Deletion Of Cytoplasmic Domain Research Articles

Two distinct mechanisms of Plexin A function in Drosophila optic lobe lamination and morphogenesis.

Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that, in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In Plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of semaphorin function indicates that Semaphorin 1a, acting in a subset of medulla neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A has little effect on the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of Plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles.

Thrombomodulin Regulates PTEN/AKT Signaling Axis in Endothelial Cells.

We recently demonstrated that deletion of thrombomodulin gene from endothelial cells results in upregulation of proinflammatory phenotype. In this study, we investigated the molecular basis for the altered phenotype in thrombomodulin-deficient (TM-/-) cells. Different constructs containing deletions or mutations in the cytoplasmic domain of thrombomodulin were prepared and introduced to TM-/- cells. The phenotype of cells expressing different derivatives of thrombomodulin and tissue samples of thrombomodulin-knockout mice were analyzed for expression of distinct regulatory genes in established signaling assays. The phosphatase and tensin homolog were phosphorylated and its recruitment to the plasma membrane was impaired in TM-/- cells, leading to hyperactivation of AKT (protein kinase B) and phosphorylation-dependent nuclear exclusion of the transcription factor, forkhead box O1. The proliferative/migratory properties of TM-/- cells were enhanced, and cells exhibited hypersensitivity to stimulation by angiopoietin 1 and vascular endothelial growth factor. Reexpression of wild-type thrombomodulin in TM-/- cells normalized the cellular phenotype; however, thrombomodulin lacking its cytoplasmic domain failed to restore the normal phenotype in TM-/- cells. Increased basal permeability and loss of VE-cadherin were restored to normal levels by reexpression of wild-type thrombomodulin but not by a thrombomodulin construct lacking its cytoplasmic domain. A thrombomodulin cytoplasmic domain deletion mutant containing 3-membrane-proximal Arg-Lys-Lys residues restored the barrier-permeability function of TM-/- cells. Enhanced phosphatase and tensin homolog phosphorylation and activation of AKT and mTORC1 (mammalian target of rapamycin complex 1) were also observed in the liver of thrombomodulin-KO mice. These results suggest that the cytoplasmic domain of thrombomodulin interacts with the actin cytoskeleton and plays a crucial role in regulation of phosphatase and tensin homolog/AKT signaling in endothelial cells.

Transcriptome profiling reveals transcriptional regulation of VISTA in T cell activation

V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a novel type of immune checkpoint. This study was performed to explore the potential mechanism by which different domains of VISTA affect T-cell activation and search for potential interacting proteins. Stably transfected Jurkat cell lines were constructed to overexpress human VISTA (VISTA-FL), cytoplasmic domain deletion mutants (VISTA-ΔECD) and extracellular domain deletion mutants (VISTA- ΔCD). Empty vector (EV) control cell lines were constructed. Four stable cell lines were subjected to transcriptome sequencing after stimulation with PMA and PHA. The differentially expressed genes (DEGs) were analysed to explore the potential pathway by which VISTA inhibits T-cell activation. Proteinprotein interaction (PPI) network analysis was used to search for potential interacting proteins of VISTA. In this study, 1256 DEGs were identified in Jurkat-VISTA-FL cells, 740 DEGs in Jurkat-VISTA-ΔCD cells, and 5605 DEGs in Jurkat-VISTA-ΔECD cells compared with Jurkat-EV cells. DEGs were mainly enriched in pathways related to T-cell differentiation, T-cell receptor signalling pathway and T-cell migration in Jurkat-VISTA-ΔECD cells; with cholesterol biosynthesis in Jurkat-VISTA-ΔCD cells; and with the inflammatory response in Jurkat-VISTA-FL cells. HHLA2 and CTH were identified as potential partners that interact directly with VISTA. The results also show an indirect interaction between VISTA and PSGL-1. This study revealed the pathways by which VISTA is involved in T-cell activation and identified the potential binding partners of VISTA through RNA-seq, providing valuable resources for developing in-depth studies of the action mechanisms of VISTA as a potential target for cancer and inflammatory diseases.

Evaluation of the Safety and Immunogenicity of Duck-Plague Virus gE Mutants.

