1-integrin regulates tumour blood vessels by both controlling PDGFRβ and basement membrane architecture. These data establish a novel dual role for pericyte α6-integrin as modulating the blood vessel phenotype during pathological angiogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Platelet-derived growth factor (PDGF)-D is a PDGF receptor β (PDGFRβ)-specific ligand implicated in a number of pathological conditions, such as cardiovascular disease and cancer, but its biological function remains incompletely understood. In this study, we demonstrate that PDGF-D binds directly to neuropilin 1 (NRP1), in a manner that requires the PDGF-D C-terminal Arg residue. Stimulation with PDGF-D, but not PDGF-B, induced PDGFRβ-NRP1 complex formation in fibroblasts. Additionally, PDGF-D induced translocation of NRP1 to cell-cell junctions in endothelial cells, independently of PDGFRβ, altering the availability of NRP1 for VEGF-A-VEGFR2 signaling. PDGF-D showed differential effects on pericyte behavior in ex vivo sprouting assays compared to PDGF-B. Furthermore, PDGF-D-induced PDGFRβ-NRP1 interaction can occur in trans between molecules located in different cells (endothelial cells and pericytes). In summary, we show that NRP1 can act as a co-receptor for PDGF-D-PDGFRβ signaling and is possibly implicated in intercellular communication in the vascular wall., (© 2017. Published by The Company of Biologists Ltd.)

Platelet-derived growth factor receptor β (PDGFRβ) is a receptor tyrosine kinase which upon activation by PDGF-BB stimulates cell proliferation, migration and angiogenesis. Ligand binding induces intracellular signaling cascades but also internalization of the receptor, eventually resulting in its lysosomal degradation. However, endocytic trafficking of receptors often modulates their downstream signaling. We previously reported that internalization of PDGFRβ occurs via dynamin-dependent and -independent pathways but their further molecular determinants remained unknown. Here we show that, in human fibroblasts expressing endogenous PDGFRβ and stimulated with 50 ng/ml PDGF-BB, ligand-receptor uptake proceeds via the parallel routes of clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE). CME involves the canonical AP2 complex as a clathrin adaptor, while CIE requires RhoA-ROCK, Cdc42 and galectin-3, the latter indicating lectin-mediated internalization via clathrin-independent carriers (CLICs). Although different uptake routes appear to be partly interdependent, they cannot fully substitute for each other. Strikingly, inhibition of any internalization mechanism impaired activation of STAT3 but not of other downstream effectors of PDGFRβ. Our data indicate that multiple routes of internalization of PDGFRβ contribute to a transcriptional and mitogenic response of cells to PDGF., (© 2017. Published by The Company of Biologists Ltd.)

Primary cilia are microtubule-based sensory organelles projecting from most quiescent mammalian cells, which disassemble in cells cultured in serum-deprived conditions upon re-addition of serum or growth factors. Platelet-derived growth factors (PDGF) are implicated in deciliation, but the specific receptor isoforms and mechanisms involved are unclear. We report that PDGFRβ promotes deciliation in cultured cells and provide evidence implicating PLCγ and intracellular Ca(2+) release in this process. Activation of wild-type PDGFRα alone did not elicit deciliation. However, expression of constitutively active PDGFRα D842V mutant receptor, which potently activates PLCγ (also known as PLCG1), caused significant deciliation, and this phenotype was rescued by inhibiting PDGFRα D842V kinase activity or AURKA. We propose that PDGFRβ and PDGFRα D842V promote deciliation through PLCγ-mediated Ca(2+) release from intracellular stores, causing activation of calmodulin and AURKA-triggered deciliation., (© 2015. Published by The Company of Biologists Ltd.)

We have shown previously that epidermal growth factor (EGF) receptor (EGFR) endocytosis is controlled by EGFR dimerization. However, it is not clear how the dimerization drives receptor internalization. We propose that EGFR endocytosis is driven by dimerization, bringing two sets of endocytic codes, one contained in each receptor monomer, in close proximity. Here, we tested this hypothesis by generating specific homo- or hetero-dimers of various receptors and their mutants. We show that ErbB2 and ErbB3 homodimers are endocytosis deficient owing to the lack of endocytic codes. Interestingly, EGFR-ErbB2 or EGFR-ErbB3 heterodimers are also endocytosis deficient. Moreover, the heterodimer of EGFR and the endocytosis-deficient mutant EGFRΔ1005-1017 is also impaired in endocytosis. These results indicate that two sets of endocytic codes are required for receptor endocytosis. We found that an EGFR-PDGFRβ heterodimer is endocytosis deficient, although both EGFR and PDGFRβ homodimers are endocytosis-competent, indicating that two compatible sets of endocytic codes are required. Finally, we found that to mediate the endocytosis of the receptor dimer, the two sets of compatible endocytic codes, one contained in each receptor molecule, have to be spatially coordinated., (© 2015. Published by The Company of Biologists Ltd.)

