Structural and Functional Properties of Platelet-Derived Growth Factor and Stem Cell Factor Receptors

The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors—including gene fusions, point mutations, and amplifications—drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma.The type III tyrosine kinase receptor family consists of platelet-derived growth factor (PDGF) receptor α and β, stem cell factor (SCF) receptor (Kit), colony-stimulating factor-1 (CSF-1) receptor, and Flt-3 (Blume-Jensen and Hunter 2001). Members of this receptor family are characterized by five Ig-like domains in their extracellular part, a single transmembrane domain, and an intracellular part consisting of a rather well-conserved juxtamembrane domain, a tyrosine kinase domain with a characteristic inserted sequence without homology with kinases, and a less well-conserved carboxy-terminal tail. The ligands for these receptors are all dimeric molecules, and on binding they induce receptor dimerization. Although the overall mechanisms for the activation of the type III tyrosine kinase receptors and the signaling pathways they induce are similar, the receptors are expressed on different cell types and thus have different functions in vivo.Here we will describe the structural and functional properties of the PDGF receptors and Kit.     

Expression of Nerve Growth Factor and Nerve Growth Factor Receptor Genes in Human Tissues and in Prostatic Adenocarcinoma Cell Lines

Nerve growth factor (NGF) mRNAs were detected and quantified in a variety of normal and neoplastic human tissues by northern blot hybridization. Human heart contained the highest NGF mRNA levels, whereas lower but comparable levels were found in the placenta, prostate, and kidney. All tissues examined coexpressed the low-affinity NGF receptor (LNGFR), whereas none of these tissues expressed the high-affinity NGF receptor encoded by the trk protooncogene. The widespread distribution of the LNGFR suggests that it plays a role in the regulation of normal cell growth. No overexpression of NGF or LNGFR mRNA was detected in neoplastic tissues, whereas LNGFR-like immunoreactivity was localized outside of tumor cells. Transforming growth factor-alpha and protooncogene c-fos expression in these tissues did not show a systematic correlation with NGF/LNGFR expression. Furthermore, regulation of the human NGF gene was studied in DU145 cells, a prostatic adenocarcinoma cell line that synthesizes significant NGF mRNA levels. Serum induced, whereas dexamethasone inhibited, NGF mRNA synthesis in these cells. Serum induction was preceded by a rapid and transient activation of the c-fos protooncogene.     

结缔组织生长因子对血管生成和细胞功能的调节

结缔组织生长因子(CTGF)是CCN家族的主要成员,是研究较多的CCN蛋白之一。CTGF能促进细胞的增殖、存活、迁移和黏附,调节血管生成分子(bFGF,VEGF)以及一些影响胞外基质完整性和稳定性的分子(胶原、MMPs和TIMPs)的活性。CTGF可以在多个控制位点采取直接或间接的机制调控细胞功能和血管生成。结合新的发现和新的视点,阐述CTGF对细胞功能和血管生成的调控作用以及与肿瘤生长的关系。     

Promotion of Plant Cell Division by an Epidermal Growth Factor

In some specified treatments, an epidermal growth factor (EGF)promoted adventitious root formation in epicotyl cuttings ofVigna angularis. The number of the roots induced in cuttingstreated with 0.1 mg liter^-1 EGF during the first 24 h and with210^-4 M IAA during the second 24 h was 15% greater than thatof the roots in cuttings treated without EGF and with IAA. Analysisof the optimum timing of EGF application was performed by dividingthe first 24 h period into three sequential 8 h periods (0–8h, 8–16 h and 16–24 h). The most effective timeperiods in terms of the root formation were 8–16 h and16–24 h. The 0–8 h period was ineffective with respectto the formation. When carrot suspension cells were culturedfor 15 days at a very low cell density (1,000 cells/3 ml Murashigeand Skoog's medium) with more than 0.1 mg liter^-1 EGF, cellnumbers were 72% higher than those cultured without EGF. Theseresults suggest that EGF promotes cell division of plants. (Received October 5, 1992; Accepted May 24, 1993)     

