Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties

Development ◽  
1997 ◽  
Vol 124 (10) ◽  
pp. 1865-1875 ◽  
Author(s):  
A. Joliot ◽  
A. Trembleau ◽  
G. Raposo ◽  
S. Calvet ◽  
M. Volovitch ◽  
...  
Keyword(s):  
Binding Sites ◽  
Cell Adhesion Molecule ◽  
Proteolytic Enzymes ◽  
Subcellular Fractionation ◽  
Low Density ◽  
Toxin Binding ◽  
Neural Cell Adhesion ◽  
Neural Tissues ◽  
Triton X ◽  
Specific Membrane

We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.

scholarly journals Cell surface modulation of the neural cell adhesion molecule resulting from alternative mRNA splicing in a tissue-specific developmental sequence.

1986 ◽  
Vol 103 (4) ◽  
pp. 1431-1439 ◽  
Author(s):  
B A Murray ◽  
G C Owens ◽  
E A Prediger ◽  
K L Crossin ◽  
B A Cunningham ◽  
...  
Keyword(s):  
Nervous System ◽  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Neural Cell Adhesion Molecule ◽  
Cytoplasmic Domain ◽  
Neural Cell ◽  
Mrna Splicing ◽  
Neural Cell Adhesion ◽  
Neural Tissues

The neural cell adhesion molecule N-CAM is an intrinsic membrane glycoprotein that is expressed in the embryonic chicken nervous system as two different polypeptide chains encoded by alternatively spliced transcripts of a single gene. Because they differ by the presence or absence of approximately 250 amino acids in their cytoplasmic domains, these polypeptides are designated ld and sd, for large and small cytoplasmic domain, respectively. We report here that the ld-specific sequences comprise a single exon in the chicken N-CAM gene and that developmental expression of the ld and sd chains occurs in a tissue-specific fashion, with the ld chain restricted to the nervous system. Comparison of the nucleotide sequences from an N-CAM genomic clone with cDNA sequences showed that a single exon of 783 base pairs corresponded to the unique cytoplasmic domain of the ld polypeptide. Sequences from this exon were absent from the single N-CAM mRNA detected in several non-neural tissues by RNA blot hybridization, and immunoblot analysis confirmed that antigenic determinants unique to the ld-specific domain were not expressed in these tissues. Immunohistochemical experiments indicated that only the sd chain was expressed on cell surfaces of non-neural tissues throughout embryonic development. The ld chain was found on cell bodies and neurites of differentiated neurons; it first appeared as neurons began to extend neurites and to express the neuron-glia cell adhesion molecule (Ng-CAM) and it was restricted to definite layers in laminar tissues such as the retina and cerebellum. These results suggest that the control of mRNA splicing may affect the regulation of N-CAM function at specific sites within the nervous system and thus influence the control of neural morphogenesis and histogenesis.

Saturable binding of [3H]phenytoin to rat brain membrane fraction

1981 ◽  
Vol 59 (4) ◽  
pp. 402-407 ◽  
Author(s):  
W. M. Burnham ◽  
L. Spero ◽  
M. M. Okazaki ◽  
B. K. Madras
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Membrane Fraction ◽  
Proteolytic Enzymes ◽  
Subcellular Fractionation ◽  
Brain Membrane ◽  
High Affinity ◽  
Number Of Binding Sites ◽  
Very High ◽  
Maximal Capacity

Preliminary studies indicate that [3H]phenytoin binds in a saturable and reversible fashion to at least two distinct sites in the membrane fraction of whole rat brain. One of these displays a high affinity (Kd = 6 nM) and a low maximal capacity (Bmax = 10 pmol/g protein). The other has a low affinity (Kd = 4.8 μM) and is estimated to have a very high maximal capacity. Phenytoin binding is reduced if the membrane fraction is preincubated with proteolytic enzymes and subcellular fractionation studies indicate that the P2 fraction has the largest number of binding sites. Competition experiments fail to reveal significant binding interactions with putative neurotransmitters or with other drugs except the hydantoins and anticonvulsant barbiturates. Although it is premature to speculate on the clinical significance of these findings, it is encouraging to note that the low affinity site has a Kd very similar to the therapeutic levels of phenytoin found in cerebrospinal fluid and that there seems to be some relationship between binding potency and anticonvulsant potency within the hydantoin series.

scholarly journals Binding of antibodies to acetylcholine receptors in Electrophorus and Torpedo electroplax membranes.

1978 ◽  
Vol 76 (3) ◽  
pp. 577-592 ◽  
Author(s):  
A Karlin ◽  
E Holtzman ◽  
R Valderrama ◽  
V Damle ◽  
K Hsu ◽  
...  
Keyword(s):  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Subcellular Fractionation ◽  
Antigenic Determinants ◽  
Electrophorus Electricus ◽  
Rabbit Igg ◽  
Immunohistochemical Methods ◽  
Triton X ◽  
Receptor Antibodies ◽  
Millipore Filters

Antisera against purified acetylcholine receptors from the electric tissues of Torpedo californica and of Electrophorus electricus were raised in rabbits. The antisera contain antibodies which bind to both autologous and heterologous receptors in solution as shown by an immunoprecipitation assay. Antibodies in both types of antisera bind specifically to the postjunctional membrane on the innervated surface of the intact electroplax from Electrophorus electric tissue as demonstrated by an indirect immunohistochemical procedure using horseradish peroxidase conjugated to anti-rabbit IgG. Only anti-Electrophorus receptor antisera, however, cause inhibition of the receptor-mediated depolarization of the intact Electrophorus electroplax. The lack of inhibition by anti-Torpedo receptor antibodies, which do bind, suggests that the receptor does not undergo extensive movement during activity. The binding of anti-Torpedo antibodies to receptor-rich vesicles prepared by subcellular fractionation of Torpedo electric tissue was demonstrated by both direct and indirect immunohistochemical methods using ferritin conjugates. These vesicles can be conveniently collected and prepared for electron microscopy on Millipore filters, a procedure requiring only 25 micrograms of membrane protein per filter. In addition, it was possible to visualize the binding of anti-Torpedo receptor antibodies directly, without ferritin. These anti-Torpedo receptor antibodies, however, do not inhibit the binding of acetylcholine or of alpha-neurotoxin to receptor in Torpedo microsacs but do inhibit binding of alpha-neurotoxin to Torpedo receptor in Triton X-100 solution. It is likely that the principal antigenic determinants on receptor are at sites other than the acetylcholine-binding sites and that inhibition of receptor function, when it occurs, may be due to a stabilization by antibody binding of an inactive conformational state.

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