Duck plague (DP) is an acute infectious disease in the duck industry. The duck plague virus (DPV) is the pathogen, a subfamily of alphaherpesvirinae. gE is a type I membrane protein that contains three parts: an extracellular domain, a transmembrane domain, and a cytoplasmic domain. gE is the major virulence determinant of α-herpesvirus. However, the functions of the gE extracellular and cytoplasmic domains have not been reported in DPV. In this study, a gE extracellular domain deletion mutant and a gE cytoplasmic domain deletion mutant were constructed from DPV. Virus replication kinetics showed that the growth titers of both the gE ectodomain-deleted mutant virus and the gE cytoplasmic domain-deleted virus in DEFs were lower than that of the parental virus CHv-50. DPV CHv-gEΔET and DPV CHv-gEΔCT were continuously passed to the 20th passage in DEFs and the 10th in ducklings. The mutant virus DNA after passage was extracted for identification. The results showed that the gE ectodomain and gE cytoplasmic domain deletion mutant viruses have good genetic stability. The ducklings in each group (n=10) were inoculated with the same titers of DPV CHv-gEΔET, DPV CHv-gEΔCT, DPV CHv-ΔgE, and parental CHv-50, respectively. Clinical symptoms and serum antibody levels were detected after inoculation. The results showed that the virulence of DPV CHv-gEΔCT to ducklings was reduced compared with parental CHv-50, while the virulence of DPV CHv-gEΔET to ducklings was significantly reduced. 105 TCID50 DPV CHv-gEΔET or DPV CHv-ΔgE can induce ducklings to produce DPV-specific antibodies, protect the ducklings from virulent CHv challenge. Therefore, DPV CHv-gEΔET may serve as a promising vaccine candidate to prevent and control duck plague.

Mitochondrial reactive oxygen species trigger metformin-dependent antitumor immunity via activation of Nrf2/mTORC1/p62 axis in tumor-infiltrating CD8T lymphocytes

BackgroundMetformin (Met) is the first-line treatment for type 2 diabetes mellitus and plays an effective role in treating various diseases, such as cardiovascular disease, neurodegenerative disease, cancer, and aging. However,...

Deletion of BST2 Cytoplasmic and Transmembrane N-Terminal Domains Results in SARS-CoV, SARS-CoV-2, and Influenza Virus Production Suppression in a Vero Cell Line.

SARS-CoV-2, which emerged in Wuhan (China), has become a great worldwide problem in 2020 and has led to more than 1,000,000 deaths worldwide. Many laboratories are searching for ways to fight this pandemic. We studied the action of the cellular antiviral protein tetherin, which is encoded by the BST2 gene. We deleted the transmembrane domain-encoding part of the gene in the Vero cell line. The transmembrane domain is a target for virus-antagonizing proteins. We showed a decrease in SARS-CoV-2 in cells with deleted transmembrane BST2 domains compared to the initial Vero cell line. Similar results were obtained for SARS-CoV and avian influenza virus. This finding may help the development of antiviral therapies competitively targeting the transmembrane domain of tetherin with viral-antagonizing proteins.

Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly

The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

Analysis of the entry mechanism of Crimean-Congo hemorrhagic fever virus, using a vesicular stomatitis virus pseudotyping system.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease causing severe hemorrhagic symptoms with a nearly 30 % case-fatality rate in humans. The experimental use of CCHF virus (CCHFV), which causes CCHF, requires high-biosafety-level (BSL) containment. In contrast, pseudotyping of various viral glycoproteins (GPs) onto vesicular stomatitis virus (VSV) can be used in facilities with lower BSL containment, and this has facilitated studies on the viral entry mechanism and the measurement of neutralizing activity, especially for highly pathogenic viruses. In the present study, we generated high titers of pseudotyped VSV bearing the CCHFV envelope GP and analyzed the mechanisms involved in CCHFV infection. A partial deletion of the CCHFV GP cytoplasmic domain increased the titer of the pseudotyped VSV, the entry mechanism of which was dependent on the CCHFV envelope GP. Using the pseudotype virus, DC-SIGN (a calcium-dependent [C-type] lectin cell-surface molecule) was revealed to enhance viral infection and act as an entry factor for CCHFV.