Loss of SIRT3 Provides Growth Advantage for B Cell Malignancies

B cell malignancies comprise a diverse group of cancers that proliferate in lymph nodes, bone marrow, and peripheral blood. SIRT3 (sirtuin 3) is the major deacetylase within the mitochondrial matrix that promotes aerobic metabolism and controls reactive oxygen species (ROS) by deacetylating and activating isocitrate dehydrogenase 2 (IDH2) and superoxide dismutase 2 (SOD2). There is controversy as to whether SIRT3 acts as an oncogene or a tumor suppressor, and here we investigated its role in B cell malignancies. In mantle cell lymphoma patient samples, we found that lower SIRT3 protein expression was associated with worse overall survival. Further, SIRT3 protein expression was reduced in chronic lymphocytic leukemia primary samples and malignant B cell lines compared to primary B cells from healthy donors. This lower level of expression correlated with hyperacetylation of IDH2 and SOD2 mitochondrial proteins, lowered enzymatic activities, and higher ROS levels. Overexpression of SIRT3 decreased proliferation and diminished the Warburg-like phenotype in SIRT3-deficient cell lines, and this effect is largely dependent on deacetylation of IDH2 and SOD2. Lastly, depletion of SIRT3 from malignant B cell lines resulted in greater susceptibility to treatment with an ROS scavenger but did not result in greater sensitivity to inhibition of the hypoxia-inducible factor-1α pathway, suggesting that loss of SIRT3 increases proliferation via ROS-dependent but hypoxia-inducible factor-1α-independent mechanisms. Our study suggests that SIRT3 acts as a tumor suppressor in B cell malignancies, and activating the SIRT3 pathway might represent a novel therapeutic approach for treating B cell malignancies.     

Synergistic Signaling of Tumor Cell Invasiveness by Hepatocyte Growth Factor and Hypoxia

Hepatocyte growth factor (HGF) signaling promotes tumor invasiveness in renal cell carcinoma (RCC) and other cancers. In clear cell RCC, VHL loss generates pseudohypoxia that exacerbates HGF-driven invasion through β-catenin deregulation. Hypoxia also enhances HGF-driven invasiveness by papillary RCC cells, but in the absence of VHL, loss signaling integration involves three parallel routes: 1) hypoxia-induced reactive oxygen species production and decreased DUSP2 expression, leading to enhanced mitogen-activated protein kinase (MAPK) cascade activation; 2) reactive oxygen species-induced diacylglycerol production by phospholipase Cγ, leading to protein kinase C activation and increased protein phosphatase-2A activity, thereby suppressing HGF-induced Akt activation; and 3) a profound shift from HGF-enhanced, proliferation-oriented metabolism to autophagy-dependent invasion and suppression of proliferation. This tripartite signaling integration was not unique to RCC or HGF; in RCC cells, invasive synergy induced by the combination of hypoxia and epidermal growth factor occurred through the same mechanism, and in estrogen receptor-positive breast cancer cells, this mechanism was suppressed in the absence of estrogen. These results define the molecular basis of growth factor and hypoxia invasive synergy in VHL-competent papillary RCC cells, illustrate the plasticity of invasive and proliferative tumor cell states, and provide signaling profiles by which they may be predicted.     

