QUANTITATIVE FEATURES OF BINDING OF RECEPTOR AND LIGAND

LIGAND-BINDING AND CELL-SIGNALING STUDIES

Different assays have been established to study the kinase activity and signaling pathways. Dependent on the phase of the study several known techniques have the potential to explore valuable information regarding cell-signaling. In the following divisions, we are going to discuss some precise approaches used for the study of cell signaling.

INHIBITOR BLOCKS SPECIFIC KINASES

Several inhibitors are currently acknowledged to study the specific interactions with receptor or specific kinases. These inhibitors may be exact for single kinase at low doses. High doses of these inhibitors may distress wider class of molecules. Most of the inhibitors act as competitive inhibitor of ATP binding site and are reversible. To avoid the distress of wider class of molecules, persistent levels of inhibitor is compulsory throughout the experimental duration. Evaluation of inhibitor potential should consist of biochemical, morphological, and behavioral analysis.

CELL BEHAVIOR ASSAYS

Cell behavior assays may define alterations in characteristics such as changes in cell shape, cytoskeletal elements, matrix binding, migration, or differentiation. Spreading, attachment, and migration assays are commonly being used to regulate up or down signal transduction proteins and their respective change in the cellular behavior (Berrier et al., 2000). Inhibitors also regulate signal transduction proteins by precise inhibitors of cell surface receptors and intracellular kinases. Transfecting or micro- injecting the cells with plasmids can also explore changes in the cellular behavior by altering the activity of specific proteins/enzymes (Berrier et ah, 2000). Blocking protein synthesis by antisense oligonucleotides specific to the mRNA of that specific protein can modulate function of the specific protein. Studies based on this technology showed that different signal transduction pathway contribute to cytoskeletal rearrangements. The ECM-stimulated fluctuations in the arrangement of actin cytoskeleton have been well recognized in tissue by using transmission electron microscopy (ТЕМ) and confocal microscopy (Chu et ah, 2000). Researchers also perform in vitro wound-healing method in which the cells grown to confluence and then a scrape/wound is positioned in the culture dish (Song et ah, 2000). The cells are detected by stirring the surface of the wound under numerous conditions to govern what proteins are essential for cell migration resulting into wound healing.

SIGNAL TRANSDUCTION EVENT VISUALIZATION

Usually, signal transduction activities like pH or change in calcium levels changes can be detected through intracellular fluorescent indicators. Techniques like fluorescent markers, immunoliistochemistry, and caged proteins can be utilized to study translocation of specific protein in a definite plasma membrane lipid bilayer. Recently, transportation of proteins has been tracked by integrating a fluorescent protein gene (green fluorescent protein (GFP)) into genetic vector encrypting the protein to be studied.

QUANTITATIVE ALTERATION IN CALCIUM AND pH

Alteration in calcium or pH levels can be accomplished by single-wavelength dyes. These fluorescent probes show a spectral reply upon binding with Ca2+. The alteration in concentrations of intracellular free Ca2+ can be detected by using FACS (fluorescence activated cell sorting) and fluorescence spectroscopy. Mostly fluorescent indicators are derivatives of Ca2+ chelators (ethylene glycol-bis (p- aminoethyl ether)-N,N,N,’N’-tetraacetic acid), o-aminophenol-N,N,0-triacetic acid, and l,2-bis(o- aminophenoxy) ethane- A,A,iV',iV'-tetraacetic acid. The mutual ratiometric dye for Ca2+ is indo-1 and fura-2. This dye alters the wavelength within a range of ion concentration. Single wavelength dyes are commercially available such as calcium orange, fluo-3, calcium green, fura red, calcium crimson, and Rliod-2 (Haugland and Johnson, 1999). These compound needs a fluorescent microscope with the capability to record moderately fast alteration in emission wavelength by using charged coupled device (CCD) cameras, enhanced video or veiy fast confocal microscopes.

IMMUNOHISTOCHEMISTRY

To locate the signaling proteins, researchers are using immunohistochem- istiy technique. This technique indicates not only the protein activation but also might confirm the state of the target protein. Many companies are also manufacturing antibodies which signal the activated state of protein. These antibodies have recognized epitope in phosphate or other activated conformation. Anti- active (antibodies against active epitopes) antibodies are also accessible for the specific signal transduction protein. In rare cases when the anti-active antibody is unavailable, a substitute method is used. In this method, the cells are twice labeled with a target antibody and another antibody that identifies all the phosphorylated amino acids like serine, threonine, or tyrosine. The outcome of method is an overlap of both the signals i.e., a site where the active protein occurs and the site where the single-labeled protein is not activated.

