Interaction of the immunoglobulin-like cell adhesion molecule hepaCAM with caveolin-1

Subsequent to our identification of the novel immunoglobulin-like cell adhesion molecule hepaCAM, we demonstrated that hepaCAM is capable of modulating cell growth and cell–extracellular matrix interactions. In this study, we examined the localization of hepaCAM in lipid rafts/caveolae as well as the interaction of hepaCAM with the caveolar structural protein caveolin-1 (Cav-1). Our results revealed that a portion of hepaCAM resided in detergent-resistant membranes and co-partitioned with Cav-1 to low buoyant density fractions characteristic of lipid rafts/caveolae. In addition, co-localization and coimmunoprecipitation assays confirmed the association of hepaCAM with Cav-1. Deletion analysis of hepaCAM showed that the extracellular first immunoglobulin domain of hepaCAM was required for binding Cav-1. Furthermore, when co-expressed, Cav-1 induced the expression of hepaCAM as well as distributed hepaCAM to intracellular Cav-1-positive caveolar structures. Taken together, our findings indicate that hepaCAM is partially localized in the lipid rafts/caveolae and interacts with Cav-1 through its first immunoglobulin domain.     

Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility

The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.     

Syndecan-4 cytoplasmic domain regulation of turkey satellite cell focal adhesions and apoptosis

Syndecan-4 is a cell membrane proteoglycan composed of a transmembrane core protein and substituted glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains. The core protein has three domains: extracellular, transmembrane and cytoplasmic domains. The GAG and N-glycosylated chains and the cytoplasmic domain of syndecan-4, especially the amino acids: Ser(178) and Tyr(187) are critical in regulation of turkey satellite cell growth and development. How these processes are regulated is still unknown. The objective of the current study was to determine whether the syndecan-4 GAG and N-glycosylated chains and the cytoplasmic domain functions through modulating focal adhesion formation and apoptosis. Twelve mutant clones were generated: a truncated syndecan-4 without the cytoplasmic domain with or without GAG and N-glycosylated chains, and Ser(178) and Tyr(187) mutants with or without GAG and N-glycosylated chains. The wild type syndecan-4 and all of the syndecan-4 mutants were transfected into turkey myogenic satellite cells after which cell apoptosis and focal adhesion formation were measured. Syndecan-4 increased cell membrane localization of β1 integrin and the activity of focal adhesion kinase (FAK) whereas the cytoplasmic domain mutation decreased the phosphorylation of FAK. However, syndecan-4 and syndecan-4 mutants did not influence cell apoptosis. They also had no effect on vinculin or paxillin-containing focal adhesion formation. These results suggested that the syndecan-4 cytoplasmic domain plays an important role in regulating FAK activity and β1 integrin cell membrane localization but not cell apoptosis and vinculin or paxillin-containing focal adhesion formation.     

Lateral flagella are required for increased cell adherence,invasion and biofilm formation by Aeromonas spp

Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf- mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces.     

Tumor suppressor in lung cancer (TSLC)1 suppresses epithelial cell scattering and tubulogenesis

The tumor suppressor in lung cancer 1 (TSLC1/IGSF4) encodes an immunoglobulin-superfamily cell adhesion molecule whose cytoplasmic domain contains a protein 4.1-binding motif (protein 4.1-BM) and a PDZ-binding motif (PDZ-BM). Loss of TSLC1 expression is frequently observed in advanced cancers implying its involvement in tumor invasion and/or metastasis. Using Madin-Darby canine kidney cells expressing a full-length TSLC1 or various cytoplasmic deletion mutants of TSLC1, we examined the role of TSLC1 in epithelial mesenchymal transitions during the hepatocyte growth factor (HGF)-induced tubulogenesis and cell scattering. In a three-dimensional culture, the full-length TSLC1, which was localized to the lateral membrane of Madin-Darby canine kidney cysts, inhibited HGF-induced tubulogenesis. In contrast, the mutants lacking either the protein 4.1-BM or the PDZ-BM abolished the inhibitory effect on tubulogenesis. In addition, these mutants showed aberrant subcellular localization indicating that lateral localization is correlated with the effect of TSLC1. In a two-dimensional culture, the full-length TSLC1, but not the mutants lacking the protein 4.1-BM or the PDZ-BM, suppressed HGF-induced cell scattering. Furthermore, the cells expressing full-length TSLC1 retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These cells showed prolonged activation of Rac and low activity of Rho, whereas the HGF-treated parental cells induced transient activation of Rac and sustained activation of Rho. Prolonged Rac activation caused by the expression of TSLC1 required its cytoplasmic tail. These findings, taken together, suggest that TSLC1 plays a role in suppressing induction of epithelial mesenchymal transitions by regulating the activation of small Rho GTPases.     