表皮生长因子对鼠胚成纤维细胞细胞周期的调节作用

本文观察了表皮生长因子（ＥＧＦ）对小鼠胚胎成纤维细胞Ｃ_３Ｈ／１０Ｔ_１／２ＣＬ_８（简称Ｃ_３Ｈ／１０）细胞周期的影响。结果表明：ＥＧＦ使Ｓ期提前,细胞周期缩短。进一步探讨了ＥＧＦ对细胞周期影响的机制,发现ＥＧＦ可活化在细胞周期调节中起重要作用的Ｐ３４~（ｃｄｃ２）激酶（简称ＣＤ２Ｋ）,使ＣＤ２Ｋ在周期中活性高峰出现的时间提前,提示ＥＧＦ对细胞周期的影响可能通过作用于ＣＤ２Ｋ实现的。     

Cell Migration Is Regulated by Platelet-Derived Growth Factor Receptor Endocytosis

Cell migration requires spatial and temporal processes that detect and transfer extracellular stimuli into intracellular signals. The platelet-derived growth factor (PDGF) receptor is a cell surface receptor on fibroblasts that regulates proliferation and chemotaxis in response to PDGF. How the PDGF signal is transmitted accurately through the receptor into cells is an unresolved question. Here, we report a new intracellular signaling pathway by which DOCK4, a Rac1 guanine exchange factor, and Dynamin regulate cell migration by PDGF receptor endocytosis. We showed by a series of biochemical and microscopy techniques that Grb2 serves as an adaptor protein in the formation of a ternary complex between the PDGF receptor, DOCK4, and Dynamin, which is formed at the leading edge of cells. We found that this ternary complex regulates PDGF-dependent cell migration by promoting PDGF receptor endocytosis and Rac1 activation at the cell membrane. This study revealed a new mechanism by which cell migration is regulated by PDGF receptor endocytosis.Chemoattractants bind to cell surface receptors, resulting in the cytoskeletal reorganization that permits the migration of cells toward a stimulus. In fibroblasts, the platelet-derived growth factor receptor β (PDGFRβ) is a cell surface receptor tyrosine kinase (RTK) that regulates cell proliferation and chemotaxis in response to PDGF. PDGF binding activates PDGF receptor autophosphorylation, which in turn mediates a series of intracellular signaling cascades initiated by the association of SH2 domain-containing adaptor proteins (25). The adaptor protein Grb2 at the plasma membrane binds to Ras exchange factor Sos1, activating mitogen-activated protein kinase (MAPK) and cell proliferation signals (19). Grb2 also plays a critical role in receptor internalization via its interaction with dynamin, an exchange factor that facilitates receptor entry into endocytic vesicles (32). Grb2 regulates ubiquitination and the degradation of the receptor via its interaction with Cbl, an E3 ubiquitin ligase (33). While the role of Grb2 in modulating receptor levels and facilitating growth factor-dependent mitogenic signals is defined, its role in coordinating receptor-dependent chemotaxis has not been elucidated.The small GTPase Rac1 plays a crucial role in PDGF-mediated chemotaxis by regulating cortical actin at the leading edge of cells. PDGF receptor activation promotes GTP loading and the translocation of Rac1 to the cell membrane via guanine exchange factors (GEFs). The DOCK family of Rac1 GEFs, also called CDM proteins (for Caenorhabditis elegans ced-5, vertebrate DOCK180, and Drosophila myoblast city), are regulators of cell migration and have been implicated in various biological processes, such as lymphocyte migration, phagocytosis, and cancer progression (6, 10, 30, 35). In migrating fibroblasts, DOCK proteins localize to the cell''s leading edge via their interaction with the phospholipid PIP3, but a direct molecular link to PDGF has not been established (5). Biochemical studies show that Rac activation requires the DHR2/docker domain of DOCK proteins and the expression of the PH domain-containing protein Ced-12/ELMO. Previously we identified DOCK4 in a screen for novel tumor suppressor genes using representational difference analysis on mouse tumor cell lines (35). DOCK4, like other CDM proteins, binds ELMO and exerts its biochemical effects on the small GTPases Rac and Rap1 (30, 35). An interesting observation is that the amino acid sequence toward the C terminus is not conserved among individual DOCK family members. The alternate splicing of the DOCK4 gene has been reported, but how amino acid sequence variation alters the signaling properties of DOCK4 for the regulation of cell migration is unknown.Members of the Nck family of adaptor proteins, CrkII and Nck, have been reported to bind to the C terminus of DOCK180 (12, 29). Here, we show that the third member of the family of Nck adaptors, namely Grb2, binds to wild-type DOCK4. We found that a ternary complex formed by Grb2-DOCK4-Dynamin2 interacts with PDGF-activated PDGFβ receptor and promotes growth factor-dependent migration without altering cell proliferation. PDGF-dependent migration requires receptor endocytosis and is regulated by the formation of a DOCK4-Grb2-Dynamin2-PDGFRβ complex at the cell''s leading edge. These studies provide novel mechanistic insights into PDGFRβ regulation and cell migration.     

