scholarly journals The Tetraspan Molecule Cd151, a Novel Constituent of Hemidesmosomes, Associates with the Integrin α6β4 and May Regulate the Spatial Organization of Hemidesmosomes

2000 ◽  
Vol 149 (4) ◽  
pp. 969-982 ◽  
Author(s):  
Lotus M.Th. Sterk ◽  
Cecile A.W. Geuijen ◽  
Lauran C.J.M. Oomen ◽  
Jero Calafat ◽  
Hans Janssen ◽  
...  
Keyword(s):  
Cell Surface ◽  
Spatial Organization ◽  
Transmembrane Domain ◽  
Focal Adhesions ◽  
Surface Protein ◽  
K562 Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Junctional Epidermolysis Bullosa ◽  
Integrin Α6β4

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with α3β1 and α6β4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with α3β1 and α6β4. In β4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and α3β1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and α3β1. Transient transfection studies of PA-JEB cells with β4 revealed that the integrin α6β4 becomes incorporated into the α3β1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of α3β1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with α6β4 in hemidesmosomes, whereas its codistribution with α3β1 in focal adhesions becomes partial. The localization of α6β4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using β4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of β4, we found that for recruitment of CD151 into hemidesmosomes, the β4 subunit must be associated with α6, confirming that integrins associate with tetraspans via their α subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface. In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin α6β4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.

scholarly journals Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor

2012 ◽  
Vol 49 (3) ◽  
pp. 237-248 ◽  
Author(s):  
Hui Huang ◽  
Ya-Xiong Tao
Keyword(s):  
Cell Surface ◽  
Structure Function ◽  
Transmembrane Domain ◽  
Mapk Pathway ◽  
Surface Expression ◽  
Camp Signaling ◽  
Cell Surface Expression ◽  
Melanocortin 4 Receptor ◽  
Function Relationship ◽  
Relationship Of

The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure–function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for α-melanocyte-stimulating hormone (α-MSH) and β-MSH binding, and six were important for signaling in the Gs–cAMP–PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure–function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders.

CD39/NTPDase1 Variants Identified in Human Neutrophils Regulate Antithrombotic Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3088-3088 ◽  
Author(s):  
Kim E. Olson ◽  
Dianne Pulte ◽  
Marinus Johan Broekman ◽  
Ashley E. Olson ◽  
Joan Drosopoulos ◽  
...  
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Western Blot ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Neutrophils ◽  
Platelet Activity ◽  
Total Rna ◽  
Cos Cells

Abstract Blood-borne cellular elements expressing ectonucleotidase activity have been shown to regulate platelet activation and recruitment in response to agonists. In particular, exposure of a platelet releasate to isolated neutrophils (PMN) results in loss of its platelet activating activity in a subsequent assay (Valles et al, J Clin Invest1993, 92:1357–1365). Whereas expression of CD39 on vascular endothelial cells has been well characterized, expression on leukocytes has been less well studied. Freshly prepared lymphocyte and PMN cell populations were evaluated for both cell surface expression of CD39 and ectonucleotidase activity. FACS analysis showed that 98% of PMN were positive for CD39 compared to only 20% of lymphocytes. In addition, neutrophils stained more intensely, indicating the presence of a higher quantity of cell surface-expressed CD39. Interestingly, neutrophils exhibited only 1/3 of the ATPase and 1/2 of the ADPase activities of the same number of lymphocytes, although the latter are thought to have greater antithrombotic capacity. RT-PCR products from total RNA isolated from lymphocytes and PMN were sequenced. This revealed alternately spliced CD39 mRNA species present in PMN at levels equal to that of CD39 mRNA. In contrast, lymphocytes, which showed much higher levels of CD39 mRNA, expressed these variants at much lower levels. RACE analyses of cDNAs generated from total RNA demonstrated two CD39 gene-derived mRNAs. Each was comprised of an alternate 3′ segment lacking the C-terminal transmembrane domain, and distinguished by an internal deletion. Myc- and Flag-tagged constructs expressed in COS cells resulted in cell surface expression of the respectively tagged variants (immunocytochemistry, western blot analyses of plasma membrane preparations). Membrane preparations assayed for enzyme activity revealed no apyrase activity for either molecule expressed alone or together. Co-transfection of CD39 with equal amounts of either construct singly or in combination resulted in a 30-50% decrease in ATPase activity compared to CD39 alone. Similarly, CD39 co-expressed with either construct alone lost 75–90% of its ADPase activity. Unexpectedly, co-transfection of CD39 with both variants together resulted in a 20–40% increase in ADPase activity. Glutaraldehyde cross-linking of membrane preparations from triply transfected COS cells followed by immunoprecipitation and western blot analyses demonstrated the presence of all three species in higher order complexes. Thus, both variants can simultaneously associate with CD39, generating hetero-multimers with altered substrate preference and catalytic efficiency compared to CD39 tetramers. These observations add to our understanding of the regulation of ectonucleotidase activity at the cell surface. The balanced expression of CD39 and its two identified variants may underlie the anti-platelet activity of neutrophils previously reported. The finding that association of CD39 with either construct alone results in near complete loss of ADPase activity with only partial diminution of ATPase activity suggests a possible etiology for a pro-thrombotic phenotype.

