scholarly journals Regulation of cellular interactions with laminin by integrin cytoplasmic domains: the A and B structural variants of the alpha 6 beta 1 integrin differentially modulate the adhesive strength, morphology, and migration of macrophages.

1994 ◽  
Vol 5 (6) ◽  
pp. 679-690 ◽  
Author(s):  
L M Shaw ◽  
A M Mercurio
Keyword(s):  
Culture Medium ◽  
Cytoplasmic Domain ◽  
Laminin Receptor ◽  
Cellular Interactions ◽  
Structural Variants ◽  
Cytoplasmic Domains ◽  
Macrophage Cell ◽  
Beta 1 Integrin ◽  
And Migration ◽  
Migration Of Macrophages

Several integrin alpha subunits have structural variants that are identical in their extracellular and transmembrane domains but that differ in their cytoplasmic domains. The functional significance of these variants, however, is unknown. In the present study, we examined the possibility that the A and B variants of the alpha 6 beta 1 integrin laminin receptor differ in function. For this purpose, we expressed the alpha 6A and alpha 6B cDNAs, as well as a truncated alpha 6 cDNA (alpha 6-delta CYT) in which the cytoplasmic domain sequence was deleted after the GFFKR pentapeptide, in P388D1 cells, an alpha 6 deficient macrophage cell line. Populations of stable alpha 6A, alpha 6B, and alpha 6-delta CYT transfectants that expressed equivalent levels of cell surface alpha 6 were obtained by fluorescence-activated cell sorter and shown to form heterodimers with endogenous beta 1 subunits. Upon attachment to laminin, the alpha 6A transfectants extended numerous pseudopodia. In contrast, the alpha 6B transfectants remained rounded and extended few processes. The transfectants were also examined for their ability to migrate toward a laminin substratum using Transwell chambers. The alpha 6A transfectants were three- to fourfold more migratory than the alpha 6B transfectants. The alpha 6-delta CYT transfectants did not attach to laminin in normal culture medium, but they did attach in the presence of Mn2+. The alpha 6-delta CYT transfectants migrated to a lesser extent than either the alpha 6A or alpha 6B transfectants in the presence of Mn2+. The alpha 6 transfectants differed significantly in the concentration of substratum bound laminin required for half-maximal adhesion in the presence of Mn2+:alpha 6A (2.1 micrograms/ml), alpha 6B (6.3 micrograms/ml), and alpha 6-delta CYT (8.8 micrograms/ml). Divalent cation titration studies revealed that these transfectants also differed significantly in both the [Ca2+] and [Mn2+] required to obtain half-maximal adhesion to laminin. These data demonstrate that the A and B variants of the alpha 6 cytoplasmic domain can differentially modulate the function of the alpha 6 beta 1 extracellular domain.

Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor

1996 ◽  
Vol 109 (13) ◽  
pp. 3139-3150 ◽  
Author(s):  
C.C. Yao ◽  
B.L. Ziober ◽  
A.E. Sutherland ◽  
D.L. Mendrick ◽  
R.H. Kramer
Keyword(s):  
Cytoplasmic Domain ◽  
Matrix Assembly ◽  
Developmentally Regulated ◽  
Laminin 5 ◽  
Alternative Mrna Splicing ◽  
Beta 1 Integrin ◽  
Alpha 7 ◽  
Myoblast Cell ◽  
And Migration

The alpha 7 beta 1 integrin is specifically expressed by skeletal and cardiac muscles, and its expression and alternative mRNA splicing at the cytoplasmic domain are developmentally regulated. We analyzed the role of alpha 7 integrin in mediating myoblast adhesion and motility on different laminin isoforms. Mouse C2C12 and MM14 myoblast cell lines were found by flow cytometry and immunoprecipitation to express high levels of the alpha 7 integrin. Overall expression of alpha 7 increased as the C2C12 myoblasts differentiated; myoblasts expressed only the alpha 7B cytoplasmic variant whereas in differentiating myotubes alpha 7A increased markedly. Function-perturbing monoclonal antibodies generated to alpha 7 integrin efficiently blocked both adhesion and migration of MM14 and C2C12 mouse myoblasts on laminin 1. Other studies with MM14 myoblasts showed that alpha 7 is also a receptor for laminin 2/4 (human placental merosins) but not for epithelial-cell-specific laminin 5. Blocking antibody to alpha 7 only partially inhibited adhesion to laminin 2/4 but almost completely blocked motility on this substrate. Finally, to assess the potential role of the alpha 7 cytoplasmic domain, CHO cells were stably transfected to expressed chimeric alpha 5 cDNA constructs containing the wild-type alpha 5 or the alpha 7A or alpha 7B cytoplasmic domain; all forms of the integrin showed identical activities for adhesion, migration, proliferation, and matrix assembly on fibronectin substrates. These results established that alpha 7 beta 1 receptor can promote myoblast adhesion and motility on a restricted number of laminin isoforms and may be important in myogenic precursor recruitment during regeneration and differentiation.

scholarly journals Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit.