Nerve Growth Factor Administration Attenuates Cognitive but Not Neurobehavioral Motor Dysfunction or Hippocampal Cell Loss Following Fluid-Percussion Brain Injury in Rats

Abstract: Lateral fluid-percussion brain injury in rats results in cognitive deficits, motor dysfunction, and selective hippocampal cell loss. Neurotrophic factors have been shown to have potential therapeutic applications in neurodegenerative diseases, and nerve growth factor (NGF) has been shown to be neuroprotective in models of excitotoxicity. This study evaluated the neuroprotective efficacy of intracerebral NGF infusion after traumatic brain injury. Male Sprague-Dawley rats received lateral fluid-percussion brain injury of moderate severity (2.1–2.3 atm). A miniosmotic pump was implanted 24 h after injury to infuse NGF (n = 34) or vehicle (n = 16) directly into the region of maximal cortical injury. Infusions of NGF continued until the animal was killed at 72 h, 1 week, or 2 weeks after injury. Animals were evaluated for cognitive dysfunction (Morris Water Maze) and regional neuronal cell loss (Nissl staining) at each of the three time points. Animals surviving for 1 or 2 weeks were also evaluated for neurobehavioral motor function. Although an improvement in memory scores was not observed at 72 h after injury, animals receiving NGF infusions showed significantly improved memory scores when tested at 1 or 2 weeks after injury compared with injured animals receiving vehicle infusions ( p < 0.05). Motor scores and CA3 hippocampal cell loss were not significantly different in any group of NGF-treated animals when compared with controls. These data suggest that NGF administration, in the acute, posttraumatic period following fluid-percussion brain injury, may have potential in improving post-traumatic cognitive deficits.     

酸性成纤维细胞生长因子的促细胞增殖作用和对实验秃毛大鼠的疗效研究

利用大肠杆菌菌株表达出高纯度的酸性成纤维细胞生长因子,并对其促3T3细胞的增殖作用和对实验秃毛大鼠的疗效进行了研究。结果表明,在体外实验中,1.95 ng/ml~1000ng/ml的aFGF溶液可以促进balb/c 3T3细胞的分裂增殖,与PBS组比较具有显著性差异(P<0.05,P<0.01);在体内实验中,在第7天,4μg/ml aFGF组大鼠毛发长度变长,与模型对照组比较有显著性差异(P<0.05);在第14天,2μg/ml 和4μg/ml aFGF组大鼠毛发长度继续变长,与模型对照组比较有显著性差异(P<0.05)。病理检查结果显示aFGF可以促进实验秃毛大鼠的毛囊数目增多,毛囊无萎缩变小现象,血管无充血现象,基本恢复到正常水平。由此得出aFGF可以促进3T3细胞的分裂增殖,以及促进实验秃毛大鼠的毛发生长。aFGF具有很好的开发前景。     