Hepatic stellate cells (HSCs) are liver-specific pericytes that are recruited to vessels and secret pro-angiogenic cytokines, and thus actively involved in pathological vascularization during liver fibrosis. Peroxisome proliferator-activated receptor-γ (PPARγ) is a switch molecule controlling HSC activation. We investigated PPARγ regulation of angiogenic signal transduction and the molecular mechanisms involved in HSCs. Primary rat HSCs and liver sinusoidal endothelial cells (LSECs) were isolated and used in this study. Boyden chamber and tubulogenesis assays, identified that focal adhesion kinase (FAK)-RhoA signaling activated by platelet-derived growth factor (PDGF) was required for HSC motility and the associated vascularization. PDGF also stimulated vascular endothelial growth factor (VEGF) expression and HSC-driven vascularization through signals mediated by extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR). Gain- and loss-of-function analyses demonstrated that activation of PPARγ interrupted FAK-RhoA, ERK and mTOR cascades and inhibited HSC-based vascularization. Molecular evidence further revealed that PPARγ attenuation of HSC angiogenic properties was dependent on inhibition of PDGF-β receptor expression. We concluded that PPARγ inhibited angiogenic signal transduction through transrepression of PDGF-β receptor leading to reduced HSC motility, reduced VEGF expression, and thereby attenuated HSC-driven angiogenesis. PPARγ could be a molecular target for preventing vascular remolding in hepatic fibrosis.

Cell migration during vascular remodelling is regulated by crosstalk between growth factor receptors and integrin receptors, which together coordinate cytoskeletal and motogenic changes. Here, we report extracellular matrix (ECM)-directed crosstalk between platelet-derived growth factor receptor (PDGFR)-β and α5β1-integrin, which controls the migration of mesenchymal stem (stromal) cells (MSCs). Cell adhesion to fibronectin induced α5β1-integrin-dependent phosphorylation of PDGFR-β in the absence of growth factor stimulation. Phosphorylated PDGFR-β co-immunoprecipitated with α5-integrin and colocalised with α5β1-integrin in the transient tidemarks of focal adhesions. Adhesion to fibronectin also strongly potentiated PDGF-BB-induced PDGFR-β phosphorylation and focal adhesion kinase (FAK) activity, in an α5β1-integrin-dependent manner. PDGFR-β-induced phosphoinositide 3-kinase (PI3K) and Akt activity, actin reorganisation and cell migration were all regulated by fibronectin and α5β1-integrin. This synergistic relationship between α5β1-integrin and PDGFR-β is a fundamental determinant of cell migration. Thus, fibronectin-rich matrices can prime PDGFR-β to recruit mesenchymal cells at sites of vascular remodelling.

Cell-surface retention sequence (CRS) binding protein (CRSBP-1) is a membrane glycoprotein identified by its ability to bind PDGF-BB and VEGF-A via their CRS motifs (clusters of basic amino acid residues). CRSBP-1 is identical to LYVE-1 and exhibits dual ligand (CRS-containing proteins and hyaluronic acid) binding activity, suggesting the importance of CRSBP-1 ligands in lymphatic function. Here, we show that CRSBP-1 ligands induce disruption of VE-cadherin-mediated intercellular adhesion and opening of intercellular junctions in lymphatic endothelial cell (LEC) monolayers as determined by immunofluorescence microscopy and Transwell permeability assay. This occurs by interaction with CRSBP-1 in the CRSBP-1-PDGFβR-β-catenin complex, resulting in tyrosine phosphorylation of the complex, dissociation of β-catenin and p120-catenin from VE-cadherin, and internalization of VE-cadherin. Pretreatment of LECs with a PDGFβR kinase inhibitor abolishes ligand-stimulated tyrosine phosphorylation of VE-cadherin, halts the ligand-induced disruption of VE-cadherin intercellular adhesion and blocks the ligand-induced opening of intercellular junctions. These CRSBP-1 ligands also induce opening of lymphatic intercellular junctions that respond to PDGFβR kinase inhibitor in wild-type mice (but not in Crsbp1-null mice) as evidenced by increased transit of injected FITC-dextran and induced edema fluid from the interstitial space into lymphatic vessels. These results disclose a novel mechanism involved in the opening of lymphatic intercellular junctions.