1993 ◽  
Vol 123 (4) ◽  
pp. 1017-1025 ◽  
Author(s):  
L M Shaw ◽  
A M Mercurio
Keyword(s):  
Point Mutations ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Serine Phosphorylation ◽  
Laminin Receptor ◽  
Site Directed Mutagenesis ◽  
Receptor Function ◽  
Serine Residue ◽  
Beta 1 Integrin

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.

scholarly journals The major laminin receptor of mouse embryonic stem cells is a novel isoform of the alpha 6 beta 1 integrin.

1991 ◽  
Vol 115 (3) ◽  
pp. 843-850 ◽  
Author(s):  
H M Cooper ◽  
R N Tamura ◽  
V Quaranta
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Cytoplasmic Domain ◽  
Laminin Receptor ◽  
Extracellular Matrix Protein ◽  
Cytoplasmic Domains ◽  
Alternative Mrna Splicing

Laminin is the first extracellular matrix protein expressed in the developing mouse embryo. It is known to influence morphogenesis and affect cell migration and polarization. Several laminin receptors are included in the integrin family of extracellular matrix receptors. Ligand binding by integrin heterodimers results in signal transduction events controlling cell motility. We report that the major laminin receptor on murine embryonic stem (ES) cells is the integrin heterodimer alpha 6 beta 1, an important receptor for laminin in neurons, lymphocytes, macrophages, fibroblasts, platelets and other cell types. However, the cytoplasmic domain of the ES cell alpha 6 (alpha 6 B) differs totally from the reported cytoplasmic domain amino acid sequence of alpha 6 (alpha 6 A). Comparisons of alpha 6 cDNAs from ES cells and other cells suggest that the alpha 6 A and alpha 6 B cytoplasmic domains derive from alternative mRNA splicing. Anti-peptide antibodies to alpha 6 A are unreactive with ES cells, but react with mouse melanoma cells and embryonic fibroblasts. When ES cells are cultured under conditions that permit their differentiation, they become positive for alpha 6 A, concurrent with the morphologic appearance of differentiated cell types. Thus, expression of the alpha 6 B beta 1 laminin receptor may be favored in undifferentiated, totipotent cells, while the expression of alpha 6 A beta 1 receptor occurs in committed lineages. While the functions of integrin alpha chain cytoplasmic domains are not understood, it is possible that they contribute to transferring signals to the cell interior, e.g., by delivering cytoskeleton organizing signals in response to integrin engagement with extracellular matrix ligands. It is therefore reasonable to propose that the cellular responses to laminin may vary, according to what alpha subunit isoform (alpha 6 A or alpha 6 B) is expressed as part of the alpha 6 beta 1 laminin receptor. The switch from alpha 6 B to alpha 6 A, if confirmed in early embryos, could then be of striking potential relevance to the developmental role of laminin.

scholarly journals A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

1994 ◽  
Vol 14 (11) ◽  
pp. 7404-7413 ◽  
Author(s):  
S Takaki ◽  
H Kanazawa ◽  
M Shiiba ◽  
K Takatsu
Keyword(s):  
Signal Transduction ◽  
Protein Tyrosine Kinase ◽  
Cytoplasmic Domain ◽  
Beta Chain ◽  
Cytoplasmic Domains ◽  
Interleukin 5 ◽  
Alpha Chain ◽  
Gm Csf ◽  
Response Expression ◽  
And Function

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.

scholarly journals Multiple regions within the cytoplasmic domains of the leukemia inhibitory factor receptor and gp130 cooperate in signal transduction in hepatic and neuronal cells.