Concanavalin-A-induced autophagy biomarkers requires membrane type-1 matrix metalloproteinase intracellular signaling in glioblastoma cells

Pre-clinical trials for cancer therapeutics support the anti-neoplastic properties of the lectin from Canavalia ensiformis (Concanavalin-A, ConA) in targeting apoptosis and autophagy in a variety of cancer cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP), a plasma membrane-anchored matrix metalloproteinase, is a glycoprotein strongly expressed in radioresistant and chemoresistant glioblastoma that mediates pro-apoptotic signalling in brain cancer cells, we investigated whether MT1-MMP could also signal autophagy. Among the four lectins tested, we found that the mannopyranoside/glucopyranoside-binding ConA, which is also well documented to trigger MT1-MMP expression, increases autophagic acidic vacuoles formation as demonstrated by Acridine Orange cell staining. Although siRNA-mediated MT1-MMP gene silencing effectively reversed ConA-induced autophagy, inhibition of the MT1-MMP extracellular catalytic function with Actinonin or Ilomastat did not. Conversely, direct overexpression of the recombinant Wt-MT1-MMP protein triggered proMMP-2 activation and green fluorescent protein-microtubule-associated protein light chain 3 puncta indicative of autophagosomes formation, while deletion of MT1-MMP's cytoplasmic domain disabled such autophagy induction. ConA-treated U87 cells also showed an upregulation of BNIP3 and of autophagy-related gene members autophagy-related protein 3, autophagy-related protein 12 and autophagy-related protein 16-like 1, where respective inductions were reversed when MT1-MMP gene expression was silenced. Altogether, we provide molecular evidence supporting the pro-autophagic mechanism of action of ConA in glioblastoma cells. We also highlight new signal transduction functions of MT1-MMP within apoptotic and autophagic pathways that often characterize cancer cell responses to chemotherapeutic drugs.

Potential Relation of Aberrant Proteolysis of Human Protein Tyrosine Kinase 7 (PTK7) chuzhoi by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) to Congenital Defects

Membrane PTK7 pseudo-kinase plays an essential role in planar cell polarity and the non-canonical Wnt pathway in vertebrates. Recently, a new N-ethyl-N-nitrosourea-induced mutant named chuzhoi (chz) was isolated in mice. chz embryos have severe birth defects, including a defective neural tube, defective heart and lung development, and a shortened anterior-posterior body axis. The chz mutation was mapped to the Ala-Asn-Pro tripeptide insertion into the junction region between the fifth and the sixth Ig-like domains of PTK7. Unexpectedly, chz reduced membrane localization of the PTK7 protein. We hypothesized and then proved that the chz mutation caused an insertion of an additional membrane type 1 matrix metalloproteinase cleavage site in PTK7 and that the resulting aberrant proteolysis of chz affected the migratory parameters of the cells. It is likely that aberrations in the membrane type 1 matrix metalloproteinase/PTK7 axis are detrimental to cell movements that shape the body plan and that chz represents a novel model system for increasing our understanding of the role of proteolysis in developmental pathologies, including congenital defects.

Abstract 423: Decipher the molecular mechanisms by which syndecan-1 in breast cancer stroma fibroblasts regulates extracellular matrix fiber organization and carcinoma cell motility

