myospheroid
Integrins as a fibrinogen receptors Integrins mediate signal transduction through interaction with multiple cellular or extracellular matrix ligands. Integrin
alphavbeta3 recognizes fibrinogen, von Willebrand factor, and vitronectin; alphavbeta1 does not. The
mechanisms for defining the ligand specificity of these integrins was studied by swapping the highly diverse sequences in the I domain-like
structure of the beta1 and beta3 subunits. When the sequence CTSEQNC (residues 187-193) of beta1 is replaced with the
corresponding CYDMKTTC sequence of beta3, the ligand specificity of alphavbeta1 is altered. The mutant
(alphavbeta1-3-1), like alphavbeta3, recognizes fibrinogen, von Willebrand factor, and vitronectin (a gain-of-function effect).
The alphavbeta1-3-1 mutant is recruited to focal contacts on fibrinogen and vitronectin, suggesting that the mutant transduces
intracellular signals on adhesion. The reciprocal beta3-1-3 mutation blocks binding of alphavbeta3 to these multiple ligands and
to LM609, a function-blocking anti-alphavbeta3 antibody. These results suggest that the highly divergent sequence is a key
determinant of integrin ligand specificity. The data also support a recent hypothetical model of the I domain of beta, in which
the sequence is located in the ligand binding site (Takagi, 1997).
Integrins as fibronectin receptors The high affinity interaction of integrin alpha5beta1 with the central cell binding domain (CCBD) of
fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III repeat) and a second
site (in the adjacent 9th type III repeat) that synergizes with RGD. The
fibronectin binding interface on alpha5beta1 was mapped using monoclonal antibodies (mAbs) that inhibit ligand
recognition. The binding of two anti-alpha5 mAbs (P1D6 and JBS5) to alpha5beta1 is strongly
inhibited by a tryptic CCBD fragment of fibronectin (containing both synergy sequence and RGD) but
not by GRGDS peptide. The synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5
to alpha5beta1. In contrast, binding of the anti-beta1 mAb P4C10 to alpha5beta1 is inhibited to a
similar extent by either the GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the
synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding. P1D6 inhibits
the interaction of a wild type CCBD fragment with alpha5beta1 but has no effect on the binding of a
mutant fragment that lacks the synergy region. The epitopes of P1D6 and JBS5 map to the
NH2-terminal repeats of the alpha5 subunit. These results indicate that the synergy region is recognized
primarily by the alpha5 subunit (in particular by its NH2-terminal repeats) but that the beta1 subunit
plays the major role in binding of the RGD sequence (Mould, 1997).
HT-29 colon carcinoma cells form liver metastases upon intrasplenic injection;
adhesion to fibronectin under the liver microvascular liver endothelium is likely to be
important for metastasis formation. Fibronectin adhesion is not affected by blocking antibodies against the beta1,
alpha3, and alpha5 integrin subunits, but it is blocked by an RGD-containing peptide,
indicating involvement of RGD-dependent non-beta1 alphaV integrins. Both
alphaVbeta5 and alphaVbeta6 are detected on HT-29 cells. Blocking mAb against
alphaV, but not against alphaVbeta5, abolishes adhesion. From a HT-29 cell lysate,
only alphaVbeta6 binds to a fibronectin-Sepharose column. Thus, alphaVbeta6 is the
main fibronectin receptor on HT-29 cells, despite the very low levels of alphaVbeta6
and the much higher levels of alphaVbeta5. The HT29 cells do not spread on
fibronectin in the absence of serum, not even after a three- to fourfold increase in
alphaVbeta6 levels, induced by interleukin 4. The cells do spread on vitronectin. Using
immunofluorescence, it has been observed ( on both vitronectin and fibronectin) that alphaVbeta5 is arranged in a striped pattern aligned with actin fibers, and not in
focal adhesions.
On fibronectin, but not on vitronectin, alphaVbeta6 is concentrated
in a punctate pattern at the periphery of cell islands (Kemperman, 1997).