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Δ) complemented the Rin1 depletion effects, and overexpression of Rin1Δ had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.Internalization of epidermal growth factor receptors (EGFR)² and their subsequent delivery to lysosomes play key roles in attenuating EGF-mediated signaling cascades (1, 2). The proper delivery of EGFR into lysosomes for degradation requires a series of highly regulated targeting and delivery events. Following ligand binding, EGFR is internalized via endocytic vesicles that are subsequently targeted to early endosomes. This targeting event is mediated by the small GTPase, Rab5 (3, 4). Once delivered to the early endosome, receptors that are destined for degradation are incorporated into vesicles that bud into the lumen of the endosome, forming the multivesicular body (reviewed in Refs. 5, 6). Sequestration of the activated cytoplasmic domain of EGFR into the intralumenal vesicles of the multivesicular body effectively terminates receptor signaling (7). Subsequent fusion of the multivesicular body with lysosomes delivers the intralumenal vesicles and their contents into the lumen of the lysosome where they are degraded (reviewed in Refs. 8–10). Inactivating mutations in Rab5 disrupt the delivery of cell surface receptors, such as EGFR, to early endosomes, thereby inhibiting receptor trafficking to the lysosome and receptor degradation (11, 12). Therefore, activation of Rab5 is a key point of regulation for EGFR signaling.Rab5 cycles between an inactive GDP-bound state and an active GTP-bound state, and Rab5 activation requires the exchange of GDP to GTP. This exchange is catalyzed by guanine nucleotide exchange factors (GEFs) that are specific to the Rab5 family of proteins (reviewed in Ref. 13). Rab5 family GEFs all contain a catalytic vacuolar protein sorting 9 (Vps9) domain that facilitates the GDP to GTP exchange (14–17). Many Rab5 GEFs contain other functional domains that are involved in cell signaling events (13). Rin1 is a good example of a multidomain Rab5 GEF. In addition to the Vps9 domain, Rin1 also contains an Src homology 2 domain, a proline-rich domain, and a Ras association domain. Rin1 was originally identified through its ability to interact with active Ras (18), and a role for Rin1 in a number of cell signaling systems has been established, including EGF-mediated signaling (19–21). Rin1 directly interacts with the activated EGFR through its Src homology 2 domain (22). Furthermore, Ras occupation of the Rin1 Ras association domain positively impacts the Rab5 GEF activity of Rin1, which promotes EGFR internalization and attenuation in fibroblasts (23). However, Rin1 expression is up-regulated in several types of cancers, including squamous cell carcinoma (24), colorectal cancer (25), and cervical cancer (26), through duplications or rearrangements of the RIN1 locus. These studies suggest that Rin1 may also play a role in enhancing cell proliferation.It is well established that a large percentage of non-small cell lung adenocarcinomas exhibit up-regulation of EGFR and aberrant signaling through the Ras/MAPK pathway (reviewed in Ref. 27). In addition, a recent study examining 188 human lung adenocarcinomas identified that 132 of 188 tumor samples exhibited mutations relating to the Ras/MAPK signaling pathway (28). Accordingly, the role of Rin1 in non-small cell lung adenocarcinoma was addressed. Examination of a panel of non-small cell lung adenocarcinoma lines (including A549) revealed enhanced Rin1 expression relative to a nontransformed lung epithelial cell line (BEAS-2B). Depletion of Rin1 from A549 cells resulted in decreased proliferation. This decrease correlated with a reduction in EGF-activated ERK phosphorylation and the stabilization of cell surface EGFR. These defects were complemented by wild type Rin1 expression but not by mutant Rin1 lacking a functional Vps9 domain, suggesting that the GEF activity of Rin1 is necessary for proper EGFR signaling in A549 cells. In addition, overexpression of Rin1Δ, dominant negative Rab5, and dynamin resulted in similar defects in cell proliferation and EGFR signaling as Rin1 depletion. These data indicate that proper EGFR internalization and trafficking are critical for robust EGFR-mediated signaling and cell proliferation in A549 cells and offer evidence that Rin1 positively regulates cell proliferation in non-small cell lung adenocarcinoma.     

