scholarly journals Ligand-dependent and -independent integrin focal contact localization: the role of the alpha chain cytoplasmic domain.

1993 ◽  
Vol 4 (6) ◽  
pp. 593-604 ◽  
Author(s):  
R Briesewitz ◽  
A Kern ◽  
E E Marcantonio
Keyword(s):  
Cell Lines ◽  
Cellular Localization ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Striking Difference ◽  
Focal Contact ◽  
Integrin Receptors ◽  
Independent Manner ◽  
Contact Sites ◽  
Focal Contacts

Many integrin receptors localize to focal contact sites upon binding their ligand. However, unoccupied integrin receptors do not localize to focal contact sites. Because the integrin beta 1 cytoplasmic domain appears to have a focal contact localization signal, there must be a mechanism by which this domain is kept inactive in the unoccupied state and becomes exposed or activated in the occupied receptor. We considered that this mechanism involves the alpha subunit cytoplasmic domain. To test this hypothesis, we have established two NIH 3T3 cell lines that express either the human alpha 1 wild-type subunit (HA1 cells) or the cytoplasmic domain deleted alpha 1 subunit (CYT cells). Both cell lines express similar levels of the human alpha 1 subunit, and there is no significant effect of the deletion on the dimerization and surface expression of the receptor. Furthermore, the deletion had no effect on the binding or adhesion via alpha 1 beta 1 to its ligand collagen IV. However, when these two cell lines are plated on fibronectin (FN), which is a ligand for alpha 5 beta 1 but not for alpha 1 beta 1, there is a striking difference in the cellular localization of alpha 1 beta 1. The HA1 cells show only alpha 5 in focal contacts, without alpha 1, demonstrating that all of the integrin localization is ligand dependent. In contrast, when the CYT cells are plated on FN, the mutant alpha 1 appears in focal contacts along with the alpha 5/beta 1. Thus, there is both ligand-dependent (alpha 5/beta 1) and ligand-independent (alpha 1/beta 1) focal contact localization in these cells. The truncated alpha 1 also localized to focal contacts in a ligand-independent manner on vitronectin. We conclude that the mutant alpha 1 no longer requires ligand occupancy for focal contact localization. These data strongly suggest that the alpha cytoplasmic domain plays a role in the normal ligand-dependent integrin focal contact localization.

Integrin-Mediated Fibroblast Adhesion Strength: Role of the β1 Subunit

1993 ◽  
Vol 331 ◽  
Author(s):  
Kelly A. Ward ◽  
Jun-Lin Guan ◽  
Daniel A. Hammer
Keyword(s):  
Extracellular Matrix ◽  
Adhesion Strength ◽  
Cytoplasmic Domain ◽  
Integrin Receptors ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Cell Substrate ◽  
Extracellular Matrix Molecules ◽  
Integrin Ligand ◽  
Cell Substrate Adhesion

AbstractCell-substratum adhesion is important in wound healing [4], embryogenic development [11], tissue architecture [6], and metastasis [7]. Integrins constitute a major class of heterodimeric cell-surface glycoproteins involved in receptor-mediated adhesion to the extracellular matrix (ECM). Focal contacts are regions of the cell-substratum adhesion in which clusters of integrin receptors connect the cytoskeleton to extracellular matrix molecules such as fibronectin. Focal contacts strengthen cell-substrate adhesion, and are sites of biochemical activity. Since cell adhesion strength in part depends on the cell's ability to cluster receptors and cytoskeleton into focal contacts, the integrity of the focal contact, and hence a cell's adhesive strength, will depend both on integrin-cytoskeletal binding as well as integrin-ligand binding.Using a centrifugation assay, we have quantified cell-substratum adhesion strength of mouse 3T3 cells transfected with the avian β1 integrin receptor (wild type), including various deletion mutants of its cytoplasmic domain, to surfaces containing varying concentrations of CSAT, a monoclonal antibody against the extracellular domain of the avian β1 subunit. For all the transfectants, adhesion strength decreases with decreasing CSAT concentration and increasing centrifugal strength. Different truncations of the cytoplasmic domain lead to different levels of adhesion. There is no simple correlation between the length of the cytoplasmic domain and the strength of adhesion.

scholarly journals Targeting Pyk2 to β1-Integrin–Containing Focal Contacts Rescues Fibronectin-Stimulated Signaling and Haptotactic Motility Defects of Focal Adhesion Kinase–Null Cells

2001 ◽  
Vol 152 (1) ◽  
pp. 97-110 ◽  
Author(s):  
Candice K. Klingbeil ◽  
Christof R. Hauck ◽  
Datsun A. Hsia ◽  
K.C. Jones ◽  
Shannon R. Reider ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Β1 Integrin ◽  
Focal Contact ◽  
Contact Sites ◽  
Focal Contacts ◽  
Tyrosine Kinase 2 ◽  
Transient Overexpression ◽  
The Impact

Focal adhesion kinase–null (FAK−/−) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK−/− cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK−/− cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a β1-integrin–containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK−/− cells, exhibit elevated FN-stimulated extracellular signal–regulated kinase 2 (ERK2) and c-Jun NH2-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH2-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to β-integrin–containing focal contact sites via interactions mediated by the FAK-CT domain.

scholarly journals Assembly and function of integrin receptors is dependent on opposing alpha and beta cytoplasmic domains.