The region of fibronectin encompassing type III repeats 4-6 contains a low affinity heparin binding
domain, but its physiological significance is not clear. This domain is able to
interact with cells as already shown for other heparin binding domains of fibronectin. A computer
search based on homologies with known active sites in fibronectin revealed the sequence KLDAPT
located in FN-III5. A synthetic peptide containing this sequence induces lymphoid cell adhesion upon
treatment with the activating anti-beta1 monoclonal antibody (mAb) TS2/16 or with Mn2+, indicating
that KLDAPT is binding to an integrin. A recombinant fragment containing repeat III5 (FN-III5)
also mediates adhesion of TS2/16/Mn2+-treated cells, while the FN-III6 fragment does not. Soluble
KLDAPT peptide inhibits cell adhesion to FN-III5 as well as to a 38-kDa fibronectin fragment and
VCAM-1, two previously known ligands for alpha4beta1 integrin. KLDAPT also competes with the
binding of soluble alkaline phosphatase-coupled VCAM-Ig to Mn2+-treated alpha4beta1.
mAbs anti-alpha4 and anti-alpha4beta7, but not mAbs to other integrins, inhibit cell adhesion to
FN-III5 and KLDAPT. These results therefore establish a cell adhesive function for the FN-III5
repeat and show that KLDAPT is a novel fibronectin ligand for activated alpha4 integrins (Moyano, 1997).
The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the alpha5beta1 integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed "matrix assembly sites," which mediate the assembly of soluble fibronectin into insoluble fibrils. The amino-terminal type I repeats of fibronectin, which are distinct from RGD sequences, bind to the alpha5beta1 integrin and support cell adhesion. The amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides are able to cross-compete for binding to the alpha5beta1 integrin, suggesting that these two separate domains of fibronectin cannot bind to the alpha5beta1 integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin is enhanced by cytochalasin D, suggesting that the ligand specificity of the alpha5beta1 integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin (Hocking, 1998).
Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, this study shows that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5β1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. It is proposed that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template (Stenzel, 2011).
Integrins as vitronectin receptors Integrin-mediated cell attachment modulates growth responses; in a complementary manner, growth factors regulate cell
attachment. Moreover, both cell attachment to extracellular matrix and mitogenic signaling by growth
factors are necessary for the proliferation of most types of normal cells, suggesting that integrin and
growth factor receptor signaling pathways meet at some downstream point. A
small, highly tyrosine-phosphorylated fraction of PDGFbeta and insulin receptors
co-immunoprecipitate with the alphavbeta3 integrin from cells. The integrin association requires
growth factor stimulation of the receptors. Several signaling molecules that are known to be associated
with activated growth factor receptors were present in the alphavbeta3 integrin complexes.
Mitogenicity and chemotaxis induced by PDGF-BB are enhanced in cells plated on the alphavbeta3
ligand vitronectin, as compared with cells plated on the beta1 integrin ligand collagen. Thus, the
engagement of the alphavbeta3 integrin in cell-matrix interactions appears to coordinate an intense
response to growth factors, helping to explain the importance of this integrin for tissue regeneration,
angiogenesis and tumor metastasis (Schneller. 1997).
Expression of full-length p16(INK4a) blocks alphavbeta3 integrin-dependent cell spreading on vitronectin but not collagen IV.
G1-associated cell cycle kinases (CDK) inhibitory (CKI) synthetic peptides derived from p16(INK4a), p18(INK4c) and p21(Cip1/Waf1)
can be delivered directly into cells from the tissue culture medium. These inhibitory peptides do not affect non-alphavbeta3-dependent spreading on collagen IV, laminin and
fibronectin at concentrations that inhibit cell cycle progression in late G1. The alphavbeta3 heterodimer remains intact after CKI peptide treatment but
is immediately dissociated from the focal adhesion contacts. Treatment with phorbol 12-myristate 13-acetate (PMA) allows alphavbeta3 to locate to
the focal adhesion contacts and the cells to spread on vitronectin in the presence of CKI peptides. The cdk6 protein is found to suppress
p16(INK4a)-mediated inhibition of spreading and is also shown to localize to the ruffling edge of spreading cells, indicating a function for cdk6 in
controlling matrix-dependent cell spreading. These results demonstrate a novel G1 CDK-associated integrin regulatory pathway that acts upstream
of alphavbeta3-dependent activation of PKC as well as a novel function for the p16(INK4a) tumour suppressor protein in regulating
matrix-dependent cell migration (Fahraeus, 1999).