Expression and function of chicken integrin beta 1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells

Chicken integrin beta 1 cDNA and its site-directed mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken beta 1 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin beta 1 subunit. The chicken beta 1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD alpha subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD beta 1 and an 140-kD alpha subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse alpha subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.     

Shortened cytoplasmic domain affects intracellular transport but not nuclear localization of a viral glycoprotein

Herpes simplex virus (HSV) buds from the inner nuclear membrane of the infected cells. The glycoprotein gB-1 of HSV contains a stretch of 69 hydrophobic amino acids near the COOH terminus and a 109-amino acid cytoplasmic domain. By oligonucleotide-directed mutagenesis, five gB-1 mutants were constructed which either lack a cytoplasmic tail or contained 3, 6, 22, or 43 amino acids in the cytoplasmic tail. When expressed in COS cells all of the mutant glycoproteins were synthesized but the rate of intracellular transport and the appearance at the cell surface of the mutant gB-1 protein lacking the cytoplasmic tail or containing 3 and 6 amino acids in the cytoplasmic domain was drastically reduced. The wild-type gB-1 as well as all of the mutants in the cytoplasmic tail were, however, located on the nuclear envelope. These results suggest that the cytoplasmic domain of the glycoprotein gB may play a role in intracellular transport but not in the nuclear localization.     

The immunoglobulin‐like cell adhesion molecule hepaCAM induces differentiation of human glioblastoma U373‐MG cells

Subsequent to our identification of a novel immunoglobulin‐like cell adhesion molecule hepaCAM, we showed that hepaCAM is frequently lost in diverse human cancers and is capable of modulating cell motility and growth when re‐expressed. Very recently, a molecule identical to hepaCAM (designated as GlialCAM) was found highly expressed in glial cells of the brain. Here, we demonstrate that hepaCAM is capable of inducing differentiation of the human glioblastoma U373‐MG cells. Expression of hepaCAM resulted in a significant increase in the astrocyte differentiation marker glial fibrillary acid protein (GFAP), indicating that hepaCAM promotes glioblastoma cells to undergo differentiation. To determine the relationship between hepaCAM expression level and cell differentiation, we established two U373‐MG cell lines expressing hepaCAM at different levels. The results revealed that high‐level hepaCAM triggered a clear increase in GFAP expression as well as morphological changes characteristic of glioblastoma cell differentiation. Furthermore, high expression of hepaCAM significantly accelerated cell adhesion but inhibited cell proliferation and migration. Concomitantly, deregulation of cell cycle regulatory proteins was detected. Expectedly, the differentiation was noticeably less apparent in cells expressing low‐level hepaCAM. Taken together, our findings suggest that hepaCAM induces differentiation of the glioblastoma U373‐MG cells. The degree of cell differentiation is dependent on the expression level of hepaCAM. J. Cell. Biochem. 107: 1129–1138, 2009. © 2009 Wiley‐Liss, Inc.     

Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells

The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.     

The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts

A number of recent reports on the trafficking of receptor proteins in MDCK epithelial cells have provided evidence that delivery to the basolateral domain requires a specific targeting sequence and that deletion of this sequence results in constitutive expression on the apical surface. To date, these studies have concentrated on receptors which are competent for internalization via the clathrin coated pits. We have examined the localization of a resident plasma membrane protein by transfecting human CD44 into MDCK cells. Using human specific and cross-species reactive antibodies, we show that in MDCK cells both the endogenous and transfected wild-type CD44 are found on the basolateral surface where they are restricted to the lateral domain. Deletion of the CD44 cytoplasmic tail reduces the half life of this mutant protein and causes it to be expressed both on the apical surface and to a significant extent within the cell. We have also used biochemical and morphological analysis to investigate the interaction of CD44 with the cytoskeleton in detergent extracted cells. Strikingly different extraction results were obtained between epithelial and fibroblast cells. However, there is no difference in the Triton X-100 solubility of the transfected wild-type and tail-less CD44 in fibroblasts and both forms of the protein remain associated with the cortical cytoskeleton after Triton X-100 extraction. These results demonstrate that the sequence present in the cytoplasmic domain of CD44 responsible for its distribution in epithelial cells is functionally and spatially separate from the ability of this protein to associate with the cytoskeleton.     