Functional Cell Surface Expression of Band 3, the Human Red Blood Cell Anion Exchange Protein (AE1), in K562 Erythroleukemia Cells: Band 3 Enhances the Cell Surface Reactivity of Rh Antigens

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4428-4438 ◽  
Author(s):  
Roland Beckmann ◽  
Jonathan S. Smythe ◽  
David J. Anstee ◽  
Michael J.A. Tanner
Keyword(s):  
Cell Surface ◽  
Chloride Transport ◽  
K562 Cells ◽  
Surface Expression ◽  
Band 3 ◽  
Surface Reactivity ◽  
Cell Surface Expression ◽  
Anion Exchanger 1 ◽  
Erythroleukemia Cells ◽  
Antigen Activity

Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe retroviral vector. Stable K562 clones expressing band 3 were isolated by flow cytometry, and surface expression was quantified by immunoblotting. The function of band 3 expressed at the cell surface was demonstrated in chloride transport assays. K562 cells expressing band 3 also displayed high levels of the Wrb blood group antigen, confirming the role of band 3 in Wrb expression, and an increase in the low levels of endogenous Rh antigen activity. We also performed coexpression experiments with K562 clones that had previously been transduced with cDNAs encoding RhD or RhcE polypeptides. The transfection and expression of band 3 in these clones substantially increased the levels of RhD and cE antigen activity expressed on the cells and also increased the reactivity of the cells with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The increased reactivity of Rh antigens may result from cell surface or intracellular interactions of band 3 with the protein complex which contains the Rh polypeptides and RhGP, or from indirect effects of band 3 on the membrane environment. This work establishes a system for cell surface expression of band 3 in a mammalian cell line, which will enable further studies of the protein and its interactions with other membrane components.

Defects of the Tpo-Receptor, Mpl, Caused by Mutations Found in CAMT Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2181-2181
Author(s):  
Marloes R. Tijssen ◽  
Franca di Summa ◽  
Sonja Van den Oudenrijn ◽  
Carlijn Voermans ◽  
C.Ellen Van der Schoot ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Amino Acid ◽  
Cell Surface ◽  
Cell Line ◽  
Mrna Level ◽  
K562 Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Amino Acid Substitutions ◽  
Bhk Cells