Integrin binding to cell adhesion molecules (CAMs) Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell ligand for two leukocyte integrins
(alpha4beta1 and alpha4beta7). A related CAM, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is recognized by
alpha4beta7 but is a poor ligand for alpha4beta1. Previous studies have revealed that all alpha4 integrin-ligand interactions are
dependent on a key acidic ligand motif centered on the CAM domain 1 C-D loop region. By generating
VCAM-1/MAdCAM-1 chimeras and testing recombinant proteins in cell adhesion assays it has been found that alpha4beta1
binds to the MAdCAM-1 adhesion motif when present in VCAM-1, but not when the VCAM-1 motif is present in
MAdCAM-1, suggesting that this region does not contain all of the information necessary to determine integrin binding
specificity. To characterize integrin-CAM specificity further, alpha4beta1 and alpha4beta7 binding was measured to a
comprehensive set of mutant VCAM-1 constructs containing amino acid substitutions within the predicted integrin adhesion
face. These data reveal the presence of key "regulatory residues" adjacent to integrin contact sites and an important
difference in the "footprint" of alpha4beta1 and alpha4beta7 that is associated with an accessory binding site located in
VCAM-1 Ig domain 2. The analogous region in MAdCAM-1 is markedly different in size and sequence; when this region is mutated, integrin binding activity is
abolished (Newham, 1997).
Intercellular adhesion molecule-3 (ICAM-3), a ligand for beta2 integrins, elicits a variety of activation responses in
lymphocytes. A functional mapping study was performed that focused on the 37-residue cytoplasmic region of ICAM-3.
Carboxyl-terminal truncations delineate portions involved in T cell antigen receptor costimulation, homotypic aggregation, and
cellular spreading. Truncation of the membrane distal 25 residues results in loss of T cell antigen receptor costimulation as
determined by interleukin 2 secretion. Aggregation and cell spreading are sensitive to truncation of the membrane distal and
proximal thirds of the cytoplasmic portion. Ser489 is a site of phosphorylation in vivo. Mutation of Ser489 or
Ser515 to alanine blocks interleukin 2 secretion, aggregation and cell spreading, while mutation of other serine residues
affects only a subset of functions. Ser489 is a phosphorylation site in vitro for recombinant protein kinase Ctheta. Treatment of Jurkat cells with a protein kinase C inhibitor prevents ICAM-3-triggered spreading (Hayflick, 1997).
A single immunoglobulin-like domain of the human neural cell
adhesion molecule L1 (Drosophila homolog: Fasciclin II) supports adhesion by multiple vascular and
platelet integrins. The L1 has been shown to function as a homophilic ligand in a variety of
dynamic neurological processes. The sixth immunoglobulin-like domain of
human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin
superfamily, including alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. The interaction
between L1-Ig6 and alphaIIbbeta3 is found to support the rapid attachment of activated human
platelets, whereas a corresponding interaction with alphavbeta3 and alphavbeta1 supports the
adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human
L1-Ig6 effectively abrogates binding by the aforementioned integrins. An L1 peptide containing this
RGD motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocks L1
integrin interactions and, as an immobilized ligand, supports adhesion via alphavbeta3, alphavbeta1,
alpha5beta1, and alphaIIbbeta3. Whereas beta3 integrin binding to L1-Ig6 is evident in the presence
of either Ca2+, Mg2+, or Mn2+, a corresponding interaction with the beta1 integrins is only observed
in the presence of Mn2+. Furthermore, such Mn2+-dependent binding by alpha5beta1 and alphavbeta1
is significantly inhibited by exogenous Ca2+. These findings suggest that physiological levels of calcium
will impose a hierarchy of integrin binding to L1 such that alphavbeta3 or active alphaIIbbeta3>alphavbeta1>alpha5beta1. Given that L1 can interact with multiple vascular or platelet integrins it is
significant that de novo L1 expression on blood vessels is associated with
certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel
role for L1 in vascular and thrombogenic processes (Felding-Habermann, 1997).
