Fibronectin variable

FIGURE 7-1 The immunoglobulin (Ig) gene family of molecules. Several varieties of Ig domain-containing molecules are contained within the Ig gene superfamily. Most are type I membrane proteins some have only Ig domains or other moieties that may convey function (see text). V, variable Ig domain C, constant Ig domain MAG, myelin-associated glycoprotein NCAM, neural cell adhesion molecule GPI, glycosylphosphatidyl-inositol EC, extracellular domain FN, fibronectin. 

The Ig-superfamily contains many proteins involved in immune recognition such as products of the MHC complex and accessory molecules [53]. In addition there are ten or more members associated mainly with nervous tissues in mature animals and several others in non-nervous tissue that are important factors in cell-cell and cell-substratum adhesion in non-immune cells. See [54] and [55] for detailed discussion of other aspects of Ig-superfamily glycoproteins. All of the cell adhesion glycoproteins in the family contain a variable number of Ig-like domains of about one hundred amino-acid residues, usually but not always defined within a pair of disulfide-bonded cysteine residues, and of the C2 type fold. In many cases the molecules contain variable numbers of another type of modular sequence known as the fibronectin type III repeat, sinee it was discovered in fibroneetin. In the following discussion, some principles of the structure and functions of this large family of cell adhesion molecules will be considered with particular emphasis on the interplay between different members in adhesion and modulation of adhesive interactions by carbohydrates. 

Fig. 8. Structures of LI, MAG and PO glycoproteins. The sequences of LI (a), MAG (b) and PO (c) glycoproteins predicted from analysis of cDNA clones as well as protein in the case of PO are shown to variably include in the extracellular portions Ig-like domains, fibronectin type III repeats (hatched), a transmembrane segment and a cytoplasmic domain. Potential A-glycosylation sites are indicated (open circle). Splicing variants in the cytoplasmic domains of LI and MAG are shown (open triangle).

The Boyden chamber is a simple apparatus used to test for chemotaxis, especially of leukocytes. It can also be used to assess tumor cell transmigration across an endo-thehal monolayer in vitro. It consists of two compartments separated by a MiUi-pore filter (3-8 pm pore size). A chemotac-tic factor is placed in one compartment, and a gradient develops across the thickness of the filter (ca. 150 pm). Cell movement into the filter is measured after an incubation period less than the time taken for the gradient to decay. Cell motility can be measured in Boyden chambers containing filters precoated with different materials, for example fibronectin or fibronectin fragments. The method, when apphed to malignant and non-mahgnant cell hnes, shows that the variable invasive potentials of these cells correlate with their abihty to disrupt the endothelial cell monolayer. 

This simple-looking inversion of the concentration-absorbance relationship has rather important consequences. It is possible, by use of the "P" matrix approach, to quantify the composition of mixtures in the presence of variable amounts of "impurities", without being able to specify the composition or concentration of those impurities. The only requirement is that the impurities must be present in the calibration standards in amounts that bracket the concentrations of these impurities in the unknown samples. This property of the "P" matrix method makes it feasible to generate calibration spectra of plasma or whole blood containing known amounts of the proteins of interest (albumin, gamma-globulins, fibrinogen, fibronectin, transferrin. . . ) and the remainder of the blood components can be treated as impurities. 

The cytoplasmic domains of all of these receptors have an intrinsic protein tyrosine kinase activity, and all the receptors have hydrophobic transmembrane sequences. Their extracellular regions are more variable in stmcture. Depending on the receptor, they may contain a range of domains, including (1) immrmoglobulin domains, (2) cysteine-rich motifs, (3) fibronectin type III repeats, and (4) EGF motifs. These can be present singly or in different combinations. Growth factor receptors are therefore examples of mosaic proteins. 

The conformation of the fibronectin blocks was not determined from these analyses. Since the fibronectin blocks are located between SLP block domains in the sequence of ProNectin F, it must be presumed that they occur at the turns between the crystallized SLP blocks. Their conformation is probably variable, but the fact that they are highly active as cell adhesion ligands, indicates that at least some of them are accessible and are exposed at the exterior of the crystalline region of the tUes . 

Figure 3. Focal adhesions in (A,B) embryonic fibroblasts and (C,D) tumor cells removed from MMTY-PyMT mice. Cells were plated on fibronectin in serum-free medium. Fibroblasts were stained with rhodamine-phalloidin, FITC conjugated anti-paxillin antibodies, and Hoechst 33258 stain. Tumor cells were stained with rhodamine-phalloidin and FITC conjugated anti-vinculin antibodies. Fluorescence images of the cells were obtained using a decovolution microscope. Paxillin and vinculin (green) are localized to ends of microfilaments (red) in focal adhesions. Mgat5 (A) fibroblasts and (C) tumor cell show focal adhesions but these structures are absent in MgatS (B) fibroblasts and (D) tumor cells. (E), T cell receptor dependent stimulation measured by H-thymi-dine incorporation in response to anti-CD3 antibodies at 48h (F) Model of responses to variable substratum adhesions for Mgat5 and Mgat5 cells.

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