Abstract Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder that presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. The disease may develop into bone marrow aplasia. In vitro, CD34-positive hematopoietic progenitor cells from CAMT patients did not show any megakaryocyte formation in a Tpo-driven expansion culture. We and others found genetic defects in the gene encoding the Tpo receptor, c-mpl (Van den Oudenrijn et al., Br J Haematol.2002, 117: 390–398 and Ballmaier et al., Ann N Y Acad Sci.2003, 996: 17–25). In our patients, we found four mutations that predicted amino-acid substitutions, of which three in the extracellular domain; Arg102Pro, Pro136His and Arg257Cys, and one in the intracellular signaling domain (Pro635Leu), which may result in either defective Tpo-binding and/or signaling. To investigate this, we transfected full-length Mpl (wt and mutants) into the erythroleukemic cell line K562 and truncated Mpl (encompassing the extracellular domain; wt and mutants) into Baby Hamster Kidney (BHK) cells. In the K562 cells, the mRNA level (RQ-PCR) of the Pro136His mutant was severely decreased compared to the wt transfectant, while the mRNA level of the other mutants was comparable to that of wt. On Western blot, wt Mpl migrated as two, presumably differently glycosylated, bands of 75 kD and 72 kD. The mutants showed an altered migration pattern, which might result from differences in glycosylation. With the Pro635Leu mutant lower signals were obtained when equal amounts of total protein were loaded. Since the Mpl mRNA level was comparable to that of wt, this suggests a higher level of protein degradation. Upon transfection of the Arg102Pro and the Arg257Cys mutants in BHK cells, we observed that these mutants did not gain endo-H resistency, which suggests an aberrant processing of these mutant Mpls through the Golgi apparatus and retention in the ER. However, in cell fractionation experiments with surface-biotinylated K562 cells, biotinylated wt Mpl and mutant Mpl (except Pro136His) could be detected. Apparently, in K562 cells, the amino-acid substitutions do not impair membrane expression completely. To examine whether the mutant receptors were still able to signal after Tpo incubation, K562 cells were serum-starved and subsequently stimulated with 50 ng/ml rhTpo for 5 to 30 minutes. All mutants, including Pro136His, showed increased ERK phosphorylation after 5 minutes. To summarize, the Pro136His mutant is hardly expressed in the K562 expression model, presumably because of instability of the mRNA, but is still able to induce signaling. In contrast to the results obtained in the BHK model, the Arg102Pro and Arg257Cys mutants, showed cell-surface expression in the K562 cell line. The obtained cell-surface expression in the K562 model may have been significantly increased compared to the in vivo situation on hematopoietic stem cells, because of artificially induced efficient expression. Finally, with a super-physiological concentration of rhTpo, we obtained evidence that all Mpl mutants were able to signal upon Tpo binding. Whether impaired signaling by the Mpl mutants in the presence of physiological levels of Tpo may contribute to the development of CAMT, will be investigated.

scholarly journals Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants.

1989 ◽  
Vol 108 (5) ◽  
pp. 1979-1985 ◽  
Author(s):  
F F Roossien ◽  
D de Rijk ◽  
A Bikker ◽  
E Roos
Keyword(s):  
Cell Surface ◽  
Surface Protein ◽  
Metastatic Potential ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Lymphocyte Function ◽  
Lymphoma Cells ◽  
Mutant Cells

Lymphocyte function-associated antigen-1 (LFA-1) is a leukocyte and lymphoma cell surface protein that promotes intercellular adhesion. We have previously shown that the invasion of hepatocyte cultures by lymphoma cells is inhibited by anti-LFA-1 antibodies (Roos, E., and F. F. Roossien. 1987. J. Cell Biol. 105:553-559). In addition, we now report that LFA-1 is also involved in invasion of lymphoma cells into fibroblast monolayers. To investigate the role of LFA-1 in metastasis of these lymphoma cells, we have generated mutants that are deficient in LFA-1 cell surface expression because of impaired synthesis of either the alpha or beta subunit precursor of LFA-1. We identified at least three distinct mutant clones. The invasive potential of the mutant cells in vitro, in both hepatocyte and fibroblast cultures, was considerably lower than that of parental cells. The metastatic potential of the mutants was much reduced, indicating that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells.

scholarly journals Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 1038-1047 ◽  
Author(s):  
Isabelle Mouro-Chanteloup ◽  
Anne Marie D'Ambrosio ◽  
Pierre Gane ◽  
Caroline Le Van Kim ◽  
Virginie Raynal ◽  
...  
Keyword(s):  
Cell Surface ◽  
K562 Cells ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Expression Vectors ◽  
Cell Surface Expression ◽  
Membrane Expression ◽  
Cmv Promoter ◽  
Transfected Cells

Abstract In most cases, the lack of Rh in Rhnull red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter–based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.

scholarly journals Hypoxia-inducible Factor Regulates αvβ3 Integrin Cell Surface Expression

2005 ◽  
Vol 16 (4) ◽  
pp. 1901-1912 ◽  
Author(s):  
Karen D. Cowden Dahl ◽  
Sarah E. Robertson ◽  
Valerie M. Weaver ◽  
M. Celeste Simon
Keyword(s):  
Cell Surface ◽  
Cell Fate ◽  
Hypoxia Inducible Factor ◽  
Focal Adhesions ◽  
Surface Expression ◽  
Melanoma Cells ◽  
Cell Surface Expression ◽  
Migration And Invasion ◽  
Αvβ3 Integrin ◽  
And Migration

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of α and β aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt-/- and Hifα-/- TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin αvβ3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O2). Culturing B16F0 melanoma cells at 1.5% O2 resulted in increased αvβ3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O2 tension influence placental invasion and tumor migration by increasing cell surface expression of αvβ3 integrin.

scholarly journals Growth of rotaviruses in continuous human and monkey cell lines that vary in their expression of integrins