Abstract The cell surface proteoglycan syndecan-1 (Sdc1) is frequently induced in the stromal fibroblasts of invasive breast carcinomas. We have recently identified a striking correlation between stromal Sdc1 expression and extracellular matrix (ECM) fiber architecture, both In vivo (in human breast carcinoma samples) and in vitro. Bioactive, cell-free 3D ECMs generated from cultures of Sdc1-positive human mammary fibroblasts (HMF), termed ECM-Sdc1, showed an organized, aligned fiber architecture, which contrasted markedly with the random fiber arrangement seen in the ECM of Sdc1-negative HMFs (ECM-mock). We further demonstrated that aligned ECM fiber architecture was responsible for the enhanced directional migration and invasion of the breast carcinoma cells when seeded into the fibroblast-free ECM-Sdc1. To decipher the molecular mechanisms responsible for the formation of an invasion-permissive ECM, a series of Sdc1 mutants was introduced into HMF. These include a heparan sulfate (HS) -deficient, a cytoplasmic domain (CD) deletion and a transmembrane domain (TMD) substitution mutant. Early experiments suggest that HS are required for the activity of Sdc1 in regulating ECM alignment, while TDM is dispensable. Integrins play a key role in the assembly of the provisional fibronectin-rich ECM and Sdc1 has been shown to be an important regulator of integrin activity. Therefore, we have begun to investigate the the role of integrins in Sdc1-mediated ECM fiber alignment. HMF expresses various integrins, as determined by Western blotting and flow cytometry. Using siRNA oligos, we are targeting different integrin subunits including α4, α5, αV and β3 to determine, which integrins are responsible for Sdc1-dependent ECM fiber alignment and consequently the invasion-permissive properties of the ECM. A better understanding of the mechanisms governing ECM organization may lead to the development of novel stroma-targeted therapy for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 423. doi:10.1158/1538-7445.AM2011-423

Domain Analysis of Protein P30 in Mycoplasma pneumoniae Cytadherence and Gliding Motility

The cell wall-less prokaryote Mycoplasma pneumoniae causes bronchitis and atypical pneumonia in humans. Mycoplasma attachment and gliding motility are required for colonization of the respiratory epithelium and are mediated largely by a differentiated terminal organelle. P30 is a membrane protein at the distal end of the terminal organelle and is required for cytadherence and gliding motility, but little is known about the functional role of its specific domains. In the current study, domain deletion and substitution derivatives of P30 were engineered and introduced into a P30 null mutant by transposon delivery to assess their ability to rescue P30 function. Domain deletions involving the extracellular region of P30 severely impacted protein stability and adherence and gliding function, as well as the capacity to stabilize terminal organelle protein P65. Amino acid substitutions in the transmembrane domain revealed specific residues uniquely required for P30 stability and function, perhaps to establish correct topography in the membrane for effective alignment with binding partners. Deletions within the predicted cytoplasmic domain did not affect P30 localization or its capacity to stabilize P65 but markedly impaired gliding motility and cytadherence. The larger of two cytoplasmic domain deletions also appeared to remove the P30 signal peptide processing site, suggesting a larger leader peptide than expected. We propose that the P30 cytoplasmic domain may be required to link P30 to the terminal organelle core, to enable the P30 extracellular domain to achieve a functional conformation, or perhaps both.

Visual Impairment in the Absence of Dystroglycan

Ocular involvement in muscular dystrophy ranges from structural defects to abnormal electroretinograms. While the mechanisms underlying the abnormal retinal physiology in patients are not understood, it is thought that alpha-dystroglycan extracellular interactions are critical for normal visual function. Here we show that beta-dystroglycan anchors dystrophin and the inward rectifying K(+) channel Kir4.1 at glial endfeet and that disruption of dystrophin and potassium channel clustering in dystroglycan mutant mice is associated with an attenuation of the electroretinogram b-wave. Glial-specific inactivation of dystroglycan or deletion of the cytoplasmic domain of beta-dystroglycan was sufficient to attenuate the electroretinogram b-wave. Unexpectedly, deletion of the beta-dystroglycan cytoplasmic domain did not disrupt the laminar structure of the retina. In contrast to the role of alpha-dystroglycan extracellular interactions during early development of the CNS, beta-dystroglycan intracellular interactions are important for visual function but not the laminar development of the retina.

Agrobacterium VirB10 domain requirements for type IV secretion and T pilus biogenesis