The structure of the I domain of integrin αLβ2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the αL I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal α7 helix with introduced disulfide bonds ratchets the β6-α7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity (Sjimaoka, 2003).
Integrins are a family of noncovalently associated, αβ heterodimeric transmembrane molecules that mediate cell-cell and cell-extracellular matrix adhesion. Lymphocyte function-associated antigen-1 (LFA-1, αLβ2) is an integrin that is critically important in antigen-specific responses and homing by lymphocytes and together with other β2 integrins in diapedesis by monocytes and neutrophils at inflammatory sites. αLβ2 recognizes intercellular adhesion molecules (ICAMs), members of the Ig superfamily (IgSF) of which ICAM-1 is the most biologically important. ICAM-1 is highly inducible on antigen-presenting cells and endothelium by cytokines in inflammation (Sjimaoka, 2003).
Although the extracellular domains of αL and β2 are each large and structurally complex, the ligand binding site is contained solely within the 180 residue inserted (I) domain of αL. The I domain is important in ligand binding in the 9 of 18 integrin α subunits in which it is present. Crystal structures of integrin I domains reveal a dinucleotide binding or Rossmann fold, with a central hydrophobic β sheet surrounded by seven amphipathic α helices. A Mg2+ ion is coordinated at the 'top' of the domain in a metal ion-dependent adhesion site (MIDAS) (Sjimaoka, 2003).
Integrins trigger 'outside-in' signals in response to ligand binding. In B and T cell responses, αLβ2 augments proliferation and protects against apoptosis. Ligand binding induces significant structural rearrangements in the I domain of the integrin α2 subunit, as seen in a complex with a collagen-like peptide. Compared to the unliganded 'closed' conformation of the α2 I domain, the liganded 'open' conformation exhibits a large 10 Å movement of the C-terminal α helix down the side of the domain, and a rearrangement in metal coordination at the MIDAS. The metal ion is central in the binding site and directly coordinates a Glu residue in the ligand. A similar conformational change has been observed in the αM I domain; however, in this case it is induced by a ligand-like contact of the metal in the MIDAS with a Glu residue of a neighboring I domain in the crystal lattice. By contrast, multiple αL I domain structures have consistently revealed the unliganded, closed conformation (Sjimaoka, 2003).
In the absence of activation, αLβ2 has low affinity for ligand. In inside-out signaling by integrins, signals received by other receptors activate intracellular signaling pathways that impinge on integrin cytoplasmic domains and make the extracellular domain competent for ligand binding on a timescale of less than 1 s. This unique property enables leukocytes to rapidly respond to signals in the environment, such as foreign antigen or chemoattractants, to activate adhesion and direct cell migration. A disulfide bond has been introduced into the αL I domain to stabilize the predicted open conformation and was found to increase the affinity for ICAM-1 by 10,000-fold. This strongly supports the idea that conformation regulates affinity; however, whether the open conformation would be stable in the absence of bound ligand has not been established because all open I domain structures determined to date are ligand bound. Two activation states have been defined functionally for αLβ2 on cells activated by differing stimuli. Both states are competent for cell adhesion, but high-affinity soluble ligand binding is only detectable for one of these states. However, the concept that I domains might exist in intermediate- as well as high-affinity states has not been tested by attempting to mutationally stabilize such states or define their structure (Sjimaoka, 2003).
