FNIII Domain Research Articles

FLRT3, a cell surface molecule containing LRR repeats and a FNIII domain, promotes neurite outgrowth

The mature peripheral nervous system has the ability to survive and to regenerate its axons following axonal injury. After nerve injury, the distal axonal and myelin segment undergoes dissolution and absorption by the surrounding cellular environment, a process called Wallerian degeneration. Using cDNA microarrays, we isolated FLRT3 as one of the up-regulated genes expressed in the distal segment of the sciatic nerve 7 days after transection relative to those of the intact sciatic nerve. FLRT3 is a putative type I transmembrane protein containing 10 leucine-rich repeats, a fibronectin type III domain, and an intracellular tail. The neurons plated on CHO cells expressing FLRT3 extended significantly longer neurites than those plated on wild-type CHO cells, demonstrating that FLRT3 promotes neurite outgrowth. FLRT3 mRNA was especially abundant in the basal ganglia, the granular layer of cerebellum, and the hippocampus, except the CA1 region in the adult rat brain. Thus, FLRT3 may contribute to regeneration following axonal injury.

Folding–unfolding of FN-III domains in tenascin: an elastically coupled two-state system

In a single-molecule atomic force microscopy (AFM) experiment, the tenascin molecule is stretched by an external force causing an elongation which is due to the unfolding of the FN-III modules. The features of the force-extension curves depend on the pulling speed and show a saw-tooth pattern (lower speeds) or a smooth pattern (higher speeds). In any case, the unfolded domains are elastically coupled to the unfolded modules, acting as transmitters of the external force. In this communication, the folding–unfolding process of the FN-III domains in tenascin is studied using reaction rate theory and a simple two-state model. The main hypothesis of the study is that, at microscopic level, the force needed to unfold a domain and the unfolding rate (unfolding velocity) can mimic the macroscopic process of measurement by AFM. As the external force is applied, the probability of unfolding increases as dictated by the reaction rate theory. Within this context, a relationship between the unfolding force and the unfolding velocity is obtained. The latter relation will describe microscopically the process in a phenomenological fashion. Moreover, while relating the results of this study with other experimental (AFM measurements) and theoretical (Monte Carlo simulations) data, we found that the graph of unfolding force-unfolding velocity is similar to that of external force-pulling velocity. The refolding process can also be studied within this model and the results show similar trends. The latter suggests a generic and universal behavior of such kind of molecular domains at least in the light of the proposed model.

Titins in C.elegans with unusual features: coiled-coil domains, novel regulation of kinase activity and two new possible elastic regions.

We report that there are previously unrecognized proteins in Caenorhabditis elegans that are similar to the giant muscle proteins called titins, and these are encoded by a single approximately 90kb gene. The gene structure was predicted by GeneMark.hmm and then experimentally verified. The Ce titin gene encodes polypeptides of 2.2MDa, 1.2MDa and 301kDa. The 2.2MDa isoform resembles twitchin and UNC-89 in that it contains multiple Ig (56) and FnIII (11) domains, and a single protein kinase domain. In addition, however, the 2.2MDa isoform contains four classes of short, 14-51 residue, repeat motifs arranged mostly in many tandem copies. One of these tandem repeat regions is similar to the PEVK regions of vertebrate and fly titins. As the PEVK region is one of the main elastic elements of the titins and is also composed of short tandem repeats, this suggests that the repeat motifs in the Ce titins may have a similar elastic function. An interesting aspect of the two largest Ce titin isoforms, is that in contrast to other members of the twitchin/titin family, there are multiple regions which are likely to form coiled-coil structure. In transgenic animals, the first approximately 100 residues of the largest isoforms targets to dense bodies, the worm analogs of Z-discs. Anti-Ce titin antibodies show localization to muscle I-bands beginning at the L2-L3 larval stages and this pattern continues into adult muscle. Ce titins may not have a role in early myofibril assembly: (1) Ce titins are too short to span half a sarcomere, and the onset of their expression is well after the initial assembly of thick filaments. (2) Ce titins are not localized to I-bands in embryonic or L1 larval muscle. The Ce titin protein kinase domain is most similar to the kinase domains of the twitchins and projectin. The Ce titin kinase has protein kinase activity in vitro, and this activity is regulated by a novel mechanism.

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.