Agrobacterium tumefaciens VirB10 couples inner membrane (IM) ATP energy consumption to substrate transfer through the VirB/D4 type IV secretion (T4S) channel and also mediates biogenesis of the virB-encoded T pilus. Here, we determined the functional importance of VirB10 domains denoted as the: (i) N-terminal cytoplasmic region, (ii) transmembrane (TM) alpha-helix, (iii) proline-rich region (PRR) and (iv) C-terminal beta-barrel domain. Mutations conferring a transfer- and pilus-minus (Tra(-), Pil(-)) phenotype included PRR deletion and beta-barrel substitution mutations that prevented VirB10 interaction with the outer membrane (OM) VirB7-VirB9 channel complex. Mutations permissive for substrate transfer but blocking pilus production (Tra(+), Pil(-)) included a cytoplasmic domain deletion and TM domain insertion mutations. Another class of Tra(+) mutations also selectively disrupted pilus biogenesis but caused release of pilin monomers to the milieu; these mutations included deletions of alpha-helical projections extending from the beta-barrel domain. Our findings, together with results of Cys accessibility studies, indicate that VirB10 stably integrates into the IM, extends via its PRR across the periplasm, and interacts via its beta-barrel domain with the VirB7-VirB9 channel complex. The data further support a model that distinct domains of VirB10 regulate formation of the secretion channel or the T pilus.

Caveolin-1 inhibits membrane-type 1 matrix metalloproteinase activity

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent proteinase found in cholesterol-rich lipid rafts on the plasma membrane. MT1-MMP hydrolyzes extracellular matrix (ECM) proteins, activates pro-matrix metalloproteinase-2 (proMMP-2) and plays an important role in ECM remodeling, cancer cell migration and metastasis. The role of caveolin-1, an integral protein of caveolae, in the activation of MT1-MMP remains largely unknown. Here, we show that the expression of caveolin-1 attenuates the activation of proMMP-2, reduces proteolytic cleavage of ECM and inhibits cell migration. We utilized the cytoplasmic tail domain deletion (DeltaCT) or the E240A mutant of MT1-MMP. Co-expression of caveolin-1 with the wild-type or the DeltaCT MT1-MMP decreased the proMMP-2 activation and inhibited collagen degradation and cell migration. Caveolin-1 had no effect on the catalytically inert E240A MT1-MMP. Our findings suggest that caveolin-1 is essential in the down-regulation of MT1-MMP activity by promoting internalization from the cell surface.

23-OR: Deletion of cytoplasmic domain impairs the HLA class I mediated signaling pathway

23-OR: Deletion of cytoplasmic domain impairs the HLA class I mediated signaling pathway

Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery

Polyethyleneimines (PEIs) are efficient non-viral vectors for gene transfer. Heparan sulfate proteoglycans have been proposed to be the cell-surface receptors for PEI.DNA complexes (polyplexes). Here, we investigated if syndecan-1 (SDC1) and syndecan-2 (SDC2) are involved in PEI-mediated transfection. Following addition of polyplexes to HEK293 cells, green fluorescent protein-tagged SDCs rapidly formed clusters with PEI that were dependent of lipid raft integrity. However, although SDC1 overexpression slightly enhanced PEI-mediated gene expression, SDC2 dramatically inhibited it. Confocal microscopy analysis showed that SDC1.polyplex endocytosis occurred within minutes after addition of polyplexes, whereas SDC2.polyplex endocytosis took hours. Expression of SDC1 cytoplasmic deletion mutants revealed that the SDC1 cytoplasmic tail is required for gene expression, but not for clustering or endocytosis, whereas overexpression of SDC1/SDC2 chimeras showed that the SDC2 ectodomain is responsible for the inhibitory effect on gene transfer. This study provides evidence that SDCs may have opposing effects on PEI-mediated transfection.

Protease Signaling in Severe Systemic Inflammation.

