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Biomedical subjects

S E LaFlamme

Publications and source records attributed to S E LaFlamme.

14 recordsLinked to original sources
PubMed1998

Endothelial cells assemble two distinct alpha6beta4-containing vimentin-associated structures: roles for ligand binding and the beta4 cytoplasmic tail.

The alpha6beta4 laminin binding integrin functions in the assembly of type I hemidesmosomes, which are specialized cell-matrix adhesion sites found in stratified epithelial cells. Although endothelial cells do not express all the components of type I hemidesmosomes, endothelial cells can express the alpha6beta4 integrin. Because endothelial cells lose expression of alpha6beta4 in culture, we expressed recombinant alpha6beta4 in the dermal microvascular endothelial cell line, HMEC-1, to test whether endothelial cells can assemble adhesion structures containing alpha6beta4. Using immunofluorescence microscopy, we found that recombinant alpha6beta4 concentrates specifically in a novel fibrillar structure on the basal surface of endothelial cells in the absence of an exogenous laminin substrate. This localization is regulated by an intracellular mechanism, because the beta4 cytoplasmic domain is sufficient to direct a reporter domain (IL-2R) to the fibrillar structures independently of recombinant alpha6beta4. In addition, this IL-2R-beta4 chimera is sufficient to recruit the intermediate filament-associated protein HD1/plectin to these fibrillar structures and this also occurs in the absence of recombinant alpha6beta4. The fibrillar localization pattern, as well as the recruitment of HD1/plectin, requires the first and second fibronectin type III repeats and the connecting segment of the beta4 tail. In addition, when endothelial cells are provided a laminin 5-rich matrix, recombinant alpha6beta4 redistributes from the fibrillar structure to type I hemidesmosome-like structures. The beta4 cytoplasmic domain can also direct a reporter domain to these type I hemidesmosome-like structures; however, this process is dependent upon the expression of recombinant alpha6beta4 Biochemical analysis indicates that both the fibrillar and the type I hemidesmosome-like structures are associated with the vimentin intermediate filament cytoskeleton. Thus, the results illustrate that endothelial cells have the essential components necessary to assemble at least two distinct alpha6beta4-containing and vimentin-associated structures on their basal surface and that the alpha6beta4 cytoplasmic tail and the availability of specific alph6beta4 ligands regulate receptor localization to these structures.

Antigens, Surface↗
PubMed1997

The role of conserved amino acid motifs within the integrin beta3 cytoplasmic domain in triggering focal adhesion kinase phosphorylation.

Integrin-mediated adhesion of cells to extracellular matrix proteins triggers a variety of intracellular signaling pathways including a cascade of tyrosine phosphorylations. In many cell types, the cytoplasmic focal adhesion tyrosine kinase, FAK, appears to be the initial protein that becomes tyrosine-phosphorylated in response to adhesion; however, the molecular mechanisms regulating integrin-triggered FAK phosphorylation are not understood. Previous studies have shown that the integrin beta1, beta3, and beta5 subunit cytoplasmic domains all contain sufficient information to trigger FAK phosphorylation when expressed in single-subunit chimeric receptors connected to an extracellular reporter. In the present study, beta3 cytoplasmic domain deletion and substitution mutants were constructed to identify amino acids within the integrin beta3 cytoplasmic domain that regulate its ability to trigger FAK phosphorylation. Cells transiently expressing chimeric receptors containing these mutant cytoplasmic domains were magnetically sorted and assayed for the tyrosine phosphorylation of FAK. Analysis of these mutants indicated that structural information in both the membrane-proximal and C-terminal segments of the beta3 cytoplasmic domain is important for triggering FAK phosphorylation. In the C-terminal segment of the beta3 cytoplasmic domain, the highly conserved NPXY motif was found to be required for the beta3 cytoplasmic domain to trigger FAK phosphorylation. However, the putative FAK binding domain within the N-terminal segment of the beta3 cytoplasmic domain was found to be neither required nor sufficient for this signaling event. We also demonstrate that the serine 752 to proline mutation, known to cause a variant of Glanzmann's thrombasthenia, inhibits the ability of the beta3 cytoplasmic domain to signal FAK phosphorylation, suggesting that a single mutation in the beta3 cytoplasmic domain can inhibit both "inside-out" and "outside-in" integrin signaling.

Amino Acid Sequence↗
PubMed1997

Integrin cytoplasmic domains as connectors to the cell's signal transduction apparatus.

