SLLISWD Sequence in the 10FNIII Domain Initiates Fibronectin Fibrillogenesis

Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N terminus spanning the A and B β-strands. Here, we experimentally confirm that the predicted 23-residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid binding. Localization of multimerization activity to the C terminus led to the discovery of a minimal 7-amino acid “multimerization sequence” (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp-6 that reduces exposure of hydrophobic sites for 8-anilino-1-napthalenesulfonic acid binding and β-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated β-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.     

Fibronectin peptides derived from two distinct regions stimulate adipocyte differentiation by preventing fibronectin matrix assembly

Here, we show that fibronectin (FN) peptides derived from two distinct regions promote the insulin-induced adipocyte differentiation of ST-13 cells by preventing FN fibrillogenesis. ST-13 cells formed numerous FN fibrils under nonadipogenic conditions, whereas this FN fibrillogenesis was suppressed by adipose induction with insulin. The insulin-induced adipocyte differentiation was promoted by an amino-terminal 24-kDa fragment of FN, accompanied by further suppression of FN fibrillogenesis. The 24 K fragment prevented FN matrix assembly by direct incorporation into the FN matrix. Like the 24 K fragment, a peptide from the 14th type III repeat, termed FNIII14, which suppressed the integrin alpha 5 beta 1-mediated adhesion of ST-13 cells to FN, accelerated the adipocyte differentiation by preventing FN fibrillogenesis without direct incorporation into the FN matrix. FNIII14 induced the conformation change of beta1 integrins of K562 cells from active to resting, as judged by FACS analysis using a monoclonal antibody AG89 directed to an active beta1 integrin-dependent epitope. Binding of a (125)I-labeled FN fragment containing the RGD cell adhesive site to ST-13 cell surface was dissociated by FNIII14, with a concomitant binding of FNIII14 itself to the cell surface. The affinity labeling of ST-13 cells using biotinylated FNIII14 showed that FNIII14 specifically bound to a nonintegrin membrane protein with M(r) of around 50 kDa. Thus, the results indicated that prevention of FN fibrillogenesis by the 24 K Fib 1 fragment and FNIII14 caused the promotion of adipocyte differentiation of ST-13 cells and that the former was due to the direct incorporation into the FN matrix and that the latter might be interpreted by negative regulation of FN receptor alpha 5 beta 1 activity.     

Ligation of the fibrin-binding domain by β-strand addition is sufficient for expansion of soluble fibronectin

How fibronectin (FN) converts from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. "Functional upstream domain" (FUD), a polypeptide based on F1 adhesin of Streptococcus pyogenes, binds by anti-parallel β-strand addition to discontinuous sets of N-terminal FN type I modules, (2-5)FNI of the fibrin-binding domain and (8-9)FNI of the gelatin-binding domain. Such binding blocks assembly of FN. To learn whether ligation of (2-5)FNI, (8-9)FNI, or the two sets in combination is important for inhibition, we tested "high affinity downstream domain" (HADD), which binds by β-strand addition to the continuous set of FNI modules, (1-5)FNI, comprising the fibrin-binding domain. HADD and FUD were similarly active in blocking fibronectin assembly. Binding of HADD or FUD to soluble plasma FN exposed the epitope to monoclonal antibody mAbIII-10 in the tenth FN type III module ((10)FNIII) and caused expansion of FN as assessed by dynamic light scattering. Soluble N-terminal constructs truncated after (9)FNI or (3)FNIII competed better than soluble FN for binding of FUD or HADD to adsorbed FN, indicating that interactions involving type III modules more C-terminal than (3)FNIII limit β-strand addition to (1-5)FNI within intact soluble FN. Preincubation of FN with mAbIII-10 or heparin modestly increased binding to HADD or FUD. Thus, ligation of FNIII modules involved in binding of integrins and glycosaminoglycans, (10)FNIII and (12-14)FNIII, increases accessibility of (1-5)FNI. Allosteric loss of constraining interactions among (1-5)FNI, (10)FNIII, and (12-14)FNIII likely enables assembly of FN into extracellular fibrils.     

