Since the first discovery of the reelin mutant mouse in by Falconer, and later discovery of the gene for reelin in , there has been an explosion of new knowledge about this important molecule.
Reelin Glycoprotein: Structure, Biology and Roles in Health and Disease , written by an international panel of experts, summarizes the state of the knowledge on various aspects of reelin. Topics include the reelin gene and its receptors, downstream effector molecules in reelin signaling cascade, chemistry and structure of reelin, comparative anatomy of reelin, presence of reelin in various body tissues, reelin mutations, and abnormalities of reelin production in neuropsychiatric disorders and cancer.
This book will serve as a foundation for analysis of this emerging novel protein for all neuroscientists and clinicians.
Reelin glycoprotein is a serine protease with important roles in embryogenesis and during adult life. This comprehensive and integrative book examines the role that reelin plays in the etiology of various neuropsychiatric disorders, including schizophrenia and autism. The book provides an unprecedented analysis of this emerging and novel protein by examining evidence from genetic, neuroanatomic, biochemical, and behavioral studies. Read more Read less. Amazon Global Store US International products have separate terms, are sold from abroad and may differ from local products, including fit, age ratings, and language of product, labeling or instructions.
Manufacturer warranty may not apply Learn more about Amazon Global Store. Shown are sensorgrams after the subtraction of background binding to the control surface. Dissociation constant K D values calculated by a global fitting of the sensorgrams using BIAsimulation 3. Next, we checked whether the mutant reelin retains biological activity in a more physiologically relevant assay system using cultured neurons. Primary cultures of neurons derived from the cortex of mouse embryonic brain were incubated with the cell culture supernatants containing wild-type or CA mutant reelin followed by evaluation of the tyrosine phosphorylation of cellular Dab1.
To our surprise, CA mutant could not induce Dab1 phosphorylation even when undiluted culture supernatant was used, whereas Dab1 phosphorylation was clearly observed with wild-type reelin upon up to 4-fold dilution Fig. Immunoblot analysis of the supernatants with G10 antibody revealed that the there was no difference in the expression level of reelin protein between wild type and CA mutant Fig.
The lack of signaling activity of the mutant prompted us to check whether CA reelin successfully interacted with the cultured neurons. Covalent dimer formation via Cys is required for inducing Dab1 phosphorylation in cultured neurons. Primary cortical neurons were incubated with a dilution series of the cell culture supernatants sup.
The whole cell lysate was prepared and analyzed by Western blotting using anti-phosphotyrosine 4G10 top or anti-Dab1 middle.leondumoulin.nl/language/hide/ovarian-cancer-you-can-not.php
Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region
The same culture supernatant set was directly subjected to Western blotting with anti-reelin G10 bottom to confirm the equivalent expression level of WT and CA reelins. Shown are data from a representative experiment of three independent experiments. Reelin CA mutant does not stably bind neurons. Strong intracellular staining observed in small number of cells arrowheads represents endogenous reelin expressed in Cajal-Retzius cells and not the binding of exogenously added reelin.
It is known that reelin forms a covalent dimer via intermolecular disulfide bond s 16 , In the present study, we determined the critical cysteine residue participating in the formation of the covalent dimer and showed that the dimer formation via this cysteine residue is required for the full biological activity of reelin.
Careful inspection of the primary sequence guided by the structural information extracted eight potential candidates for the intermolecular disulfide formation of cysteines, and mutational experiments identified Cys present in the fifth reelin repeat as the sole responsible residue. As Cys included in the candidate list was the only cysteine residue present in the epitope segment for the CR, an antibody that is known to disrupt oligomerization of reelin 17 , we initially expected this residue to be responsible for the interchain disulfide.
However, mutation of Cys to Ala did not affect the covalent dimer formation of full-length reelin. In fact, this residue is unlikely to be functionally important because it is not conserved in human reelin. In contrast, Cys is conserved across species from sea urchin, Ciona , and lancelets to humans.
Furthermore, structural consideration also supports the involvement of Cys in the homodimer formation. In the crystal structure of R5—6 fragment, this residue is located on a surface-exposed loop at the top ridge of R5A domain with its side chain pointing outward 9. Although R5—6 region seems to adopt a stiff rodlike structure inferred by electron microscopy 8 , the location of Cys is compatible with the approach of two reelin molecules in either an antiparallel or diagonal fashion to form the disulfide-linked dimer.
Moreover, Cys is located at a molecular face opposite from the receptor-binding site i. Lys 9 , 20 , allowing the resultant dimer to bear well exposed receptor-binding sites. The fact that CA mutant reelin still oligomerizes Fig. As CA mutation led to a great reduction in the signaling activity, however, it is clear that a multimeric state alone is insufficient to keep the reelin fully active. It seems that a special higher order structure maintained by both covalent and non-covalent intermolecular interactions is required for the full activity.
Consistent with this view, it has been reported that the complete cleavage of the N-t site in full-length reelin results in greatly reduced activity There may be a structural or chemical mechanism that limits the multimer size during the biosynthesis of reelin.
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Therefore, it is not clear whether the apparent lack of signaling and binding activity toward neurons in mutant reelin Figs. A possibility remains that reelin utilizes an interaction with an as yet identified co-receptor s to achieve productive binding to neurons, which is critically dependent on the fine structure maintained by the intermolecular disulfide bond. The highly basic C-terminal region of reelin may mediate such a secondary interaction because removal of this segment is known to reduce signaling activity of reelin 38 , The physiological importance of the higher order structure of reelin described above may seem to contradict with the recently proposed hypothesis that the R3—6 fragment released by N-t and C-t cleavage functions as a major signaling molecule in tissues However, these two views are not mutually exclusive because these two molecular forms may constitute a mechanism of functional differentiation of reelin.
According to the model of Jossin et al. It is possible that the intact i. Although a detailed characterization of the molecular architecture of the functional reelin multimer awaits further study, the present biochemical characterization will help to design an experimental strategy to test the new model of reelin signaling.
We thank Drs. You'll be in good company. Journal of Lipid Research. Previous Section Next Section. Construction of Multiple Sequence Alignment and Homology Model Building Based on the sequence alignment for the reelin repeat segments made by Ichihara et al.
Protein Expression, Immunoprecipitation, and Western Blotting The expression vector for the N-terminally tagged full-length reelin was prepared as described previously Solid-phase Binding Assay A solid-phase receptor binding assay was carried out as described previously 8. In Vivo Biotinylation of Reelin Receptors and Surface Plasmon Resonance Analysis In vivo biotinylation of soluble ectodomain fragments of reelin receptors within the secretory pathway of mammalian cells was carried out using the experimental design described by Mize et al.
Dab1 Phosphorylation Assay Induction of tyrosine phosphorylation of Dab1 in mouse cortical neurons was measured as described previously Determination of Cysteine Residue s Responsible for Intermolecular Disulfide Bond Formation As reelin exists as a covalently linked oligomer in vivo , at least one cysteine residue should form a specific intermolecular disulfide bridge. Analysis of Purified Full-length Reelin The critical involvement of Cys in the interchain disulfide bond formation provided a way to investigate the molecular organization of reelin maintained by disulfide bridges.
Immunoblot Analyses of Tryptic Fragments from Wild-type Full-length Reelin To determine the disulfide bond pairing involving Cys , we needed to analyze the behavior of the fragments containing this residue. Biological Activity of Dimerization-deficient Reelin The disappearance of covalently linked multimer in CA mutant suggests that the mutation may affect the overall multimeric assembly.
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Reelin Glycoprotein: Structure, Biology and Roles in Health and Disease / Edition 1
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