Crk family adaptors, consisting of Src homology 2 (SH2) and SH3 protein-binding domains, mediate assembly of protein complexes in signaling. CrkI, an alternately spliced form of Crk, lacks the regulatory phosphorylation site and C-terminal SH3 domain present in CrkII and CrkL. We used gene silencing combined with mutational analysis to probe the role of Crk adaptors in platelet-derived growth-factor receptor beta (PDGFbetaR) signaling. We demonstrate that Crk adaptors are required for formation of focal adhesions, and for PDGF-stimulated remodeling of the actin cytoskeleton and cell migration. Crk-dependent signaling is crucial during the early stages of PDGFbetaR activation, whereas its termination by Abl family tyrosine kinases is important for turnover of focal adhesions and progression of dorsal-membrane ruffles. CrkII and CrkL preferentially activate the small GTPase Rac1, whereas variants lacking a functional C-terminal SH3 domain, including CrkI, preferentially activate Rap1. Thus, differences in the activity of Crk isoforms, including their effectors and their ability to be downregulated by phosphorylation, are important for coordinating dynamic changes in the actin cytoskeleton in response to extracellular signals.

At sites of vessel-wall injury, vascular smooth muscle cells (VSMCs) can dedifferentiate to express an invasive and proliferative phenotype, which contributes to the development of neointimal lesions and vascular disorders. Herein, we demonstrate that the loss of the VSMC differentiated phenotype, as the repression of contractile-protein expression, is correlated with a dramatic upregulation of the membrane-anchored matrix metalloproteinase MT1-MMP (also known as MMP14 and membrane-type 1 matrix metalloproteinase). Matrix metalloproteinase (MMP) inhibitors or MT1-MMP deficiency led to attenuated VSMC dedifferentiation, whereas the phenotypic switch was re-engaged following the restoration of MT1-MMP activity in MT1-MMP(-/-) cells. MT1-MMP-dependent dedifferentiation was mediated by the PDGF-BB-PDGFRbeta pathway in parallel with the proteolytic processing of the multifunctional LDL receptor-related protein LRP1 and the dynamic internalization of a PDGFRbeta-beta3-integrin-MT1-MMP-LRP1 multi-component complex. Importantly, LRP1 silencing allowed the PDGF-BB-induced dedifferentiation program to proceed in the absence of MT1-MMP activity, supporting the role of unprocessed LRP1 as a gatekeeper of VSMC differentiation. Hence, MT1-MMP and LRP1 serve as a new effector-target-molecule axis that controls the PDGF-BB-PDGFRbeta-dependent VSMC phenotype and function.

The platelet-derived growth factor beta receptor (PDGFbetaR) plays an important role in proliferation and motility of fibroblasts. We have been investigating the effects of sustained PDGFbetaR activation in mortal human diploid fibroblasts (HDFs), which are typically difficult to transform. We have previously shown that the bovine papillomavirus E5 protein, through its ability to crosslink and constitutively activate the PDGFbetaR, induces morphological transformation, enhanced growth and loss of contact inhibition (focus formation) in HDFs. Here, we characterized two E5 mutants as being severely defective for focus formation but still competent for enhanced growth, suggesting that proliferation is insufficient for loss of contact inhibition. These E5 mutants were then used in a comparative study to distinguish the PDGFbetaR signaling intermediates required for the enhanced growth phenotype from those required for focus formation. Our data suggested that a PI 3-kinase (PI3K)-AKT-cyclin D3 pathway, a Grb2-Gab1-SHP2 complex and JNK played a role in the enhanced growth phenotype. However, a SHP2-p66Shc-p190BRhoGAP complex and ROCK were implicated exclusively in focus formation. We speculate that a SHP2-p66Shc-p190BRhoGAP signaling complex recruited to the activated PDGFbetaR promotes a distinct Rho-dependent process required for focus formation but not growth of HDFs.

Combined stimulation with VEGF-A, FGF-2, or PDGF-BB has emerged as a potent strategy for therapeutic angiogenesis, although the mechanisms underlying the synergism of these factors are not well understood. In the present study, we investigated the mechanism of synergism between VEGF-A and FGF-2 by using Matrigel plug assay in vivo and embryonic stem cell (ESC)-derived VEGF receptor 2 (VEGFR2)-positive cells in vitro. Experiments in vitro revealed that, in addition to having direct mitogenic effects, these molecules enhance intercellular PDGF-B signaling in a cell-type specific manner: VEGF-A enhances endothelial PDGF-B expression, whereas FGF-2 enhances mural PDGF receptor beta (PDGFRbeta) expression. Co-stimulation with VEGF-A and FGF-2 caused significant mural cell recruitment in vitro and formation of functional neovasculature in vivo, compared with single-agent stimulation. These effects were abrogated not only by anti-PDGFRbeta neutralizing antibody, but also by exogenous PDGF-BB, which could overwhelm the endogenous PDGF-BB distribution. These findings indicated the importance of preservation of the periendothelial PDGF-BB gradient. Thus, we demonstrated that the directional enhancement of endogenous PDGF-B-PDGFRbeta signaling is indispensable for the synergistic effect of VEGF-A and FGF-2 on neoangiogenesis in adults. The findings provide insights into the mechanisms underlying the effects of co-stimulation by growth factors, which could lead to rational design of therapeutic angiogenic strategies.