Crystal structure of a hemojuvelin-binding fragment of neogenin at 1.8Å

Neogenin is a type I transmembrane glycoprotein with a large ectodomain containing tandem immunoglobulin-like and fibronectin type III (FNIII) domains. Closely related to the tumor suppressor gene DCC, neogenin functions in critical biological processes through binding to various ligands, including netrin, repulsive guidance molecules, and the iron regulatory protein hemojuvelin. We previously reported that neogenin binds to hemojuvelin through its membrane-proximal fifth and sixth FNIII domains (FN5-6), with domain 6 (FN6) contributing the majority of critical binding interactions. Here we present the crystal structure of FN5-6, the hemojuvelin-binding fragment of human neogenin, at 1.8?. The two FNIII domains are orientated nearly linearly, a domain arrangement most similar to that of a tandem FNIII-containing fragment within the cytoplasmic tail of the β4 integrin. By mapping surface-exposed residues that differ between neogenin FN5-6 and the comparable domains from DCC, which does not bind hemojuvelin, we identified a potential hemojuvelin-binding site on neogenin FN6. Neogenin FN5, which does not bind hemojuvelin in isolation, exhibits a highly electropositive surface, which may be involved in interactions with negatively-charged polysaccharides or phospholipids in the membrane bilayer. The neogenin FN5-6 structure can be used to facilitate a molecular understanding of neogenin's interaction with hemojuvelin to regulate iron homeostasis and with hemojuvelin-related repulsive guidance molecules to mediate axon guidance.     

The mechanical hierarchies of fibronectin observed with single-molecule AFM

Mechanically induced conformational changes in proteins such as fibronectin are thought to regulate the assembly of the extracellular matrix and underlie its elasticity and extensibility. Fibronectin contains a region of tandem repeats of up to 15 type III domains that play critical roles in cell binding and self-assembly. Here, we use single-molecule force spectroscopy to examine the mechanical properties of fibronectin (FN) and its individual FNIII domains. We found that fibronectin is highly extensible due to the unfolding of its FNIII domains. We found that the native FNIII region displays strong mechanical unfolding hierarchies requiring 80 pN of force to unfold the weakest domain and 200 pN for the most stable domain. In an effort to determine the identity of the weakest/strongest domain, we engineered polyproteins composed of an individual domain and measured their mechanical stability by single-protein atomic force microscopy (AFM) techniques. In contrast to chemical and thermal measurements of stability, we found that the tenth FNIII domain is mechanically the weakest and that the first and second FNIII domains are the strongest. Moreover, we found that the first FNIII domain can acquire multiple, partially folded conformations, and that their incidence is modulated strongly by its neighbor FNIII domain. The mechanical hierarchies of fibronectin demonstrated here may be important for the activation of fibrillogenesis and matrix assembly.     

The heparin III-binding domain of fibronectin (III4–5 repeats) binds to fibronectin and inhibits fibronectin matrix assembly

Fibronectin matrix assembly involves interactions among various regions of the molecule, which contribute to elongation and stabilization of the fibrils. In this study, we examined the possible role of the heparin III domain of fibronectin (repeats III4-5) in fibronectin fibrillogenesis. We show that a recombinant fragment comprising these repeats (FNIII4-5 fragment) blocked fibronectin fibril formation and the incorporation of 125I-fibronectin into cell layers. Binding assays using a biosensor revealed that FNIII4-5 bound fibronectin and the amino-terminal 70 kDa and 29 kDa fragments. It also bound to itself, indicating a previously unidentified self-association site in repeats III4-5. These interactions were specific since FNIII4-5 did not bind to the FNIII7-10 fragment, representing a central region in fibronectin. The fibronectin-binding property of the III4-5 domain, but not its matrix assembly inhibitory function, was apparently cryptic in larger fragments. By mutating the arginine residues in the WTPPRAQITGYRLTVGLTRR proteoglycan-binding sequence (HBP/III5 site) of FNIII4-5 [Moyano, J.V., Carnemolla, B., Albar, J.P., Leprini, A., Gaggero, B., Zardi, L., Garcia-Pardo, A., 1999. Cooperative role for activated alpha4beta1 integrin and chondroitin sulfate proteoglycans in cell adhesion to the heparin III domain of fibronectin. Identification of a novel heparin and cell binding sequence in repeat III5. J. Biol. Chem. 274, 135-142.], we found that the first two arginine residues in HBP/III5 were involved in the fibronectin-binding property of FNIII4-5, while the last two arginine residues in HBP/III5 were required for inhibition of matrix assembly and the binding of 125I-fibronectin to cell layers. Both properties appear to function independently from each other, depending on the conformation of the fibronectin dimer.     