Because of the key role of the interaction between LFA-1 and ICAM-1 in immune responses, defining the structural basis for this interaction is of great interest. Furthermore, development of pharmaceutical antagonists of this interaction is of great importance for treatment of autoimmune disease. Crystal structures have been determined for IgSF molecules recognized by integrins, i.e., ICAM-1, ICAM-2, VCAM-1, and MAdCAM-1, but not for IgSF complexes with integrins. In this study, the crystal structure of the I domain:ICAM-1 complex reveals an atomic view of an integrin-IgSF ligand interface for the first time. Furthermore, multiple I domain structures reveal an intermediate state in the shape-shifting pathway and show that the I domain can be stabilized in the open conformation in the absence of bound ligand (Sjimaoka, 2003).
Intergrins, agrin and the acetylcholine receptor Agrin, a basal lamina-associated proteoglycan, is a crucial nerve-derived organizer of
postsynaptic differentiation at the skeletal neuromuscular junction. Because integrins
serve as cellular receptors for many basal lamina components, agrin
interaction with integrins was examined. Agrin-induced aggregation of acetylcholine receptors on
cultured myotubes is completely blocked by antibodies to the beta1 integrin subunit
and partially blocked by antibodies to the alpha(v) integrin subunit. Agrin-induced
clustering is also inhibited by antisense oligonucleotides to alpha(v) and a peptide
that blocks the alpha(v) binding site. Non-muscle cells that express alpha(v) and
beta1 integrin subunits adher to immobilized agrin; this adhesion is blocked by
anti-alpha(v) and anti-beta1 antibodies. Integrin alpha(v)-negative cells that do not
adhere to agrin are rendered adherent by introduction of alpha(v). Together, these
results implicate integrins, including alpha(v)beta1, as components or modulators of
agrin's signal transduction pathway. It is concluded that integrins are likely to be agrin receptors. One striking parallel between integrins and agrins is the presence of G repeats in both agrin and laminin alpha chains. Binding sites for heparin/heparin sulfate alpha-dystroglycan and several integrins have been mapped to distinct G repeats of laminins (proteins that signal through integrins). Three similar repeats, separated from each other by EGF-like repeats, form much of the C-terminal half of agrin. The integrin-dependent adhesion to agrin described here raises the possibliity that agrin's effects on neurons, like its effects on muscle, involves integrins (Martin, 1997).
The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies
show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, the participation of this integrin in laminin and/or agrin-induced AChR clustering was studied. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the
extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of
C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced
AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the
physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as
well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster
formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms
containing the alpha7X2 extracellular domain are active. These results demonstrate (1) that the alpha7beta1 integrin has a physiologic role in laminin-induced
AChR clustering; (2) that alternative splicing is integral to this function of the alpha7 chain, and (3) that laminin, agrin, and the alpha7beta1 integrin interact in a
common or convergent pathway in the formation of neuromuscular junctions (Burkin, 1998).
Reelin binds integrin and inhibits neuronal migration Mice that are mutant for Reelin or Dab1, or doubly mutant for the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), show disorders of cerebral cortical lamination. How Reelin and its receptors regulate laminar organization of cerebral cortex is unknown. Reelin is shown here to inhibit migration of cortical neurons and enables detachment of neurons from radial glia. Recombinant and native Reelin associate with alpha3beta1 integrin, which regulates neuron–glia interactions and is required to achieve proper laminar organization. The effect of Reelin on cortical neuronal migration in vitro and in vivo depends on interactions between Reelin and alpha3beta1 integrin. Absence of alpha3beta1 leads to a reduction of Dab1, a signaling protein acting downstream of Reelin. Thus, Reelin may arrest neuronal migration and promote normal cortical lamination by binding alpha3beta1 integrin and modulating integrin-mediated cellular adhesion (Dulabon, 2000).