1994 ◽  
Vol 14 (1) ◽  
pp. 138-146 ◽  
Author(s):  
H Baumann ◽  
A J Symes ◽  
M R Comeau ◽  
K K Morella ◽  
Y Wang ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Amino Acid ◽  
Leukemia Inhibitory Factor ◽  
Cytoplasmic Domain ◽  
Inhibitory Factor ◽  
Receptor Protein ◽  
Human Neuroblastoma Cell ◽  
Chimeric Receptor ◽  
Cytoplasmic Domains ◽  
Human Neuroblastoma

The receptor for leukemia inhibitory factor (LIFR), in combination with the signal-transducing subunit for interleukin-6-type cytokine receptors, gp130, and LIF, activates transcription of acute-phase plasma protein genes in human and rat hepatoma cells and the vasoactive intestinal peptide gene in a human neuroblastoma cell line. To identify the regions within the cytoplasmic domain of LIFR that initiate signal transduction independently of gp130, we constructed a chimeric receptor by linking the extracellular domain of the granulocyte colony-stimulating factor receptor (G-CSFR) to the transmembrane and cytoplasmic domain of human LIFR. The function of the chimeric receptor protein in transcriptional activation was assessed by G-CSF-mediated stimulation of cotransfected cytokine-responsive reporter gene constructs in hepatoma and neuroblastoma cells. By using the full-length cytoplasmic domain and mutants with progressive carboxy-terminal deletions, internal deletions, or point mutations, we identified the first 150 amino acid residues of LIFR as the minimal region necessary for signaling. The signaling reaction appears to involve a cooperativity between the first 70-amino-acid region containing the two sequence motifs conserved among hematopoietin receptors (box 1 and box 2) and a critical sequence between residues 141 and 150 (box 3). Analogous analyses of the cytoplasmic domains of G-CSFR and gp130 indicated similar arrangements of functional domains in these receptor subunits and the requirement of a box 3-related motif for signaling.

Expression of alpha 7 integrin cytoplasmic domains during skeletal muscle development: alternate forms, conformational change, and homologies with serine/threonine kinases and tyrosine phosphatases

1993 ◽  
Vol 106 (4) ◽  
pp. 1139-1152 ◽  
Author(s):  
W.K. Song ◽  
W. Wang ◽  
H. Sato ◽  
D.A. Bielser ◽  
S.J. Kaufman
Keyword(s):  
Skeletal Muscle ◽  
Untranslated Region ◽  
Muscle Development ◽  
Myogenic Differentiation ◽  
Cytoplasmic Domain ◽  
Cytoplasmic Domains ◽  
Coding Region ◽  
Common Coding ◽  
Alternate Forms ◽  
Alpha 7

We recently reported the cloning and sequencing of the alpha 7 integrin chain and its regulated expression during the development of skeletal muscle (Song et al. (1992) J. Cell Biol. 117, 643–657). The alpha 7 chain is expressed during the development of the myogenic lineage and on adult muscle fibers and this suggests that it participates in multiple and diverse functions at different times during muscle development. One interesting portion of this isoform is its cytoplasmic domain; comprised of 77 amino acids it is the largest in the alpha chains thus reported. In these experiments we begin to study the potential functions of the alpha 7 cytoplasmic domain by analyzing homologies between the rat and human sequences, by immunologic studies using an anti-cytoplasmic domain antiserum, and by identifying two alternate forms. In keeping with the nomenclature used to describe the alpha 3 and alpha 6 alternate cytoplasmic domains, we refer to the originally reported species as alpha 7B and the two additional forms as alpha 7A and alpha 7C. These three cytoplasmic domains likely arise as a consequence of alternate splicing. A splice site at the junctions of the transmembrane and cytoplasmic domains is used to generate the alpha 3, alpha 6 and alpha 7 A and B forms. The alpha 7A form RNA contains an additional 113 nucleotides compared to the B form, and a common coding region in the A and B form RNAs is used in alternate reading frames. Part of the coding region of alpha 7B appears to be used as the 3′-untranslated region of the alpha 7A form. The alpha 7C mRNA is 595 nucleotides smaller than the alpha 7B RNA and part of the 3′-untranslated region of the alpha 7B isoform is used as coding sequence in alpha 7C. There is developmental specificity in expression of these alternate mRNAs: alpha 7A and alpha 7C transcripts are found upon terminal myogenic differentiation whereas alpha 7B is present earlier in replicating cells and diminishes upon differentiation. We suggest this selective expression of the alpha 7 cytoplasmic domains underlies the diversity in function of the alpha 7 beta 1 integrin at different stages of muscle development. Immunochemical analyses indicate that the alpha 7B cytoplasmic domain undergoes a change in conformation in response to binding laminin or upon crosslinking initiated with antibody reactive with the integrin extracellular domain. Crosslinking also promotes association of the integrin with the cell cytoskeleton.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a large complex containing the integrin alpha 6 beta 1 laminin receptor in neural retinal cells