Integrins mediate the bidirectional transfer of signals across the plasma membrane. Integrin cytoplasmic domains provide one pathway linking integrin engagement with the cell's signal transduction apparatus. Recent structure-function studies have defined regions of beta cytoplasmic domains required for integrin function and have identified distinct roles for individual alpha cytoplasmic domains in regulating cell behavior. Newly identified proteins that bind to integrin alpha and beta cytoplasmic domains have provided new insights and new questions into the mechanisms involved in integrin signaling.

Amino Acid Sequence↗
PubMed1996

Integrin signaling.

Integrins are a family of adhesion receptors used by cells to interact with their extracellular matrix. Integrins also function as signaling receptors, integrating information from the extracellular matrix and other environmental cues including growth factors and hormones. Signals triggered by integrins direct cell adhesion and regulate other aspects of cell behavior including cell proliferation and differentiation, and determine cell survival. The biochemical pathways initiated by integrin engagement are now being identified.

Animals↗
PubMed1995

Integrin beta 3 cytoplasmic tail is necessary and sufficient for regulation of alpha 5 beta 1 phagocytosis by alpha v beta 3 and integrin-associated protein.

Using a K562 cell transfection model, we have previously described a novel relationship between the integrins alpha v beta 3 and alpha 5 beta 1. alpha v beta 3 ligation was able to inhibit alpha 5 beta 1-mediated phagocytosis without effect on alpha 5 beta 1-mediated adhesion. The alpha v beta 3-dependent inhibition apparently required a signal transduction cascade as it was reversed by inhibitors of serine/threonine kinases. Now, we have studied the mechanisms of signal transduction in this system and have found that the beta 3 cytoplasmic tail is both necessary and sufficient for initiation of the signal leading to inhibition of alpha 5 beta 1 phagocytosis. Ligation of integrin-associated protein (IAP), which has been implicated in alpha v beta 3 signal transduction, mimics the effects of alpha v beta 3 ligation only when the beta 3 integrin with an intact cytoplasmic tail is present. Although fibronectin-mediated phagocytosis requires the high affinity conformation of alpha 5 beta 1, ligation of alpha v beta 3/IAP does not prevent acquisition of this high affinity state. We conclude that alpha v beta 3/IAP ligation initates a signal transduction cascade, dependent upon the beta 3 cytoplasmic tail, which inhibits the phagocytic function of alpha 5 beta 1 at a step subsequent to modulation of integrin affinity.

Antigens, CD↗
PubMed1994

"Inside-out" signal transduction inhibited by isolated integrin cytoplasmic domains.

The affinities of integrin alpha beta heterodimers for extracellular ligands are important regulators of cell adhesion. Intracellular signals provoke changes in the integrin extracellular domain resulting in "activation," as manifested by an increase in affinity. Interactions of integrin cytoplasmic domains with intracellular elements may mediate this "inside-out signaling." Here we report that overexpression of chimeras of the cytoplasmic domain of integrin beta 3 or beta 1 subunits, joined to the extracellular and transmembrane domains of the Tac subunit of the interleukin-2 receptor, reduced integrin affinity. In contrast, chimeras containing the cytoplasmic domain of alpha 5 or alpha IIb or of beta 3 bearing a mutation that disrupts inside-out signaling lacked inhibitory activity. These data suggest that limiting quantities of intracellular factors bind to integrin beta 3 and beta 1 cytoplasmic domains to modulate ligand binding affinity. Structural mimics of these domains may provide a novel means to alter cell adhesion.

Amino Acid Sequence↗
PubMed1994

Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras.

Integrins are heterodimeric, transmembrane cell adhesion receptors that have recently been shown to function in transmembrane signal transduction. To examine the specific role of integrin intracellular domains in signal transduction, chimeric receptors containing various integrin intracellular domains coupled to a reporter consisting of the transmembrane and extracellular domains of the small, non-signaling subunit of the interleukin-2 receptor were expressed in cultured human fibroblasts and assayed for their ability to trigger tyrosine phosphorylation of the 125-kDa cytoplasmic tyrosine kinase, pp125FAK. Tyrosine phosphorylation of pp125FAK was induced in cultured fibroblasts that transiently expressed chimeric receptors containing either the beta 1, beta 3, or beta 5 integrin intracellular domain and were selected by magnetic bead sorting. However, expression of chimeric receptors containing either the alpha 5 or an alternatively spliced form of the beta 3 intracellular domain (beta 3B), as well as those lacking an intracellular domain, failed to induce tyrosine phosphorylation of pp125FAK. These results indicate that information contained in the beta 1, beta 3, or beta 5 integrin intracellular domain is sufficient to stimulate integrin-mediated tyrosine phosphorylation of specific intracellular proteins and that integrin extracellular and transmembrane domains are not required for inducing tyrosine phosphorylation. Our results also indicate that alternative splicing can regulate the ability of beta integrin intracellular domains to participate in signal transduction, and they further suggest that the carboxyl-terminal region of specific beta integrins may play a role in the signal transduction pathway involving extracellular matrix molecules.