Sequence Conservation in Ig-like Domains: The Role of Highly Conserved Proline Residues in the Fibronectin Type III Superfamily

The role of conserved proline residues in fibronectin type III (fnIII) domains is investigated. Surprisingly, none of the standard set of explanations for residue conservation applies. The proline residues are not apparently conserved for function, or stability, or to nucleate folding, or to promote stabilising interactions across domain boundaries. However, when the most highly conserved proline residues are mutated to alanine there is an increase in the rate of aggregation of a fnIII double-module construct. The results suggest that proline residues may be conserved at domain–domain boundaries in fnIII domains to prevent aggregation in multi-modular proteins.

The folding nucleus of a fibronectin type III domain is composed of core residues of the immunoglobulin-like fold

To identify the contacts that stabilise the rate-limiting transition state for folding of FNfn10 (the tenth fnIII domain of human fibronectin), 42 mutants have been analysed at 29 positions across this domain. An anomalous response to mutation means that structure formation in the A, B and G strands cannot be evaluated by this method. In all the residues analysed, phi-values are fractional and no completely structured region is observed. The analysis reveals that hydrophobic residues from the central strands of the beta-sandwich form a large core of interactions in the transition state. Brønsted analysis shows that the stabilisation energy from the amino acid side-chains in the transition state is ∼40% of that in the native state. The protein folds by a nucleation-condensation mechanism, and tertiary interactions within the core make up the folding nucleus. Local interactions, in turns and loops, are apparently much less significant. Comparison with an homologous domain from human tenascin (TNfn3), shows that FNfn10 has a more extended, structured transition state spanning three different “layers” of the beta-sandwich. The results support the hypothesis that interactions in the common structural core guide the folding of these domains.

Receptor protein-tyrosine phosphatases: origin of domains (catalytic domain, Ig-related domain, fibronectin type III module) based on the sequence of the sponge Geodia cydonium

Reversible tyrosine phosphorylation of proteins is one of the major regulatory physiological events in response to cell-cell- and cell-matrix contact in Metazoa. Previously it was documented that the tyrosine phosphorylating enzymes, the tyrosine kinases (TKs), are autapomorphic characters of Metazoa, including sponges. In this paper the tyrosine dephosphorylating enzymes, the protein-tyrosine phosphatases (PTPs), are studied which can be grouped into two subfamilies, the soluble PTPs and the receptor PTPs (RPTPs). PTPs are characterized by one PTPase domain which interestingly comprises sequence similarity to yeast PTPs. In contrast to the PTPs, the RPTPs – which have been found only in Metazoa – are provided with two PTPase domains. To study the evolution of the RPTPs the full-length size RPTP was cloned from the marine demosponge Geodia cydonium, the phylogenetic oldest metazoan taxon. The 3253 bp long sequence has a putative open reading frame coding for a 999 aa long RPTP which is characterized by two fibronectin (type III; FN-III) domains in the extracellular portion, one intracellular immunoglobulin (Ig)-related domain, and two PTPase domains. Phylogenetic analysis revealed that the sponge FN-III domains form the basis of the metazoan FN-III domain with the common metazoan ancestor. The Ig-related, typical metazoan, module is classified to the disulphide lacking Ig members and represents the phylogenetic earliest member of this group. The β-sheet propensity was calculated and the characteristic amino acids are present in the seven β-sheets. The analysis of the two PTPase domains of the sponge RPTP demonstrates that the first domain is closely related to the PTPase domains present in the soluble PTPs, while the second PTPase domain is only distantly related to them. By constructing a rooted phylogenetic cladogram it became overt that the duplication of the PTPase domains must have occurred already in yeast. This interesting finding indicates that two conserved PTPase domains originated from a common ancestor in yeast while the evolutionary novelties, the FN-III domains and the Ig-related module, were added during the transition to the Metazoa. Hence, the tyrosine dephosphorylating enzyme, RPTP, is an example for a modular protein which is composed of ancient modules (PTPase domain[s]) and two metazoan novelties, while the tyrosine phosphorylating enzymes, the TKs, evolved only in Metazoa.