IGFBP-3 and IGFBP-5 associate with the cell binding domain (CBD) of fibronectin

We have used Surface Plasmon Resonance (SPR) - based biosensor technology to investigate the interaction of the six high affinity insulin-like growth factor binding proteins (IGFBP 1-6) with the cell binding domain (CBD) of fibronectin. Using a biotinylated derivative of the ninth and tenth TypeIII domains of FN (^9-10FNIII), we show that IGFBP-3 and -5 bind to FN-CBD. We show that this binding is inhibited by IGF-I and that, for IGFBP-5, binding occurs through the C-terminal heparin binding domain of the protein. Using site-directed mutagenesis of ^9-10FNIII, we show both the “synergy” and RGD sites within these FN domains are required for maximum binding of both IGFBPs. We discuss the possible biological consequences of our results.     

A novel consensus motif in fibronectin mediates dipeptidyl peptidase IV adhesion and metastasis

Lung endothelial dipeptidyl peptidase IV (DPPIV/CD26) is a vascular address for cancer cells decorated with cell-surface polymeric fibronectin (poly-FN). Here, we identified the DPPIV-binding sites in FN and examined the effect of binding site peptides on DPPIV/poly-FN adhesion and metastasis. Using proteolytic fragments and maltose-binding protein fusion proteins that together span full-length FN, we found DPPIV-binding sites in type III repeats 13, 14, and 15 (FNIII13, -14, and -15, respectively). DPPIV binding was mediated by the consensus motif T(I/L)TGLX(P/R)G(T/V)X and was confirmed by swapping this motif in FNIII13, -14, and -15 with the corresponding region in FNIII12, which did not bind DPPIV. DPPIV binding was lost in swapped FNIII13, -14, and -15 and gained in swapped FNIII12 (FNIII12(14)). Peptides containing the DPPIV-binding domain of FNIII14 blocked DPPIV/poly-FN adhesion and impeded pulmonary metastasis. This study adds to the classes of cell-surface adhesion receptors for FN and will help in the further characterization of the functional implications of the DPPIV/poly-FN adhesion in metastasis and possibly in cell-mediated immunity involving DPPIV-expressing lymphocytes.     

Contribution of leptin receptor N-linked glycans to leptin binding

The extracellular domain of the human leptin receptor (Ob-R) contains 20 potential N-glycosylation sites whose role in leptin binding remains to be elucidated. We found that a mammalian cell-expressed sOb-R (soluble Ob-R) fragment (residues 22-839 of the extracellular domain) bound leptin with a dissociation constant of 1.8 nM. This binding was inhibited by Con A (concanavalin A) or wheatgerm agglutinin. Treatment of sOb-R with peptide N-glycosidase F reduced leptin binding by approximately 80% concurrently with N-linked glycan removal. The human megakaryoblastic cell line, MEG-01, expresses two forms of the Ob-R, of approx. 170 and 130 kDa molecular mass. Endo H (endoglycosidase H) treatment and cell culture with alpha-glucosidase inhibitors demonstrated that N-linked glycans are of the complex mature type in the 170 kDa form and of the high-mannose type in the 130 kDa form. Both isoforms bound leptin, but not after peptide N-glycosidase F treatment. An insect-cell-expressed sOb-R fragment, consisting of the Ig (immunoglobulin), CRH2 (second cytokine receptor homology) and FNIII (fibronectin type III) domains, bound leptin with affinity similar to that of the entire extracellular domain, but this function was abolished after N-linked glycan removal. The same treatment had no effect on the leptin-binding activity of the isolated CRH2 domain. Our findings show that N-linked glycans within Ig and/or FNIII domains regulate Ob-R function, but are not involved in essential interactions with the ligand.     