1995 ◽  
Vol 6 (8) ◽  
pp. 997-1010 ◽  
Author(s):  
R Briesewitz ◽  
A Kern ◽  
E E Marcantonio
Keyword(s):  
Cytoplasmic Domain ◽  
Beta Subunits ◽  
Focal Contact ◽  
Wild Type ◽  
Cytoplasmic Domains ◽  
Integrin Receptors ◽  
Integrin Receptor ◽  
And Function ◽  
Intracellular Domains ◽  
Receptor Assembly

The membrane proximal regions of integrin alpha and beta subunits are highly conserved in evolution. In particular, all integrin alpha subunits share the KXGFFKR sequence at the beginning of their cytoplasmic domains. Previous work has shown that this domain is important in integrin receptor assembly. Using chimeric integrin alpha and beta subunits, we show that the native cytoplasmic domains of both subunits must be present for efficient assembly. Most strikingly, chimeric alpha 1 and beta 1 subunits with reciprocally swapped intracellular domains dimerize selectively into collagen IV receptors expressed at high levels on the surface. However, these receptors, which bind ligand efficiently, are deficient in a variety of post-ligand binding events, including cytoskeletal association and induction of tyrosine phosphorylation. Furthermore, deletion of the distal alpha cytoplasmic domain in the swapped heterodimers leads to ligand-independent focal contact localization, which also occurs in wild-type subunits when the distal cytoplasmic domain is deleted. These results show that proper integrin assembly requires opposed alpha and beta cytoplasmic domains, and this opposition prevents ligand-independent focal contact localization. Our working hypothesis is that these two domains may associate during receptor assembly and provide the mechanism for integrin receptor latency.

scholarly journals Mapping of the functional determinants of the integrin beta 1 cytoplasmic domain by site-directed mutagenesis.

1990 ◽  
Vol 1 (8) ◽  
pp. 597-604 ◽  
Author(s):  
E E Marcantonio ◽  
J L Guan ◽  
J E Trevithick ◽  
R O Hynes
Keyword(s):  
Consensus Sequence ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Focal Contact ◽  
Wild Type ◽  
3T3 Cells ◽  
Transmembrane Region ◽  
High Level Expression ◽  
Focal Contacts ◽  
High Level

We describe here the expression of deletion mutants of the cytoplasmic domain of the avian integrin beta 1 subunit. These mutants, which contain termination codons at positions 767, 776, 791, and 800, were transfected into mouse 3T3 cells to determine which sequences were essential for localization of integrins into focal contact sites. In all cases, high-level expression of the truncated avian integrins was obtained. Heterodimers were formed between the exogenous truncated avian beta 1 subunits and endogenous mouse alpha subunits, and these heterodimers were efficiently exported to the cell surface. The longest truncated beta 1 subunit tested, which is only four amino acids shorter than the wild type, does localize to focal contacts. In contrast, beta 1 subunits with moderately long truncations of the cytoplasmic domain failed to localize to focal contacts, including one which contains the consensus sequence for tyrosine phosphorylation. Surprisingly, a mutant subunit in which the bulk of the cytoplasmic domain was missing (but the segment nearest the membrane including the dibasic residues (RR) remained) did localize weakly to focal contacts. These results implicate the peptide segment nearest to the transmembrane region in focal contact localization. In addition, mutant subunits that included this segment together with a larger portion of the cytoplasmic domain did not localize as well as the shorter form, suggesting that these cytoplasmic domain segments are defective, presumably because of abnormal folding.

scholarly journals Integrin beta 1 cytoplasmic domain dominant negative effects revealed by lysophosphatidic acid treatment.

1994 ◽  
Vol 5 (11) ◽  
pp. 1215-1223 ◽  
Author(s):  
L Smilenov ◽  
R Briesewitz ◽  
E E Marcantonio
Keyword(s):  
Lysophosphatidic Acid ◽  
Actin Polymerization ◽  
Interleukin 2 ◽  
Chimeric Protein ◽  
Cytoplasmic Domain ◽  
Cytoskeletal Proteins ◽  
Dominant Negative ◽  
Focal Contact ◽  
Negative Effects ◽  
Contact Sites