Axl receptor tyrosine kinase expression in human lung cancer cell lines correlates with cellular adhesion

Axl is a receptor tyrosine kinase (RTK) with oncogenic potential and transforming activity. Since Axl bears structural similarities to cell adhesion molecules such as neural cell adhesion molecule (NCAM) (FNIII domains), it is thought that Axl might play a role in adhesion. In this study, we have analysed the expression of the Axl protein and its ligand, Gas6, in human lung cancer cell lines of different histological origin. Axl expression occurred in approximately 60% of non-small cell lung cancer (NSCLC) cell lines, which grow adherently, and in normal bronchial epithelial cells (NHBE), but not in cell lines of small cell lung cancer origin (SCLC), which grow in suspension. A number of SCLC sublines, which could be selected spontaneously or after oncogene transfection for adherent growth, all expressed Axl protein. Overexpression of Axl per se, however, did not induce any change in the adhesion phenotype. All Axl-expressing cell lines demonstrated a membrane-bound 140 kD form, as well as a soluble 85 kD form, detectable in supernatant, of Axl-RTK. Expression of the Axl ligand Gas6 was detected in approximately 80% of all cell lines investigated. We conclude from these data that loss of Axl expression is a feature of SCLC tumour cells. Axl expression appears to be a consequence of cellular adhesion and possibly influences differentiation in human lung cancers.

Two proteins with the same structure respond very differently to mutation: the role of plasticity in protein stability

As part of a systematic study of the folding of protein structural families we compare the effect of mutation in two closely related fibronectin type III (fnIII) domains, the tenth fnIII domain of human fibronectin (FNfn10) and the third fnIII domain of human tenascin (TNfn3). This comparison of the two related proteins allows us to distinguish any anomalous response to mutation. Although they have very similar structures, the effect of mutation is very different. TNfn3 behaves like a “typical” protein, with changes in free energy correlated to the number of contacts lost on mutation. The loss of free energy upon mutation is significantly lower for FNfn10, particularly mutations of residues in the A, B and G strands. Remarkably, some of the residues involved are completely buried and closely packed in the core. In FNfn10 the regions of the protein that can accommodate mutation have previously been shown to be mobile. We propose that there is a “plasticity” in the peripheral regions of FNfn10 that allows it to rearrange to minimise the effect of mutations. This study emphasises the difficulties that might arise when making generalisations from a single member of a protein family.

Posttranslational processing and differential glycosylation of Tractin, an Ig-superfamily member involved in regulation of axonal outgrowth

Tractin is a novel member of the Ig-superfamily which has a highly unusual structure. It contains six Ig domains, four FNIII-like domains, an acidic domain, 12 repeats of a novel proline- and glycine-rich motif with sequence similarity to collagen, a transmembrane domain, and an intracellular tail with an ankyrin and a PDZ domain binding motif. By generating domain-specific antibodies, we show that Tractin is proteolytically processed at two cleavage sites, one located in the third FNIII domain, and a second located just proximal to the transmembrane domain resulting in the formation of four fragments. The most NH 2-terminal fragment which is glycosylated with the Lan3-2, Lan4-2, and Laz2-369 glycoepitopes is secreted, and we present evidence which supports a model in which the remaining fragments combine to form a secreted homodimer as well as a transmembrane heterodimer. The extracellular domain of the dimers is mostly made up of the collagen-like PG/YG-repeat domain but also contains 11/2 FNIII domain and the acidic domain. The collagen-like PG/YG-repeat domain could be selectively digested by collagenase and we show by yeast two-hybrid analysis that the intracellular domain of Tractin can interact with ankyrin. Thus, the transmembrane heterodimer of Tractin constitutes a novel protein domain configuration where sequence that has properties similar to that of extracellular matrix molecules is directly linked to the cytoskeleton through interactions with ankyrin.

Folding of beta-sandwich proteins: three-state transition of a fibronectin type III module.

An analysis of the folding of the 94 residue tenth fibronectin type III (fnIII) domain of human fibronectin (FNfn10) is presented. Use of guanidine isothiocyanate as a denaturant allows us to obtain equilibrium and kinetic data across a broad range of denaturant concentrations that are unavailable in guanidine hydrochloride. Equilibrium unfolding experiments show that FNfn10 is significantly more stable than has been reported previously. Comparison of equilibrium and kinetic parameters reveals the presence of an intermediate that accumulates at low denaturant concentrations. This is the first demonstration of three-state folding kinetics for a fnIII domain. We have previously shown that a homologous domain from human tenascin (TNfn3) folds by a two-state mechanism, but this does not necessarily indicate that the two proteins fold by different folding pathways.