Epidermal Growth Factor Induces Internalization But Not Degradation of the Epidermal Growth Factor Receptor in a Human Breast Cancer Cell Line

Abstract

Metabolism of the epidermal growth factor (EGF) receptor was studied in the MDA-MB-231 human breast cancer cell line. As in normal fibroblasts the EGF receptor from MDA-MB-231 cells was synthesized from a M_r =160,000 precursor and tunicamycin treatment of cells resulted in accumulation of a M_r =130,000 polypeptide. Unlike normal fibroblasts in which a M_r =170,000 mature form of the EGF receptor was found, MDA-MB-231 cells contained a M_r =172,000 mature form. Addition of EGF to MDA-MB-231 cells led to rapid internalization of EGF receptors, however, internalization did not affect receptor half-life and receptors did not recycle to the cell surface. EGF receptors could be visualized by immunofluorescence and remained sequestered in intracellular membranous structures following internalization. EGF was degraded slowly by MDA-MB-231 cells relative to degradation of EGF by normal cells. A high endogenous level of in vivo phosphorylation of threonine 654 of the EGF receptor was found in MDA-MB-231 cells and treatment of cells with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further stimulated phosphorylation of this residue. EGF induced receptor internalization resulted in dephosphorylation of threonine 654. The significance of these unusual properties of EGF receptor metabolism in MDA-MB-231 cells is discussed.     

Cell Density Modulates Receptor-Mediated Internalization of Nerve Growth Factor in Pheochromocytoma Cells

Abstract

Binding and fate of the nerve growth factor (NGF) in pheochromocytom a cells (clone PC12) have been measured with the use of iodine-labeled ligand and with ¹²⁵I-NGF antibodies. With such double approach it is possible to distinguish between surface bound and total NGF bound to PC12 cells. It is found that NGF-receptor complexes undergo down-regulation. This process is noticeable at low but not at high cell densities, and only in PC12 cells never exposed to NGF. Previous incubation with growth factor leads to the disappearance of down-regulation of NGF-receptor complexes. Assuming that this process is an indirect measure of NGF-receptor internalization, it is concluded that it is modulated by the cell density or by previous exposure to the factor. These findings are postulated to be relevant to the mechanism of action of NGF and to its multiple effects on target cells.     

Regulation of Platelet-derived Growth Factor Receptor Function by Integrin-associated Cell Surface Transglutaminase