Abstract The tissue factor (TF) pathway drives disseminated intravascular coagulation in sepsis. Although extensive in vitro data demonstrate proinflammatory effects of protease activated receptor (PAR) signaling, it remains unclear whether PAR signaling plays a significant role in severe systemic inflammation. In a murine model of severe, 75–90% lethal endotoxemia, we addressed the interplay between intravascular coagulation activation (based on thrombin-antithrombin complexes) and inflammatory exacerbation (based on inflammatory cytokines IL6, IL1b and MCP1) downstream of PAR signaling. TF cytoplasmic domain deleted mice displayed increased TF expression in macrophages and enhanced systemic coagulation activation. However, these mice resolved inflammation earlier than wild-type controls. Thus, intravascular coagulation activation per se does not predict survival. Rather, genetic deletion of PAR2 demonstrated that PAR2 signaling increased survival in TF cytoplasmic domain-deleted mice. PAR2 deficiency alone initially attenuated inflammatory cytokine production, but did not prevent progressive exacerbation of coagulation and inflammation, further evidence for protective PAR2 signaling in advanced, severe endotoxemia. In contrast, PAR1-deficiency had no effect on initial inflammation and coagulation, but at late stages attenuated inflammation and prolonged survival. In addition, PAR1-deficiency produced a synergistic survival benefit with TF cytoplasmic domain deletion. These experiments show that coagulation protease signaling both regulates and amplifies severe systemic inflammation. Strategies to target TF cytoplasmic domain signaling may therefore be beneficial to reduce sepsis lethality in combination with therapeutic blockade of proinflammatory PAR1 signaling.

Junctional adhesion molecule-A-induced endothelial cell migration on vitronectin is integrin αvβ3 specific

Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin superfamily, and is mainly expressed in the tight junctions of both epithelial and endothelial cells. We have recently shown that JAM-A is involved in basic fibroblast growth factor (bFGF)-induced angiogenesis. Here, we show that, when ectopically expressed in human umbilical vein endothelial cells (HUVECs), JAM-A induced enhanced cell migration on vitronectin, but had no effect on fibronectin. Use of antibodies that block integrin function indicated that the migration on vitronectin is specific to integrin alpha(v)beta(3) and not to integrin alpha(v)beta(5). JAM-A-induced migration was inhibited by anti-JAM-A antibody. Additionally, overexpression of a JAM-A cytoplasmic domain deletion mutant failed to induce HUVEC migration. Addition of phosphoinositide 3-kinase and protein kinase C inhibitors blocked JAM-A-induced migration, suggesting that these kinases act downstream of JAM-A. Immunoprecipitation analysis showed that JAM-A interacts with integrin alpha(v)beta(3), and this association was increased by engagement of the ligand-binding site of the integrin by Arg-Gly-Asp-Ser (RGDS) peptide. Furthermore, activation of both focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) on vitronectin was enhanced by JAM-A overexpression but not by its cytoplasmic domain deletion mutant. Taken together, these results suggest that signaling through JAM-A is necessary for alpha(v)beta(3)-dependent HUVEC migration and implicate JAM-A in the regulation of vascular function.

Integrin alpha2beta1 modulates EGF stimulation of Rho GTPase-dependent morphological changes in adherent human rhabdomyosarcoma RD cells.

The ability of cells to undergo shape changes is essential for diverse cellular functions including cell growth, differentiation, and movement. The present study examines how an integration of the function of alpha2beta1 integrin with that of the receptor for epidermal growth factor (EGFR) modulates EGF-stimulated morphological changes in human rhabdomyosarcoma RD transfectant cells. Upon EGF stimulation, RD transfectant cells that lacked alpha2beta1 integrin expression (RDpF) underwent contraction; in contrast, expression of alpha2beta1 on RD cells (RDX2C2) resulted in transient cell spreading. Integrin alpha2 cytoplasmic domain played a critical role in the observed alpha2beta1-mediated conversion from a cell rounding to a cell spreading phenotype. Thus, the expression of an alpha2 cytoplasmic domain deletion variant (X2C0) or a chimeric alpha2beta1 containing the cytoplasmic domain of alpha4 (X2C4) or alpha5 (X2C5), instead of alpha2, failed to mediate spreading upon EGF stimulation. Using dominant negative (DN) mutants of RhoGTPases, results revealed that RhoA activation was required for both EGF-stimulated responses of cell rounding and spreading, Cdc42 functioned in the re-spreading of cells after undergoing EGF-stimulated contraction, and Rac1 was required in alpha2beta1-mediated RD cell spreading. Therefore, alpha2beta1 integrin function can switch the Rho GTPase-dependent cell shape changes in RD cells from an EGF-stimulated cell contraction to a spreading morphology. Together, results show that integrin alpha2 cytoplasmic domain plays an indispensable role in the ability of integrin alpha2beta1 to modulate EGF stimulation of Rho-GTPase-dependent morphological changes in RD cells.