The Compact Conformation of Fibronectin Is Determined by Intramolecular Ionic Interactions

Fibronectin exists in a compact or extended conformation, depending upon environmental pH and salt concentration. Using recombinant fragments expressed in bacteria and baculovirus, we determined the domains responsible for producing fibronectin's compact conformation. Our velocity and equilibrium sedimentation data show that FN2-14 (a protein containing FN-III domains 2 through 14) forms dimers in low salt. Experiments with smaller fragments indicates that the compact conformation is produced by binding of FN12-14 of one subunit to FN2-3 of the other subunit in the dimer. The binding is weakened at higher salt concentrations, implying an electrostatic interaction. Furthermore, segment FN7-14+A, which contains the alternatively spliced A domain between FN11 and 12, forms dimers, whereas FN7-14 without A does not. Segment FN12-14+A also forms dimers, but the isolated A domain does not. These data imply an association of domain A with FN12-14, and the presence of A may favor an open conformation by competing with FN2-3 for binding to FN12-14.

Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule.

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

Molecular composition of the node of Ranvier: identification of ankyrin-binding cell adhesion molecules neurofascin (mucin+/third FNIII domain-) and NrCAM at nodal axon segments.

Neurofascin, NrCAM, L1, and NgCAM are a family of Ig/FNIII cell adhesion molecules that share ankyrin-binding activity in their cytoplasmic domains, and are candidates to form membrane-spanning complexes with members of the ankyrin family of spectrin-binding proteins in a variety of cellular contexts in the nervous system. Specialized forms of ankyrin, 270 kD and/or 480 kD ankyrinG are components of the membrane undercoat of axons at the node of Ranvier. This paper focuses on definition of the isoforms of ankyrin-binding cell adhesion molecules localized with ankyrinG at the nodal axon segment. The exon usage of two major forms of neurofascin was determined by isolation of full-length cDNAs and used to prepare isoform-specific antibodies. An isoform of neurofascin containing a mucin-like domain and lacking the third FNIII domain was concentrated at axon initial segments and colocalized at nodes of Ranvier with ankyrinG and the voltage-dependent sodium channel. An alternative form of neurofascin lacking the mucin-like domain and containing the third FNIII domain was present in unmyelinated axons. The antibody initially raised against neurofascin was used to screen a rat brain cDNA expression library. In addition to neurofascin, this screen yielded a clone with 80% sequence identity to NrCAM from chicken. The sequences of two full-length cDNAs are presented. NrCAM is most closely related to neurofascin among the other members of the L1/neurofascin/NgCAM family, with over 70% identity between cytoplasmic domains. NrCAM, visualized with antibodies specific for the ecto-domain, also was found to be coexpressed with neurofascin at nodes of Ranvier and at axon initial segments. This is the first characterization of defined neuronal cell adhesion molecules localized to axonal membranes at the node of Ranvier of myelinated axons.

A novel alternative splice domain in zebrafish tenascin-C

We have identified and sequenced clones from a zebrafish library that correspond to tenascin-C (TN-C). The 2036-bp sequence covers the C-terminal segment of the protein. Comparison of this sequence to TN-C from other vertebrates indicates that our sequence starts in FN-III domain 5, continues with one alternative splice domain, then with FN-III domains 6–8 and, finally, the complete fibrinogen-like domain. The alternative splice domain, designated TNfnQ, is not closely identified to any of the domains in other species, and may represent a novel descendent of the alternative splice-domain precursor.

The proto-oncogene of v-eyk (v-ryk) is a novel receptor-type protein tyrosine kinase with extracellular Ig/GN-III domains.

v-ryk is the oncogene in the avian acute oncogenic retrovirus RPL30, its cellular counterpart, c-ryk, is described in this report. To avoid the confusion caused by another gene with the same name, we renamed v-ryk to be v-eyk, thereby changing the proto-oncogene's name to c-eyk. c-eyk is expressed highly in chicken spleen and moderately in many other tissues including embryonic tissues. Its cDNA is 3061 base pairs encoding a receptor-type protein tyrosine kinase (PTK) of 974 amino acids. Compared to c-Eyk, v-Eyk is a truncated PTK lacking the extracellular and transmembrane regions of c-Eyk in addition to two amino acid changes. c-Eyk has two C2-type Ig domains and two FN-III domains in its extracellular region, forming a new subfamily of receptor-type PTK together with UFO/Axl/Ark. As suggested by Ig/Fn-III domains in many cell adhesion molecules, this subfamily of PTK may mediate cell-cell interaction.