Binding of the extreme carboxyl-terminus of PAK-interacting exchange factor β (βPIX) to myosin 18A (MYO18A) is required for epithelial cell migration

The PAK2/βPIX/GIT1 (p21-activated kinase 2/PAK-interacting exchange factor-β/G protein-coupled receptor kinase-interactor 1) complex has been shown to distribute to both membrane ruffles and focal adhesions of cells, where it plays an important role in regulating focal adhesion turnover. However, the detailed mechanism underlying this regulation is largely unknown. We previously reported that MYO18Aα interacts via its carboxyl terminus with the PAK2/βPIX/GIT1 complex through direct binding to βPIX, and that knockdown of MYO18Aα in epithelial cells causes accumulation of the complex in focal adhesions and decreased cell migration ability (Hsu et al., 2010). The current study characterized the detailed MYO18Aα–βPIX interaction mechanism and the biological significance of this interaction. We found that deletion of the carboxyl-terminal globular domain of MYO18Aα profoundly altered the cellular localization of βPIX and inhibited cell migration. βPIX interacts through its most carboxyl-terminus, PAWDETNL (639–646), with MYO18Aα and partially colocalized with MYO18Aα in membrane ruffles of cells, whereas βPIX^1–638, a mutant with deletion of PAWDETNL, accumulated in focal adhesions. Both focal adhesion numbers and area in βPIX^1–638-expressing cells were greater than those in cells expressing wild-type βPIX^FL. Further experiments using deletion mutants of MYO18A and βPIX showed that disruption of MYO18A–βPIX interaction not only impaired cell motility but also decreased Rac1 activity. Collectively, our data unravel the interaction regions between MYO18A and βPIX and provide evidence for the critical role of this interaction in regulating cellular localization of βPIX, Rac1 activity, and adhesion and migration in epithelial cells.     

Fibroblast growth factor 2 and protein kinase C alpha are involved in syndecan-4 cytoplasmic domain modulation of turkey myogenic satellite cell proliferation

Syndecan-4 core protein is composed of extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain functions in transmitting signals into the cell through the protein kinase C alpha (PKCα) pathway. The glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains attached to the extracellular domain influence cell proliferation. The current study investigated the function of syndecan-4 cytoplasmic domain in combination with GAG and N-glycosylated chains in turkey muscle cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Syndecan-4 or syndecan-4 without the cytoplasmic domain and with or without the GAG and N-glycosylated chains were transfected or co-transfected with a small interfering RNA targeting syndecan-4 cytoplasmic domain into turkey muscle satellite cells. The overexpression of syndecan-4 mutants increased cell proliferation but did not change differentiation. Syndecan-4 mutants had increased cellular responsiveness to FGF2 during proliferation. Syndecan-4 increased PKCα cell membrane localization, whereas the syndecan-4 mutants decreased PKCα cell membrane localization compared to syndecan-4. However, compared to the cells without transfection, syndecan-4 mutants increased cell membrane localization of PKCα. These data indicated that the syndecan‐4 cytoplasmic domain and the GAG and N-glycosylated chains are critical in syndecan-4 regulating satellite cell proliferation, responsiveness to FGF2, and PKCα cell membrane localization.     