The expression of the PDGF beta-receptor is tightly regulated during a normal cell cycle. c-Myc and p73alpha repress transcription of the receptor through interaction with NF-Y. In ST15A cells which stably express the temperature-sensitive SV40 large T antigen (LT) the receptor expression and ligand binding decreased under the permissive condition. Transient expression of the LT, but not small t, decreased the endogenous receptor expression at mRNA and protein levels in NIH3T3 cells but not in the myc-null HO15.19 cells. The wild-type LT, but not the various pRb or p53 binding defective LT mutants, represses the PDGF beta-receptor promoter activity. Moreover, the inability of the LT-mediated repression in the myc-null cells, the Rb-null 3T3 cells, and the Saos-2 cells lacking pRb and p53, indicates that Myc, pRb and p53 are all necessary elements. PDGF beta-receptor promoter-luciferase assays revealed that the CCAAT motif is important for the repression. Furthermore, p53 was found to increase the promoter activity mainly via the upstream Sp1 binding sites together with the CCAAT motif in the NIH 3T3 cells. This was confirmed by Schneider's Drosophila line (SL2) cells deficient in both endogenous NF-Y and Sp1. Chromatin immunoprecipitation using ST15A cells revealed that both LT and p53 bound the PDGF beta-receptor promoter and the binding of p53 diminished when LT was expressed in the permissive condition. However, LT binds the promoter in the absence of pRb and p53 in Saos-2 cells stably expressing LT. These results suggest that LT binds the promoter and interferes with NF-Y and Sp1 to repress it in the presence of Myc, pRb and p53.

The Na(+)/H(+) exchanger regulatory factor, NHERF, is a multifunctional adapter protein involved in a wide range of physiological activities. NHERF associates with merlin and the ezrin/radixin/moesin (MERM) family of membrane-actin cytoskeletal linker proteins through its C-terminus and is capable of interacting via its PDZ1 domain to the betaPDGF receptor (betaPDGFR). Thus, NHERF, potentially links the betaPDGFR to the actin cytoskeleton through its interaction with MERM proteins. In the present study, we have examined whether abolishing the interaction of betaPDGFR with NHERF results in actin cytoskeletal rearrangements. We have stably expressed a wild-type betaPDGFR, a mutant betaPDGFR (L1106A) that is incapable of interacting with NHERF, as well as a kinase defective mutant receptor (K634R), in PDGFR-deficient mouse embryonic fibroblasts. Our observations indicate that cells expressing betaPDGFR (L1106A) were impaired in their ability to spread and migrate on fibronectin compared with wild-type and K634R cells. L1106A mutant cells also revealed an increased number of focal adhesions, a condensed F-actin ring at the cell periphery and a decrease in total focal adhesion kinase (FAK) tyrosine phosphorylation. Further, we show that NHERF and MERM proteins could act as intermediary bridging proteins between betaPDGFR and FAK. Thus, the interaction of betaPDGFR with NHERF may provide an essential link between the cell membrane and the cortical actin cytoskeleton independent of receptor activity.

c-Myc plays a key role in the cell cycle dependent control of the PDGF beta-receptor mRNA. The mouse platelet-derived growth factor (PDGF) beta-receptor promoter contains a CCAAT motif, and NF-Y plays an essential role in its transcription. Coexpression of c-Myc represses PDGF beta-receptor luciferase reporter activity, and the CCAAT motif in the promoter is indispensable for this repression. Here we show that c-Myc binds NF-Y subunits, YB and YC, by immunoprecipitation from cotransfected COS-1 cells. The in vitro-translated c-Myc also binds the glutathione S-transferase (GST)-NF-YB fusion protein and GST-NF-YC, but not GST-NF-YA. The most C-terminal region of HAP domains of NF-YB and NF-YC, and the Myc homology boxes, but not the C-terminal bHLHZip domain, are indispensable for the coimmunoprecipitation, and also for the repression of PDGF beta-receptor. c-Myc binds NF-Y complex without affecting the efficiency of NF-Y binding to DNA. However, the expression of Myc represses the transcriptional activation of NF-YC when fused to the GAL4 DNA binding domain. Furthermore, this repression was seen only when Myc homology boxes are present, and NF-YC contains the c-Myc binding region.