Modeling and experimental validation of the binary complex of the plectin actin-binding domain and the first pair of fibronectin type III (FNIII) domains of the beta4 integrin

The binding of plectin to the beta4 subunit of the alpha6beta4 integrin is a critical step in the formation of hemidesmosomes. An important interaction between these two proteins occurs between the actin-binding domain (ABD) of plectin and the first pair of fibronectin type III (FNIII) domains and a small part of the connecting segment of beta4. Previously, a few amino acids, critical for this interaction, were identified in both plectin and beta4 and mapped on the crystal structures of the ABD of plectin and the first pair of FNIII domains of beta4. In the present study, we used this biochemical information and protein-protein docking calculations to construct a model of the binary complex between these two protein domains. The top scoring computational model predicts that the calponin-homology 1 (CH1) domain of the ABD associates with the first and the second FNIII domains of beta4. Our mutational analysis of the residues at the proposed interface of both the FNIII and the CH1 domains is in agreement with the suggested interaction model. Computational simulations to predict protein motions suggest that the exact model of FNIII and plectin CH1 interaction might well differ in detail from the suggested model due to the conformational plasticity of the FNIII domains, which might lead to a closely related but different mode of interaction with the plectin-ABD. Furthermore, we show that Ser-1325 in the connecting segment of beta4 appears to be essential for the recruitment of plectin into hemidesmosomes in vivo. This is consistent with the proposed model and previously published mutational data. In conclusion, our data support a model in which the CH1 domain of the plectin-ABD associates with the groove between the two FNIII domains of beta4.     

Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin.

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.     

Alternative Splicing of the Angiogenesis Associated Extra-Domain B of Fibronectin Regulates the Accessibility of the B-C Loop of the Type III Repeat 8

Background

Fibronectin (FN) is a multi-domain molecule involved in many cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. The biological activities of FN are mediated by exposed loops located mainly at the interdomain interfaces that interact with various molecules such as, but not only, integrins. Different FN isoforms arise from the alternative splicing of the pre-mRNA. In malignancies, the splicing pattern of FN pre-mRNA is altered; in particular, the FN isoform containing the extra-domain B (ED-B), a complete FN type III repeat constituted by 91 residues, is undetectable in normal adult tissues, but exhibits a much greater expression in fetal and tumor tissues, and is accumulated around neovasculature during angiogenic processes, thus making ED-B one of the best markers and targets of angiogenesis. The functions of ED-B are still unclear; however, it has been postulated that the insertion of an extra-domain such as ED-B modifies the domain-domain interface and may unmask loops that are otherwise cryptic, thus giving FN new potential activities.

Methodology

We used the mAb C6, which reacts with ED-B containing FN, but not with ED-B-free FN and various recombinant FN fragments containing mutations, to precisely localize the epitopes recognized by the mAb C6.

Conclusion

We formally demonstrated that the inclusion of the alternatively spliced angiogenesis-associated ED-B leads to the unmasking of the FNIII 8 B-C loop that is cryptic in FN molecules lacking ED-B. Thus, the mAb C6, in addition to providing a new reagent for angiogenesis targeting, represents a new tool for the study of the potential biological functions of the B-C loop of the repeat FNIII 8 that is unmasked during angiogenic processes.     

Interaction of heparin with fibronectin and isolated fibronectin domains.