2000 ◽  
Vol 81 (9) ◽  
pp. 2203-2213 ◽  
Author(s):  
Sarah L. Londrigan ◽  
Marilyn J. Hewish ◽  
Melanie J. Thomson ◽  
Georgina M. Sanders ◽  
Huseyin Mustafa ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
K562 Cells ◽  
Surface Expression ◽  
Cell Entry ◽  
Cell Surface Expression ◽  
Intestinal Epithelial ◽  
Susceptibility To Infection ◽  
Rd Cells

Rotavirus replication occurs in vivo in intestinal epithelial cells. Cell lines fully permissive to rotavirus include kidney epithelial (MA104), colonic (Caco-2) and hepatic (HepG2) types. Previously, it has been shown that cellular integrins α2β1, α4β1 and αXβ2 are involved in rotavirus cell entry. As receptor usage is a major determinant of virus tropism, the levels of cell surface expression of these integrins have now been investigated by flow cytometry on cell lines of human (Caco-2, HepG2, RD, K562) and monkey (MA104, COS-7) origin in relation to cellular susceptibility to infection with monkey and human rotaviruses. Cells supporting any replication of human rotaviruses (RD, HepG2, Caco-2, COS-7 and MA104) expressed α2β1 and (when tested) αXβ2, whereas the non-permissive K562 cells did not express α2β1, α4β1 or αXβ2. Only RD cells expressed α4β1. Although SA11 grew to higher titres in RD, HepG2, Caco-2, COS-7 and MA104 cells, this virus still replicated at a low level in K562 cells. In all cell lines tested, SA11 replicated to higher titres than did human strains, consistent with the ability of SA11 to use sialic acids as alternative receptors. Levels of cell surface α2 integrin correlated with levels of rotavirus growth. The α2 integrin relative linear median fluorescence intensity on K562, RD, COS-7, MA104 and Caco-2 cells correlated linearly with the titre of SA11 produced in these cells at 20 h after infection at a multiplicity of 0·1, and the data best fitted a sigmoidal dose–response curve (r 2=1·00, P=0·005). Thus, growth of rotaviruses in these cell lines correlates with their surface expression of α2β1 integrin and is consistent with their expression of αXβ2 and α4β1 integrins.

scholarly journals Trafficking of Kv1.4 potassium channels: interdependence of a pore region determinant and a cytoplasmic C-terminal VXXSL determinant in regulating cell-surface trafficking

2003 ◽  
Vol 375 (3) ◽  
pp. 761-768 ◽  
Author(s):  
Jing ZHU ◽  
Itaru WATANABE ◽  
Barbara GOMEZ ◽  
William B. THORNHILL
Keyword(s):  
Cell Surface ◽  
Cell Function ◽  
Cell Signalling ◽  
Surface Protein ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Cell Surface Expression ◽  
Pore Region ◽  
Expression Levels ◽  
Protein Levels

Kv1.4 and Kv1.1 potassium channel homomers have been shown to exhibit different intracellular trafficking programmes and cell-surface expression levels in cell lines: a determinant in the pore region of Kv1.4 and Kv1.1 [Zhu, Watanabe, Gomez and Thornhill (2001) J. Biol. Chem. 276, 39419–39427] and a cytoplasmic C-terminal VXXSL determinant on Kv1.4 [Li, Takimoto and Levitan (2000) J. Biol. Chem. 275, 11597–11602] have been described, which affected trafficking and cell-surface expression levels. In the present study, we examined whether trafficking pore determinants influenced any cytoplasmic C-terminal trafficking determinant. We found that removal of VXXSL from a Kv1.4 chimaera that contained the pore of Kv1.1 did not affect cell-surface trafficking. Therefore removal of the C-terminal VXXSL of Kv1.4 inhibited protein surface levels only in the presence of the Kv1.4 pore. In contrast, truncating the cytoplasmic C-terminus of Kv1.1 or truncating a Kv1.1 chimaera with the pore of Kv1.4, had little effect on surface protein levels. Furthermore, the subregion of the Kv1.4 pore trafficking determinant that was required for the inhibitory effect of VXXSL removal was mapped to a threonine residue in the deep pore region. Therefore the Kv1.4 pore determinant affected the trafficking and cell-surface levels directed by the C-terminal VXXSL determinant. Different Kv1 trafficking programmes would affect cell-surface expression levels either positively or negatively and also cell signalling. Cells may use differential trafficking programmes of membrane proteins as a post-translational mechanism to regulate surface protein levels and cell function.

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