A functional collaboration between growth factor receptors such as platelet derived growth factor receptor (PDGFR) and integrins is required for effective signal transduction in response to soluble growth factors. However, the mechanisms of synergistic PDGFR/integrin signaling remain poorly understood. Our previous work showed that cell surface tissue transglutaminase (tTG) induces clustering of integrins and amplifies integrin signaling by acting as an integrin binding adhesion co-receptor for fibronectin. Here we report that in fibroblasts tTG enhances PDGFR-integrin association by interacting with PDGFR and bridging the two receptors on the cell surface. The interaction between tTG and PDGFR reduces cellular levels of the receptor by accelerating its turnover. Moreover, the association of PDGFR with tTG causes receptor clustering, increases PDGF binding, promotes adhesion-mediated and growth factor-induced PDGFR activation, and up-regulates downstream signaling. Importantly, tTG is required for efficient PDGF-dependent proliferation and migration of fibroblasts. These results reveal a previously unrecognized role for cell surface tTG in the regulation of the joint PDGFR/integrin signaling and PDGFR-dependent cell responses.Adhesion of cells to the extracellular matrix (ECM)² regulates a wide range of cellular processes, including cell survival, growth, migration, and differentiation. A central paradigm in the field entails both physical association and functional collaboration between integrins and growth factor receptors (GFRs) in the regulation of cell responses to the ECM and soluble growth factors (1). In particular, the engagement of β1 and αvβ3 integrins with ECM ligands transiently activates platelet-derived growth factor (PDGF) receptor-tyrosine kinase even in the absence of its soluble ligands and promotes and sustains growth factor-initiated signaling by PDGFR (2). Despite a significance of this synergistic signaling, the molecular mechanisms underlying the cross-talk between the two receptor systems remain unknown. A direct or indirect association between these two types of signaling receptors may be enhanced by their co-sequestering in cholesterol-enriched membrane microdomains (3). Because integrins and receptor-tyrosine kinases share many downstream signaling targets, integrin-ECM interaction may also increase availability of signal relay enzymes and adapter proteins to receptor-tyrosine kinases by promoting their recruitment from cytosol to the plasma membrane (4).PDGF is a major survival factor, mitogen, and motogen for mesenchymal cells (5). This ligand-receptor pair is implicated in tumor-associated processes, including autocrine growth stimulation of tumor cells, tumor angiogenesis, and regulation of stromal fibroblasts (6). Atherosclerosis in the vessel wall and restenosis after angioplasty also involve hyperactivation of the PDGF-PDGFR signaling axis in vascular smooth muscle cells (7). Likewise, skin wound healing and liver, lung, and kidney fibrosis depend on PDGF-mediated signaling and cell responses (8). Importantly, ECM composition and cell-matrix interactions modulate cell responsiveness to PDGF (9).Upon binding a dimeric PDGF molecule, PDGFR undergoes dimerization and autophosphorylation of tyrosine residues in trans because of the juxtaposition of cytoplasmic tails of the receptor. Phosphorylation of the conserved tyrosine residue in the kinase domain (Tyr-849 of PDGFRα and Tyr-857 of PDGFRβ) increases catalytic activity of the kinases, whereas autophosphorylation of tyrosine residues outside the kinase domain creates docking sites for signal transduction proteins containing Src homology 2 domains. The latter include various enzymes such as phosphatidylinositol 3-kinase, phospholipase Cγ, the Src family tyrosine kinases, the tyrosine phosphatase Shp-2, and the GTPase activating protein for Ras, RasGAP. Other PDGFR binding partners including Grb2, Grb7, Nck, Shc, and Crk lacking enzymatic activity but serve adapter functions in the downstream signaling pathways (10).Previous studies revealed a transient PDGF-independent tyrosine phosphorylation of PDGFRβ in human fibroblasts during adhesion on fibronectin or collagen type I, whereas similar PDGFRβ activation response was reproduced by application of external strain to quiescent cells (2). Clustering of integrins with fibronectin-coated beads was shown to stimulate PDGFR phosphorylation in fibroblasts (11). Furthermore, fibronectin was found to promote PDGF-mediated signaling in fibroblasts by increasing association of phosphatase Shp-2 with PDGFR and limiting the time that the negative signaling regulator, RasGAP, interacts with the receptor (4). Whereas these results implicate cell-ECM interactions and integrin function in the regulation of PDGFR activity, many details of this functional cross-talk remain unknown.Tissue transglutaminase (tTG) is a multifunctional protein that possesses Ca²⁺-dependent transamidating and GTPase activities (12). On the surface of various cells, all the tTG forms stable non-covalent complexes with β1 and β3 integrins and functionally collaborates with these receptors by acting as a co-receptor for fibronectin (13). This adhesive function of tTG is involved in the assembly of fibronectin matrices and cell migration on fibronectin (14–16). tTG broadly affects integrin signaling by promoting their clustering and increasing activation of focal adhesion kinase and RhoA (13, 17). Thus, we set to examine whether signaling mediated by GFRs, which depends on the integrin function, is altered by tTG.Here we present a novel mechanistic insight into the cross-talk between integrin and PDGFR signaling pathways. We provide evidence that tTG interacts with PDGFR on the cell surface and mediates its physical association with integrins. In turn, the formation of stable integrin-tTG-PDGFR ternary complexes promotes PDGFR activation and downstream signaling, regulates the receptor turnover, and amplifies PDGFR-mediated cellular responses. These studies reveal a novel function of tTG in coupling the adhesion-mediated and growth factor-dependent signaling pathways. They suggest that this tTG activity might be involved in pro-inflammatory function of this protein in normal wound healing and tissue fibrosis (18), vascular remodeling (19), and tumor metastasis (20).