1994 ◽  
Vol 107 (11) ◽  
pp. 3165-3172 ◽  
Author(s):  
I. de Curtis ◽  
G. Gatti
Keyword(s):  
Biochemical Properties ◽  
Cytoskeletal Proteins ◽  
Laminin Receptor ◽  
Retinal Neurons ◽  
Differential Distribution ◽  
Gradient Distribution ◽  
Large Complex ◽  
Beta 1 Integrin ◽  
Integrin Alpha 6 ◽  
And Function

Integrin alpha 6 beta 1 is a laminin receptor involved in adhesion and neurite extension of retinal neurons on laminin. The present study was carried out to identify potential interactions between the alpha 6 beta 1 receptor and cellular proteins that may be involved in integrin signaling and function. For this purpose we have used a biochemical approach based on the solubilization of retinal neurons cultured on laminin with nonionic detergents, followed by centrifugation on sucrose velocity gradients. Analysis of the distribution of the alpha 6 and beta 1 integrin subunits in the gradients showed that they migrate as a large complex after extraction of cells with octylglucoside, but not after Triton X-100 extraction. Cytoskeletal proteins known to localize in adhesion plaques did not comigrate with alpha 6 beta 1 in octylglucoside gradients, while a set of polypeptides whose tyrosine phosphorylation was enhanced by culture on laminin colocalized with alpha 6 beta 1 on the gradients after octylglucoside solubilization. Culture of retinal neurons on bovine serum albumin, a nonadhesive substratum, partially affected the gradient distribution of the receptor after octylglucoside extraction. Furthermore, analysis of the gradient distribution of two alternatively spliced isoforms of the alpha 6 subunit, alpha 6-cytoA and alpha 6-cytoB, showed that the effect of non-adhesion on the sedimentation properties of the two integrin alpha 6 isoforms was more dramatic for alpha 6-cytoB than alpha 6-cytoA. These differences in the sedimentation behaviour indicate distinct biochemical properties of the two alpha 6 isoforms that, together with previous observations on their differential distribution in the developing retina, may reflect functional specificities.

The subcellular distribution of the high molecular mass protein, HD1, is determined by the cytoplasmic domain of the integrin beta 4 subunit

1997 ◽  
Vol 110 (2) ◽  
pp. 169-178 ◽  
Author(s):  
P. Sanchez-Aparicio ◽  
A.M. Martinez de Velasco ◽  
C.M. Niessen ◽  
L. Borradori ◽  
I. Kuikman ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Subcellular Distribution ◽  
Cytoplasmic Domain ◽  
High Molecular Mass ◽  
Integrin Subunit ◽  
Type Ii ◽  
Structural Protein ◽  
Focal Contacts ◽  
Cell Substrate ◽  
Beta 1 Integrin

The high molecular mass protein, HD1, is a structural protein present in hemidesmosomes as well as in distinct adhesion structures termed type II hemidesmosomes. We have studied the distribution and expression of HD1 in the GD25 cells, derived from murine embryonal stem cells deficient for the beta 1 integrin subunit. We report here that these cells possess HD1 but not BP230 or BP180; two other hemidesmosomal constituents, and express only traces of the alpha 6 beta 4 integrin. By immunofluorescence and interference reflection microscopy HD1 was found together with vinculin at the end of actin filaments in focal contacts. In OVCAR-4 cells, derived from a human ovarian carcinoma which, like GD25 cells, only weakly express alpha 6 beta 4, HD1 was also localized in focal contacts. Upon transfection of both GD25 and OVCAR-4 cells with cDNA for the human beta 4 subunit the subcellular distribution of HD1 changed significantly. HD1 is then no longer present in focal contacts but in other structures at cell-substrate contacts, colocalized with alpha 6 beta 4. These junctional complexes are probably the equivalent of the type II hemidesmosomes. Transfection of GD25 cells with beta 1 cDNA did not affect the distribution of HD1, which indicates that the localization of HD1 in focal contacts was not due to the absence of beta 1. Moreover, in GD25 cells transfected with cDNA encoding a beta 4/beta 1 chimera, in which the cytoplasmic domain of beta 4 was replaced by that of beta 1, the distribution of HD1 was unaffected. Our findings indicate that the cytoplasmic domain of beta 4 determines the subcellular distribution of HD1 and emphasize the important role of alpha 6 beta 4 in the assembly of hemidesmosomes and other junctional adhesive complexes containing HD1.

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