CONSTRUCTED FUSION PROTEINS

Living cells can also be identified with GFP-tagged proteins. Vectors with GFP attached to the protein of interest are transfected into tissues, cells, or transgenic animals to follow the pattern of protein expression. GFPs has been altered to produce several emission wavelength like yellow FP (YFP), cyan FP (CFP) and DsRed so that it could be utilized in combination, to label the multiple proteins (Ayoob et al., 2001). GFP-tagged fusion protein transfection is a predominant method, if the GFP tagged protein alters cellular location after activation. In a study, a section of MBP (myelin basic protein) that possesses a single consensus PRTP (ERK/MAP kinase phosphorylation motif) was combined with the GFP. The protein that has been fused and transfected inside the mammalian cell behaves as a substrate kinase. The GFP-MBP fused protein gets phosphorylated following serum stimulation while a MEK inhibitor obstructed this deviation in phosphorylation (Mandell and Gocan, 2001).

FRET AND FRAP

FRAP (fluorescence recovery/redistribution after photobleaching) and FRET (fluorescence resonance energy transfer) require protein/organelle to be studied with fluorescently labeled either in live cells or in fixed preparations. FRET is the nonradioactive shift in energy from a donor that is in an excited state to an adjacent acceptor (Matyus, 1992). A fluorescent molecule that gets excited by a particular wavelength of light is the donor here. The donor emits a higher light wavelength that excites the fluorescence acceptor molecule. Most of the fluorescent molecules can be used as a donor/acceptor pair. Donor/acceptor pair examples are the Cy3, Cy5, CFP, and YFP. Energy transfer is reliant on the space between the fluorescent molecules. As the acceptor sinks in the donor fluorescence, the donor will quench the energy and its lifetime will decrease. FRET is a very influential light microscopic technique to determine whether two proteins are within 10-70A of each other rather than co-localized with confocal microscopy.

FRAP determines the energy kinetics of diffusion through cells or tissues. It is accomplished by measuring the lateral diffusion in two dimensions, of a thin film consisting of probes that are fluorescently labeled or to scan single cells. Before the targeted region is photo-bleached, the cells are formerly loaded with the fluorescent molecule of concern and the movement of fluorescent molecules back into the bleached part is measured (Mochizuki et al., 2001). This system permits the determination of the diffusion and mobility of small molecules in the living cells’ cytoplasm. It also records the movement of macromolecules such as RNA, heavy protein or dmg outside and inside the cell organelles (nucleus).

WESTERN BLOTS

In immune-genetics and molecular biology, the protein immunoblot or western blot is a broadly used technique to analyze specific proteins from an extract or tissue homogenate. The benefit of this kind of blotting is that it specifies all of the proteins that may be tyrosine phosphorylated and can document how to separate proteins that may increase or decrease the signal. The blot does not identify the precise protein. Either a sister blot can be probed with the definite antibody or the same blot can be exposed and re-probed with additional primary antibody for the identification of the single protein.

MMUNOPRECIPITATION

Immunoprecipitation is comparatively easy and involves the same apparatus as western blot. Beads are made-up of a diversity of substances. Studies showed that cross-link primary antibody of a specific protein or all tyrosine- phosphorylated proteins to the beads may occur. The cells are lysed in a buffer comprising protease inhibitor and incubated with the antibody-coated beads. The proteins get separated by using SDS electrophoresis and recognized by the same procedures of western blot. The protein-protein interactions confirmed by western blots and immune-precipitation from cell lysate may not disclose the actual situation in vivo thus additional experiments are essential to determine precise interactions.

GST BINDING OR "PULL DOWN" ASSAYS

The glutathione S-transferase (GST) binding or “pull-down” assay is equivalent to imnrunoprecipitation. It also detects direct protein-protein interactions. Trash of proteins is produced thr ough a bacterial expression system with a GST tag. In a study, the researcher has used GST-labeled proteins like Rho GDP, Rho GTP, and the domain which binds Rho in protection (RBD-GST), and proper GST control verified that the corneal epithelial cells show a biphasic reaction to collagen. A reduction in Rho GTP after 15 min of collagen stimulation was done and after that, an augmentation at 30 min similar to the response in endothelial cells was carried out (Ren et al., 2000).