The mammalian Ced-1 ortholog MEGF10/KIAA1780 displays a novel adhesion pattern

Ced-1 protein is a Caenorhabditis elegans cell surface receptor involved in phagocytosis of dead cells. The gene encoding the mammalian ortholog of Ced-1 is yet to be identified. Here, we describe a potential candidate: human MEGF10. MEGF10 has the overall domain organization of Ced-1, containing a signal peptide, a EMI domain, 17 atypical EGF-like repeats, a transmembrane domain, and a cytoplasmic domain with NPXY and YXXL motifs. MEGF10-EGFP fusion protein expressed in HEK293 cells produced an irregular, mosaic-like pattern on the surface of coated glass. Protruded MEGF10 bound tightly to the glass, in effect "pinning" the cytoplasmic membrane firmly onto the glass, thereby restricting cell motility. These cells also took on a flat appearance. Although MEGF10-EGFP localized throughout the cytoplasmic membrane, no MEGF10-EGFP was found in lamellipodia. The MEGF10-EGFP signal was surrounded by a 1-2-microm-wide dark strip lacking EGFP. Expression analyses of various MEGF10 deletion mutants revealed that the irregular, mosaic-like adhesion pattern characteristic of MEGF10 family members is due to concerted interactions between the EMI and 17 atypical EGF-like domains. Co-culturing of MEGF10-EGFP-expressing cells with apoptotic cells revealed that MEGF10 protein accumulated around the contact region during engulfment of apoptotic cells.     

Cleavage of Notch1 by granzyme B disables its transcriptional activity

AE1 (anion exchanger 1) and protein 4.2 associate in a protein complex bridging the erythrocyte membrane and cytoskeleton; disruption of the complex results in unstable erythrocytes and HS (hereditary spherocytosis). Three HS mutations (E40K, G130R and P327R) in cdAE1 (the cytoplasmic domain of AE1) occur with deficiencies of protein 4.2. The interaction of wild-type AE1, AE1HS mutants, mdEA1 (the membrane domain of AE1), kAE1 (the kidney isoform of AE1) and AE1SAO (Southeast Asian ovalocytosis AE1) with protein 4.2 was examined in transfected HEK (human embryonic kidney)-293 cells. The HS mutants had wild-type expression levels and plasma-membrane localization. Protein 4.2 expression was not dependent on AE1. Protein 4.2 was localized throughout the cytoplasm and co-localized at the plasma membrane with the HS mutants mdAE1 and kAE1, but at the ER (endoplasmic reticulum) with AE1SAO. Pull-down assays revealed diminished levels of protein 4.2 associated with the HS mutants relative to AE1. The mdAE1 did not bind protein 4.2, whereas kAE1 and AE1SAO bound wild-type amounts of protein 4.2. A protein 4.2 fatty acylation mutant, G2A/C173A, had decreased plasma-membrane localization compared with wild-type protein 4.2, and co-expression with AE1 enhanced its plasma-membrane localization. Subcellular fractionation showed the majority of wild-type and G2A/C173A protein 4.2 was associated with the cytoskeleton of HEK-293 cells. The present study shows that cytoplasmic HS mutants cause impaired binding of protein 4.2 to AE1, leaving protein 4.2 susceptible to loss during erythrocyte development.     

The proamphiregulin cytoplasmic domain is required for basolateral sorting, but is not essential for constitutive or stimulus-induced processing in polarized Madin-Darby canine kidney cells

In this study, the role of the amphiregulin precursor (pro-AR) cytoplasmic domain in the basolateral sorting and cell-surface processing of pro-AR in polarized epithelial cells has been investigated using Madin-Darby canine kidney cells stably expressing various human pro-AR forms. Our results demonstrate that newly synthesized wild-type pro-AR (50 kDa) is delivered directly to the basolateral membrane domain with >95% efficiency, where it is sequentially cleaved within the ectodomain to release several soluble amphiregulin (AR) forms. Analyses of a pro-AR cytoplasmic domain truncation mutant (ARTL27) and two pro-AR secretory mutants (ARsec184 and ARsec190) indicated that the pro-AR cytoplasmic domain is not required for efficient delivery to the plasma membrane, but does contain essential basolateral sorting information. We show that the pro-AR cytoplasmic domain truncation mutant (ARTL27) is not sorted in polarized Madin-Darby canine kidney cells, with approximately 65% of the newly synthesized protein delivered to the apical cell surface. Under base-line conditions, ARTL27 was preferentially cleaved from the basolateral surface with 4-fold greater efficiency compared with cleavage from the apical membrane domain. However, ARTL27 ectodomain cleavage could be stimulated equivalently from either membrane domain by a variety of different stimuli. The metalloprotease inhibitor BB-94 could inhibit both base-line and stimulus-induced ectodomain cleavage of wild-type pro-AR and ARTL27. These results indicate that the pro-AR cytoplasmic domain is required for basolateral sorting, but is not essential for ectodomain processing. Preferential constitutive cleavage of ARTL27 from the basolateral cell surface also suggests that the metalloprotease activity involved in base-line and stimulus-induced ARTL27 ectodomain cleavage may be regulated differently in the apical and basolateral membrane domains of polarized epithelial cells.     

Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.     

Synemin interacts with the LIM domain protein zyxin and is essential for cell adhesion and migration

Synemin is a unique cytoplasmic intermediate filament protein for which there is limited understanding of its exact cellular functions. The single human synemin gene encodes at least two splice variants named α-synemin and β-synemin, with the larger α-synemin containing an additional 312 amino acid insert within the C-terminal tail domain. We report herein that, by using the entire tail domain of the smaller β-synemin as the bait in a yeast two-hybrid screen of a human skeletal muscle cDNA library, the LIM domain protein zyxin was identified as an interaction partner for human synemin. The synemin binding site in human zyxin was subsequently mapped to the C-terminal three tandem LIM-domain repeats, whereas the binding site for zyxin within β-synemin is within the C-terminal 332 amino acid region (SNβTII) at the end of the long tail domain. Transient expression of SNβTII within mammalian cells markedly reduced zyxin protein level, blocked localization of zyxin at focal adhesion sites and resulted in decreased cell adhesion and increased motility. Knockdown of synemin expression with siRNAs within mammalian cells resulted in significantly compromised cell adhesion and cell motility. Our results suggest that synemin participates in focal adhesion dynamics and is essential for cell adhesion and migration.     

Cellular distribution of constitutively active mutant parathyroid hormone (PTH)/PTH-related protein receptors and regulation of cyclic adenosine 3',5'-monophosphate signaling by beta-arrestin2

PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruiting beta-arrestin2. We examined the role of beta-arrestin2 in regulating the cellular distribution and cAMP signaling of two constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of a beta-arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-dependent manner, as well as the response to PTH of both mutant and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent ligands, and ssarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization of beta-arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell membrane, whereas intracellular trafficking of T410P was also observed. While agonists promoted beta-arrestin2-mediated endocytosis of bot PTH1Rc mutants, antagonists were rapidly internalized only with T410P. The protein kinases inhibitor, staurosporine, significantly decreased internalization of ligand-PTH1Rc mutant complexes, although the recruitment of beta-arrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, agonists stimulated translocation of beta-arrestin2 to the cell membrane followed by ligand-receptor complex internalization without associated beta-arrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction with beta-arrestin2 on the cell membrane, possibly leading to uncoupling from G(s)alpha. This phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the cellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.     

Scapinin,the Protein Phosphatase 1 Binding Protein,Enhances Cell Spreading and Motility by Interacting with the Actin Cytoskeleton

Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.     

Role of syndecan-4 side chains in turkey satellite cell apoptosis and focal adhesion formation

S4 (syndecan-4) is a cell membrane heparan sulfate proteoglycan that functions in muscle growth and development. It is composed of a central core protein and two types of side chains: GAGs (glycosaminoglycans) and N-glycosylated (N-linked glycosylated) chains. The N-glycosylated chains and GAG chains are required for S4 to regulate turkey myogenic satellite cell proliferation. The objective of the current study was to determine whether the S4 side chains regulate cell proliferation through muscle cell focal adhesion formation and apoptosis. S4 mutants with only one or without any N-glycosylated chains attached to the core protein with or without GAG chains were generated to study the function of N-glycosylated chains and the interaction between N-glycosylated chains and GAG chains. The wild-type S4 and all of the S4 side chain mutants were transfected into turkey myogenic satellite cells. Cell apoptosis and focal adhesion formation were measured, and PKCα (protein kinase Cα) cell membrane localization was investigated. S4 increased FAK (focal adhesion kinase) activity and the deletion of the side chains decreased this effect. S4 and the S4 mutants had no effect on β1-integrin expression, but increased the cell membrane localization of β1-integrin and PKCα. Furthermore, cell apoptosis and vinculin containing focal adhesions were not affected by S4 and its mutants. The results suggest that S4 and its side chains play important roles in regulating FAK activity, and PKCα and β1-integrin cell membrane localization, but not cell apoptosis and vinculin-containing focal adhesion formation.