Amino Acid Sequence↗
PubMed1994

Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly.

The ability of single subunit chimeric receptors containing various integrin beta intracellular domains to mimic and/or inhibit endogenous integrin function was examined. Chimeric receptors consisting of the extracellular and transmembrane domains of the small subunit of the human interleukin-2 receptor connected to either the beta 1, beta 3, beta 3B, or beta 5 intracellular domain were transiently expressed in normal human fibroblasts. When expressed at relatively low levels, the beta 3 and beta 5 chimeras mimicked endogenous ligand-occupied integrins and, like the beta 1 chimera (LaFlamme, S. E., S. K. Akiyama, and K. M. Yamada. 1992. J. Cell Biol. 117:437), concentrated with endogenous integrins in focal adhesions and sites of fibronectin fibril formation. In contrast, the chimeric receptor containing the beta 3B intracellular domain (a beta 3 intracellular domain modified by alternative splicing) was expressed diffusely on the cell surface, indicating that alternative splicing can regulate integrin receptor distribution by an intracellular mechanism. Furthermore, when expressed at higher levels, the beta 1 and beta 3 chimeric receptors functioned as dominant negative mutants and inhibited endogenous integrin function in localization to fibronectin fibrils, fibronectin matrix assembly, cell spreading, and cell migration. The beta 5 chimera was a less effective inhibitor, and the beta 3B chimera and the reporter lacking an intracellular domain did not inhibit endogenous integrin function. Comparison of the relative levels of expression of the transfected beta 1 chimera and the endogenous beta 1 subunit indicated that in 10 to 15 h assays, the beta 1 chimera can inhibit cell spreading when expressed at levels approximately equal to the endogenous beta 1 subunit. Levels of chimeric receptor expression that inhibited cell spreading also inhibited cell migration, whereas lower levels were able to inhibit alpha 5 beta 1 localization to fibrils and matrix assembly. Our results indicate that single subunit chimeric integrins can mimic and/or inhibit endogenous integrin receptor function, presumably by interacting with cytoplasmic components critical for endogenous integrin function. Our results also demonstrate that beta intracellular domains, expressed in this context, display specificity in their abilities to mimic and inhibit endogenous integrin function. Furthermore, the approach that we have used permits the analysis of intracellular domain function in the processes of cell spreading, migration and extracellular matrix assembly independent of effects due to the rest of integrin dimers. This approach should prove valuable in the further analysis of integrin intracellular domain function in these and other integrin-mediated processes requiring the interaction of integrins with cytoplasmic components.

Amino Acid Sequence↗
PubMed1992

Regulation of fibronectin receptor distribution.

To determine the role of each intracellular domain of the fibronectin receptor in receptor distribution, chimeric receptors were constructed containing the human interleukin-2 receptor (gp55 subunit) as the extracellular and transmembrane domains, in combination with either the alpha 5 or beta 1 intracellular domain of the fibronectin receptor as the cytoplasmic domain. These chimeric receptors were transiently expressed in normal fibroblasts, and their localization on the cell surface was determined by immunofluorescence using antibodies to the human interleukin-2 receptor. The alpha 5 chimera was expressed diffusely on the plasma membrane. The beta 1 chimera, however, colocalized with the endogenous fibronectin receptor at focal contacts of cells spread on fibronectin. On cells spread in the presence of serum, the beta 1 chimera colocalized both with the fibronectin receptor at sites of extracellular fibronectin fibrils and with the vitronectin receptor at focal contacts. The beta 1 intracellular domain alone, therefore, contains sufficient information to target the chimeric receptor to regions of the cell where ligand-occupied integrin receptors are concentrated. The finding that the beta 1 chimeric protein behaves like a ligand-occupied receptor, even though the beta 1 chimera cannot itself bind extracellular ligand, suggests an intracellular difference between occupied and unoccupied receptors, and predicts that the distribution of integrin receptors can be regulated by ligand occupancy. We tested this prediction by providing a soluble cell-binding fragment of fibronectin to cells spread on laminin. Under conditions preventing further ligand adsorption to the substrate, this treatment nevertheless resulted in the relocation of diffuse fibronectin receptors to focal contacts. Similarly, a redistribution of diffuse vitronectin receptors to focal contacts occurred on cells spread on laminin after the addition of the small soluble peptide GRGDS. We conclude that the propensity for receptor redistribution to focal contacts driven by the beta 1 cytoplasmic domain alone is suppressed in heterodimeric unoccupied fibronectin receptors, and that ligand occupancy can release this constraint. This redistribution of integrin receptors after the binding of a soluble substrate molecule may provide a direct means of assembling adhesion sites.