Fluorescence polarization, gel exclusion chromatography and affinity chromatography were used to characterize the interaction of heparins of different size with human plasma fibronectin (Fn) and several of its isolated domains. The fluid-phase interaction of Fn with heparin was dominated by the 30 kDa and 40 kDa Hep-2 domains located near the C-terminal ends of the A and B chains respectively. The 30 kDa Hep-2A domain from the heavy chain was indistinguishable from the 40 kDa Hep-2B domain in this respect; the presence of an additional type III homology unit in the latter had no effect on the binding. Evidence was provided that each Hep-2 domain has two binding sites for heparin. The N-terminal Hep-1 domain reacted weakly in fluid phase even though it binds strongly to immobilized heparin. Fn and Hep-2 fragments were rather undiscriminating in their reaction with fluoresceinamine-labelled heparins of different sizes. However, oligosaccharides smaller than the tetradecasaccharide (14-mer) bound Fn with a 5-10-fold lower affinity. These results suggest that the Hep-2 domains of Fn are able to recognize a broad spectrum of oligosaccharides that presumably vary significantly with respect to the amount and spatial distribution of charge.     

Altered rate of fibronectin matrix assembly by deletion of the first type III repeats

The assembly of fibronectin (FN) into a fibrillar matrix is a complex stepwise process that involves binding to integrin receptors as well as interactions between FN molecules. To follow the progression of matrix formation and determine the stages during which specific domains function, we have developed cell lines that lack an endogenous FN matrix but will form fibrils when provided with exogenous FN. Recombinant FNs (recFN) containing deletions of either the RGD cell- binding sequence (RGD-) or the first type III repeats (FN delta III1-7) including the III1 FN binding site were generated with the baculovirus insect cell expression system. After addition to cells, recFN matrix assembly was monitored by indirect immunofluorescence and by insolubility in the detergent deoxycholate (DOC). In the absence of any native FN, FN delta III1-7 was assembled into fibrils and was converted into DOC-insoluble matrix. This process could be inhibited by the amino- terminal 70 kD fragment of FN, showing that FN delta III1-7 follows an assembly pathway similar to FN. The progression of FN delta III1-7 assembly differed from native FN in that the recFN became DOC-insoluble more quickly. In contrast, RGD- recFNs were not formed into fibrils except when added in combination with native FN. These results show that the RGD sequence is essential for the initiation step but fibrils can form independently of the III1-7 modules. The altered rate of FN delta III1-7 assembly suggests that one function of the missing repeats might be to modulate an early stage of matrix formation.     

The ihog cell-surface proteins bind Hedgehog and mediate pathway activation

The ihog gene (interference hedgehog), identified by RNA interference in Drosophila cultured cells, encodes a type 1 membrane protein shown here to bind and to mediate response to the active Hedgehog (Hh) protein signal. ihog mutations produce defects characteristic of Hh signaling loss in embryos and imaginal discs, and epistasis analysis places ihog action at or upstream of the negatively acting receptor component, Patched (Ptc). The first of two extracellular fibronectin type III (FNIII) domains of the Ihog protein mediates a specific interaction with Hh protein in vitro, but the second FNIII domain is additionally required for in vivo signaling activity and for Ihog-enhanced binding of Hh protein to cells coexpressing Ptc. Other members of the Ihog family, including Drosophila Boi and mammalian CDO and BOC, also interact with Hh ligands via a specific FNIII domain, thus identifying an evolutionarily conserved family of membrane proteins that function in Hh signal response.     

A fibronectin fragment induces tumor necrosis factor production of rat basophilic leukemia cells

Proteolytic digest of fibronectin (FN), but not intact FN, induced TNF-alpha secretion of rat basophilic leukemia (RBL-2H3) cells. As a result of the identification of FN fragment responsible for TNF-alpha secretion, a 30-kDa fragment derived from the carboxyl-terminal heparin-binding (Hep 2) domain of FN was isolated from the FN digest. The TNF-alpha secretion was abrogated by treatment of RBL-2H3 cells with cycloheximide, indicating the de novo synthesis of TNF-alpha, but not with polymyxin B, excluding the possible TNF-alpha induction by some contaminated lipopolysaccharides. A 22-mer synthetic peptide originated from the Hep 2 domain, termed FNIII14, which has been found to negatively modulate the beta1 integrin activation, had the ability to induce TNF-alpha production, whereas this activity of FNIII14 disappeared by shuffling a YTIYVIAL sequence essential for the integrin-inactivating activity. FNIII14 suppressed the spreading of RBL-2H3 cells on FN substrate, wherein RBL-2H3 cell proliferation was inhibited with FNIII14 in a dose-dependent manner. Thus, it appears that FN fragments containing the YTIYVIAL anti-adhesive site affect the activation status of RBL-2H3 mast cells, characterized by the stimulation of TNF-alpha production and growth suppression, probably due to negative regulation of beta1 integrin activity.     