Reelin's effect on cortical layering is hypothesized to result from three distinctly different cellular effects. (1) Reelin may regulate cortical plate organization by initiating the splitting of preplate into marginal zone and subplate. Failure of this process in reeler mutants leads to the accumulation of cortical neurons underneath the preplate neurons. (2) A Reelin gradient may act as an attractant of neurons to the top of the cortical plate, thus enabling newly generated neurons to migrate past earlier generated ones in the developing cortical plate. (3) Reelin may induce detachment of neurons from their radial glial guides and thus stop neuronal migration at the marginal zone-developing cortical plate interface and initiate the differentiation of neurons into distinct layers. A direct demonstration of the effect of Reelin protein upon migrating cerebral cortical neurons is provided in this study. Reelin retards neuronal migration and induces neuronal detachment from radial glial guides. This particular function of Reelin depends on Reelin-alpha3beta1 integrin interactions, since in the absence of alpha3beta1 integrin, Reelin does not measurably affect neuronal migration in vitro and in vivo. Neurons have to stop their migration, detach from their radial glial guides, and begin elaborating neurites in order to generate the synaptic connectivity characteristic of distinct layers. By triggering the appropriate termination of neuronal migration and initiation of postmigratory neuronal differentiation, Reelin controls cortical layer formation. The functional role of Reelin in modulating integrin-mediated adhesion is reflected in the overly adhesive phenotype of early born neurons in the reeler cortex and the persistent apposition of reeler mutant neurons with radial glial fibers (Dulabon, 2000).
These data do not exclude alternate or additional functions for Reelin during layer formation, since other cell surface molecules, such as VLDLR and ApoER2, or members of the cadherin-related neuronal receptor family (CNRs) are found to bind Reelin and mediate signaling to Dab1. In addition, APP and APLP1 bind to Dab1 and may be targets of Reelin as well. The endocytic compartment is increasingly recognized as a critical site at which adhesion molecules converge with LDL receptors, and Reelin may thus in principle regulate a number of adhesion receptors simultaneously if they are complexed in sufficiently tight proximity to the VLDL/ApoE receptor that contains the endocytic motif. Reelin concentration, like laminin concentration, may determine the surface levels of integrins by regulating the rate at which integrin receptor is removed from the cell surface. Ligands can also regulate polarized flow of integrins toward or away from growth cone membranes. Reelin, released from the layer 1 cortical neurons, is likely to be present in a top-down gradient in the developing cortical plate. Thus, Reelin gradient-mediated modulation or endocytosis of a large adhesion complex, including alpha3beta1 integrins, might be necessary to 'switch' migrating neurons from a gliophilic adhesive preference to a neurophilic adhesive preference (Dulabon, 2000).
While it is clear that other proteins in addition to alpha3beta1 integrin bind Reelin, interactions between Reelin and alpha3beta1 integrin appear to be crucial for the effects of Reelin upon migrating neurons. Reelin induces arrest of migrating cells and release from radial glia, and mutations in Reelin prevent normal dissociation of migratory neurons from radial glia, which has been described morphologically. Inhibition of beta1 integrins with blocking antibodies releases neurons from radial glia, and inhibition of beta1 integrins with antisense retroviruses also blocks radial migration. Similarly, targeted mutation of the alpha3 integrin gene causes inhibition of radial migration, possibly via premature release from radial glia. These data suggest that alpha3beta1 integrin, either alone or in association with other partners, is necessary for radial glial guided migration and that inhibition of the alpha3beta1 integrin complex is sufficient to induce release of neurons from radial glia (Dulabon, 2000 and references therein).
The elevated levels of cleaved Reelin (180 kDa) observed in the absence of alpha3beta1 integrin suggest that a normal function of alpha3beta1 integrin is to inhibit the production or enhance the clearance of the Reelin fragment. alpha3beta1 integrin could inhibit production of 180 kDa Reelin by modulating the activity of the zinc-dependent metalloproteinase that normally cleaves Reelin. Attractive candidates for the metalloproteinase would include brain-expressed members of the metalloproteinase-disintegrin family (ADAMs), which are zinc dependent and have integrin binding sites. In addition, alpha3beta1 integrins may also bind to the 180 kDa fragment and promote its clearance. Although it is unclear at this juncture whether the 180 kDa Reelin fragment is functionally active, upregulation of this Reelin fragment may contribute to the aberrant cortical lamination observed in alpha3beta1 integrin mutant cerebral cortex (Dulabon, 2000 and references therein).