ACTIVITY ASSAYS

To identify the activity of a particular kinase, the activity assay exposes the cell lysate to a known substrate for the enzyme in the existence of radioactive phosphate. The partition of the product is accomplished by electrophoresis and bared to the x-ray film to detect the incorporated protein to the isotope. Apoptosis results in susceptible cells to undertake a cascade of morphologic and enzymatic changes. Various signal transduction pathways, as well as several specific degradative enzymes, become activated during apoptosis. These enzymes may chop crucial structural apparatus of the cell containing small nucleoproteins, actin cytoskeletal elements as well as nuclear lanrins (De Laurenzi and Melino, 2000). To establish that the cells encompass active caspase-3 or not, caspase-3 substrates consist of caspase-3 recognition sequence DEVD (aspartic acid-glutamic acid valine-aspartic acid) was used in a fluorophore-derivatized peptide which behaves same as the structural loop conformation existing in the globular proteins' indigenous protease cleavage sites. The compound is not fluorescent unless it is chopped by endogenous caspase-3. To check the reaction is particular for the caspase-3 activity. Certain tissues can be formerly treated with the caspase-3 inhibitor such as Z-VAD-FMK.

KEYWORDS

  • • enzyme coupled receptors
  • • Ephrin receptor
  • • epidermal growth factor
  • • fibroblast growth factor receptor
  • • fluorescence-activated cell sorting
  • • heterotrimeric G-protein

REFERENCES

Ayoob. J. C., Slianer, N. C., Sanger, J. W., & Sanger, J. M., (2001). Expression of green or red fluorescent protein (GFP or DsRed) linked proteins in nonmuscle and muscle cells. Mol. Biotechnol., 17(1), 65-71.

Berrier. A. L., Mastrangelo. A. M, Downward, J., Ginsberg, M., & LaFlamme, S. E., (2000). Activated R-Ras. Racl, PI 3-kinase and PKCe can each restore cell spreading inhibited by isolated integrin pi cytoplasmic domains. J. Cell Biol, 151(1), 1549-1560.

Chu, C. L.. Reenstra, W. R„ Orlow, D. L.. & Svoboda, К. К. H., (2000). ERK and PI-3 kinase are necessary for collagen binding and actin reorganization in corneal epithelia. Invest. Ophthalmol. Vis. Sci., 41(11), 3374-3382.

De Laurenzi, V., & Melino, G., (2000). Apoptosis: The little devil of death. Nature, 406 (6792), 135, 136.

Fredriksson. R., Lagerstrom. M. C., Lundin, L. G., & Sckioth. H. B., (2003). The G-protein- coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol., 63(6), 1256-1272.

Haugland, R. P.. & Johnson. I. D.. (1999). Intracellular ion indicators. Fluorescent and Luminescent Probes for Biological Activity (pp. 40-50). Mason WT. Academic Press, Cambridge.

Lappano, R.. & Maggiolini, M., (2011). G protein-coupled receptors: Novel targets for drug discovery in cancer. Nat. Rev. Drug Discov., 10(1), 47-60.

Mandell, J. W., & Gocan, N. C., (2001). A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity. Anal. Biochem., 293(2), 264-268.

Matyus, L., (1992). New trends in photobiology: Fluorescence resonance energy transfer measurements on cell surfaces. A spectroscopic tool for determining protein interactions. J. Photochem. Photobiol. B„ 12(4), 323-337.

Mochizuki. N.. Yamashita, S., Kurokawa, K., Oliba, Y., Nagai, T., Miyawaki. A.. & Matsuda. M., (2001). Spatio-temporal images of growth-factor-induced activation of Ras and Rap 1. Nature, 411(6841), 1065-1068.

Ren, X. D., Kiosses, W. B., Sieg. D. J., Otey, C. A., Schlaepfer, D. D., & Schwartz, M. A., (2000). Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover. J. Cell Scl., 113(20), 3673-3678.

Song, Q. H., Singh, R. R, Richardson, T. P„ Nugent, M. A., Trinkaus-Randall, V., (2000). Transforming growth factor-pl expression in cultured corneal fibroblasts in response to injury. J. Cell Biochem., 77(2), 186-199.

 
Source
< Prev   CONTENTS   Source   Next >