Ubiquitous SPRY domains and their role in the skeletal type ryanodine receptor

We recently identified the second of three SPRY domains in the skeletal muscle ryanodine receptor type 1 (RyR1) as a potential binding partner in the RyR1 ion channel for the recombinant II–III loop of the skeletal muscle dihydropyridine receptor, for a scorpion toxin, Imperatoxin A and for an interdomain interaction within RyR1. SPRY domains are structural domains that were first described in the fungal Dictyostelium discoideum tyrosine kinase spore lysis A and all three isoforms of the mammalian ryanodine receptor (RyR). Our studies are the first to assign a function to any of the three SPRY domains in the RyR. However, in other systems SPRY domains provide binding sites for regulatory proteins or intramolecular binding sites that maintain the structural integrity of a protein. In this article, we review the general characteristics of a range of SPRY domains and discuss evidence that the SPRY2 domain in RyR1 supports interactions with binding partners that contain a structural surface of aligned basic residues.     

A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1-7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNDeltaIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9-10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4-7, had no effect on assembly. In contrast, two deletions that included repeat III2, DeltaIII1-2 and DeltaIII2-5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN-FN interactions during fibril growth.     

Molecular analysis of receptor protein tyrosine phosphatase mu-mediated cell adhesion

Type IIB receptor protein tyrosine phosphatases (RPTPs) are bi-functional cell surface molecules. Their ectodomains mediate stable, homophilic, cell-adhesive interactions, whereas the intracellular catalytic regions can modulate the phosphorylation state of cadherin/catenin complexes. We describe a systematic investigation of the cell-adhesive properties of the extracellular region of RPTPmu, a prototypical type IIB RPTP. The crystal structure of a construct comprising its N-terminal MAM (meprin/A5/mu) and Ig domains was determined at 2.7 A resolution; this assigns the MAM fold to the jelly-roll family and reveals extensive interactions between the two domains, which form a rigid structural unit. Structure-based site-directed mutagenesis, serial domain deletions and cell-adhesion assays allowed us to identify the four N-terminal domains (MAM, Ig, fibronectin type III (FNIII)-1 and FNIII-2) as a minimal functional unit. Biophysical characterization revealed at least two independent types of homophilic interaction which, taken together, suggest that there is the potential for formation of a complex and possibly ordered array of receptor molecules at cell contact sites.     

Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain

The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region ((I)1-9) is commonly accepted as one of the assembly sites. We previously found that (I)1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that (I)1-9 bound to the aggregate formed by anastellin and a small FN fragment, (III)1-2. An engineered disulfide bond in (III)2, which stabilizes folding, inhibited aggregation, but a disulfide bond in (III)1 did not. A gelatin precipitation assay showed that (I)1-9 did not interact with anastellin, (III)1, (III)2, (III)1-2, or several (III)1-2 mutants including (III)1-2KADA. (In contrast to previous studies, we found that the (III)1-2KADA mutant was identical in conformation to wild-type (III)1-2.) Because (I)1-9 only bound to the aggregate and the unfolding of (III)2 played a role in aggregation, we generated a (III)2 domain that was destabilized by deletion of the G strand. This mutant bound (I)1-9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in (III)2, (III)3, and (III)11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in (III)2 reduced the FN matrix. These results suggest that the unfolding of (III)2 is one of the key factors for FN aggregation and assembly.