Integrin receptors localize to focal contact sites and interact with the cytoskeleton via the beta 1 cytoplasmic domain. To study the role of this domain in adhesion, we have expressed in NIH 3T3 cells a cDNA consisting of the interleukin 2 receptor alpha subunit extracellular and transmembrane domains, connected to the integrin beta 1 cytoplasmic domain (IL2R-beta 1). Since the extracellular domain of the chimeric protein has no role in adhesion, this protein could uncouple adhesion from intracellular events. As expected, in a cell line expressing IL2R-beta 1, this chimera was directed to focal contact sites. Unexpectedly, the cells exhibited normal adhesion to fibronectin (FN). However, when a rapid reorganization of the cytoskeleton was induced using lysophosphatidic acid (LPA), IL2R-beta 1 cells detached from FN in contrast to wild-type cells. The detachment in response to LPA could be prevented with cytochalasin D, an inhibitor of actin polymerization. These results imply that a beta 1 cytoplasmic domain, which is uncoupled from adhesion, can compete with the cytoplasmic domain of native integrin beta 1 for cytoskeletal proteins. As a consequence, the IL2R-beta 1 protein acts as a dominant negative effector of adhesion by disrupting the integrin-cytoskeleton connection.

scholarly journals Aquaglyceroporin-3’s Expression and Cellular Localization Is Differentially Modulated by Hypoxia in Prostate Cancer Cell Lines

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 838
Author(s):  
Andreia de Almeida ◽  
Dimitris Parthimos ◽  
Holly Dew ◽  
Oliver Smart ◽  
Marie Wiltshire ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Lines ◽  
Crucial Role ◽  
Cellular Localization ◽  
Texture Features ◽  
Stress Factors ◽  
Adaptation To Hypoxia ◽  
Machine Learning Classification ◽  
Hypoxia Exposure ◽  
Pc3 Cells

Aquaporins are required by cells to enable fast adaptation to volume and osmotic changes, as well as microenvironmental metabolic stimuli. Aquaglyceroporins play a crucial role in supplying cancer cells with glycerol for metabolic needs. Here, we show that AQP3 is differentially expressed in cells of a prostate cancer panel. AQP3 is located at the cell membrane and cytoplasm of LNCaP cell while being exclusively expressed in the cytoplasm of Du145 and PC3 cells. LNCaP cells show enhanced hypoxia growth; Du145 and PC3 cells display stress factors, indicating a crucial role for AQP3 at the plasma membrane in adaptation to hypoxia. Hypoxia, both acute and chronic affected AQP3′s cellular localization. These outcomes were validated using a machine learning classification approach of the three cell lines and of the six normoxic or hypoxic conditions. Classifiers trained on morphological features derived from cytoskeletal and nuclear labeling alongside corresponding texture features could uniquely identify each individual cell line and the corresponding hypoxia exposure. Cytoskeletal features were 70–90% accurate, while nuclear features allowed for 55–70% accuracy. Cellular texture features (73.9% accuracy) were a stronger predictor of the hypoxic load than the AQP3 distribution (60.3%).

scholarly journals Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

1983 ◽  
Vol 96 (1) ◽  
pp. 37-50 ◽  
Author(s):  
E Schmid ◽  
DL Schiller ◽  
C Grund ◽  
J Stadler ◽  
WW Franke
Keyword(s):  
Mammary Gland ◽  
Epithelial Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Intermediate Filament ◽  
Striking Difference ◽  
Intermediate Filament Proteins ◽  
Cell Clones ◽  
Vimentin Filaments

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.

Syndesmos, a protein that interacts with the cytoplasmic domain of syndecan-4, mediates cell spreading and actin cytoskeletal organization

2000 ◽  
Vol 113 (2) ◽  
pp. 315-324 ◽  
Author(s):  
P.C. Baciu ◽  
S. Saoncella ◽  
S.H. Lee ◽  
F. Denhez ◽  
D. Leuthardt ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Cellular Protein ◽  
Actin Stress Fiber ◽  
Independent Manner ◽  
Intracellular Proteins ◽  
Central Regions ◽  
A Cell

Syndecan-4 is a cell surface heparan sulfate proteoglycan which, in cooperation with integrins, transduces signals for the assembly of focal adhesions and actin stress fibers in cells plated on fibronectin. The regulation of these cellular events is proposed to occur, in part, through the interaction of the cytoplasmic domains of these transmembrane receptors with intracellular proteins. To identify potential intracellular proteins that interact with the cytoplasmic domain of syndecan-4, we carried out a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait. As a result of this screen, we have identified a novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three syndecan family members. The interaction involves both the membrane proximal and variable central regions of the cytoplasmic domain. We have named this cDNA and encoded protein syndesmos. Syndesmos is ubiquitously expressed and can be myristylated. Consistent with its myristylation and syndecan-4 association, syndesmos colocalizes with syndecan-4 in the ventral plasma membranes of cells plated on fibronectin. When overexpressed in NIH 3T3 cells, syndesmos enhances cell spreading, actin stress fiber and focal contact formation in a serum-independent manner.

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