Amino Acid Sequence↗
PubMed1990

Differential keratin gene expression during the differentiation of the cement gland of Xenopus laevis.

The expression of both epidermal and nonepidermal keratins has been detected in the cement gland of Xenopus laevis by antibody staining. Northern blot and in situ hybridizations with gene-specific probes indicated the expression of the nonepidermal keratin, XK endo B, and the embryonic epidermal keratin, XK70, in the cement gland. Furthermore, since explanted animal pole cells can be induced to differentiate into cement gland cells in vitro by incubation in NH4Cl, we have demonstrated the in vitro induction of XK endo B, maintenance of XK70, and repression of another embryonic epidermal keratin, XK81. This is the first report of keratin gene expression in the cement gland.

Animals↗
PubMed1990

XK endo B is preferentially expressed in several induced embryonic tissues during the development of Xenopus laevis.

XK endo B is a type I keratin that was originally identified by its preferential expression in the embryonic notochord of the amphibian Xenopus laevis. A peptide identical to a short region of its predicted amino acid sequence was used to generate antibodies against the XK endo B protein. This paper reports an immunocytochemical study of the spatial expression pattern of XK endo B during development. The protein was observed in the notochord and endoderm as predicted from previous RNA analysis. In addition, XK endo B was detected in the cement gland, in the pituitary, olfactory and pharyngeal pouch rudiments, and in a nonuniform distribution in the neural tube as well as the inner sensorial layer of the ectoderm. XK endo B expression is not limited to any germ layer or any particular cell type, but is nevertheless highly restricted in its distribution in the embryo. Its expression in several different embryonic tissues requiring inductive interactions for differentiation makes XK endo B a valuable tool with which to study the regulation of induced gene expression during embryogenesis.

Amino Acid Sequence↗
PubMed1988

Xenopus endo B is a keratin preferentially expressed in the embryonic notochord.

Screening of a cDNA library from neurula stage Xenopus laevis for notochord-specific sequences led to the isolation of a cDNA clone, XK endo B (Xenopus keratin endo B), which encodes a nonepidermal type I keratin. In situ and Northern blot hybridizations indicate that expression of XK endo B RNA is concentrated in the notochord, whereas expression in the endoderm is 5-10 times lower. XK endo B mRNA is present in the oocyte and increases from late gastrula. Accumulation peaks by late neurula and is greatly reduced by the tadpole stage; in the adult, a low level of XK endo B RNA is present in the liver. XK endo B shows sequence homology to mouse endo B; genomic Southern blots show that XK endo B is the most similar sequence to mouse endo B in the Xenopus genome, and vice versa, indicating that XK endo B and mouse endo B are homologs. The use of endo B as a marker and the germ layer derivation of the notochord are discussed in light of these results.

Amino Acid Sequence↗
PubMed1985

Comparison of pausing during transcription and replication.

Pausing during the transcription of MDV-1 cDNA by Escherichia coli RNA polymerase was compared with pausing during the replication of MDV-1 RNA by Q beta replicase. MDV-1 RNA is able to form many strong hairpin structures, and Q beta replicase pauses after the synthesis of each [Mills et al. (1978) Cell 15, 541-550]. Although the transcripts were virtually identical to MDV-1 RNA, the locations at which RNA polymerase paused were different and apparently were not related to sequences that can form hairpins. These results indicate that hairpin stability, per se, cannot be used to predict the occurrence of pausing during transcription. Four pauses that occur within a 5-nucleotide region were studied in detail. Insertions and deletions were made in the template DNA to determine the contribution made by the surrounding sequences to these pauses. The results indicate that some of the pauses require the presence of particular upstream sequences, while others are unaffected by the template modifications. Thus, there are at least two different transcriptional pausing mechanisms: one depends on the nature of upstream sequences, while the other is independent of upstream sequences.

Base Sequence↗