The analysis of alpha3beta1 integrin deficiency is complicated by the fact that alpha3beta1 can function as a trans-dominant inhibitor of other integrin receptor functions. In keratinocytes, alpha3beta1 deficiency leads to increased activity of fibronectin and collagen type IV receptors, possibly including alpha5beta1 integrins, which can bind Reelin. Similar upregulation of other integrin receptor activity may also occur in alpha3beta1 mutant cerebral cortex and contribute partially to the aberrant phenotype. Increased levels of cleaved Reelin, release of trans-dominant inhibition of other integrins capable of interacting with Reelin, and the availability of multiple other cell surface receptors for Reelin may account for the downregulation of Dab1 protein levels in the absence of alpha3beta1 integrins (Dulabon, 2000 and references therein).
These data allow a tentative model of Reelin function. Migration along radial glial cells is mediated by alpha3beta1 integrin in addition to other adhesive or signaling molecules. Neuronal alpha3beta1 integrin may interact with ligands such as laminin, distributed along the radial glial strands, during neuronal translocation from the ventricular zone to the cortical plate. In the cortical plate, when migrating neurons encounter Reelin, the ligand preference of alpha3beta1 integrins in neurons changes from a radial glial cell surface molecule such as laminin to Reelin. Reelin binds to alpha3beta1 integrin in a complex that includes the VLDLR, the ApoER2, and alpha3beta1 integrin, and the formation of this complex leads to altered integrin-mediated adhesion, perhaps due to internalization of the integrin receptor along with the ApoER2 and VLDLR. The altered ligand preference of alpha3beta1 integrins, coupled with the endocytosis-driven removal of alpha3beta1 integrins from the cell surface, may then cause rapid dissociation of migrating neurons from radial glia. Reelin undoubtedly also induces other downstream effects, exemplified by its effects on the phosphorylation of Tau, that may also be crucial to its inhibitory effects on migration; however, regulation of availability, function, and ligand preference of integrins appears to be critical for the dissociation of neurons from radial glia. Cultured cells rapidly regulate the surface expression of integrins in response to ligand, and neurons regulate integrin surface expression to alter adhesion and neurite extension. Regulation of ligand preference and endocytosis may be a general means for rapid, regionally specific removal of adhesive or signaling molecules from the leading edge of migrating neurons as they terminate their migration and begin their aggregation into distinct layers in cerebral cortex (Dulabon, 2000).
Animals capable of adult regeneration require specific signaling to control injury-induced cell proliferation, specification and patterning, but comparatively little is known about how the regeneration blastema assembles differentiating cells into well-structured functional tissues. Using the planarian Schmidtea mediterranea as a model, β1-integrin (see Drosophila Myospheroid) as a critical regulator of blastema architecture. beta1-integrin(RNAi) animals formed small head blastemas with severe tissue disorganization, including ectopic neural spheroids containing differentiated neurons normally found in distinct organs. By mimicking aspects of normal brain architecture but lacking normal cell-type regionalization, these spheroids bore a resemblance to mammalian tissue organoids synthesized in vitro. One of four planarian integrin-α subunits was identified, whose inhibition phenocopied these effects, suggesting a specific receptor controls brain organization through regeneration. Neoblast stem cells and progenitor cells were mislocalized in β1-integrin(RNAi) animals without significantly altered body-wide patterning. Furthermore, tissue disorganization phenotypes were most pronounced in animals undergoing brain regeneration and not homeostatic maintenance or regeneration-induced remodeling of the brain. These results suggest that integrin signaling ensures proper progenitor recruitment after injury, enabling the generation of large-scale tissue organization within the regeneration blastema (Bonar, 2017).
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