Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site.

Agrin is a basal lamina protein that induces aggregation of acetylcholine receptors (AChRs) and other molecules at the developing neuromuscular junction. Alternative splicing of chick agrin mRNA at two sites, A and B, gives rise to eight possible isoforms of which five are expressed in vivo. Motor neurons express high levels of isoforms with inserts at sites A and B, muscle cells synthesize isoforms that lack amino acids at the B-site. To obtain further insights into the mechanism of agrin-induced AChR aggregation, we have determined the EC50 (effective concentration to induce half-maximal AChR clustering) of each agrin isoform and of truncation mutants. On chick myotubes, EC50 of the COOH-terminal, 95-kD fragment of agrinA4B8 was approximately 35 pM, of agrinA4B19 approximately 110 pM and of agrinA4B11 approximately 5 nM. While some AChR clusters were observed with 64 nM of agrinA4B0, no activity was detected for agrinA0B0. Recombinant full-length chick agrin and a 100-kD fragment of ray agrin showed similar EC50 values. A 45-kD, COOH-terminal fragment of agrinA4B8 retained high activity (EC50 approximately equal to 130 pM) and a 21-kD fragment was still active, but required higher concentrations (EC50 approximately equal to 13 nM). Unlike the 45-kD fragment, the 21-kD fragment neither bound to heparin nor did heparin inhibit its capability to induce AChR aggregation. These data show quantitatively that agrinA4B8 and agrinA4B19, expressed in motor neurons, are most active, while no activity is detected in agrinA0B0, the dominant isoform synthesized by muscle cells. Furthermore, our results show that a fragment comprising site B8 and the most COOH-terminal G-like domain is sufficient for this activity, and that agrin domains required for binding to heparin and those for AChR aggregation are distinct from each other.

An amino-terminal extension is required for the secretion of chick agrin and its binding to extracellular matrix.

Agrin is an extracellular matrix (ECM) protein with a calculated relative molecular mass of more than 200 kD that induces the aggregation of acetylcholine receptors (AChRs) at the neuromuscular junction. This activity has been mapped to its COOH terminus. In an attempt to identify the functions of the NH2-terminal end, we have now characterized full-length chick agrin. We show that chick agrin encoded by a previously described cDNA is not secreted from transfected cells. Secretion is achieved with a construct that includes an additional 350 bp derived from the 5' end of chick agrin mRNA. Recombinant agrin is a heparan sulfate proteoglycan (HSPG) of more than 400 kD with glycosaminoglycan side chains attached only to the NH2-terminal half. Endogenous agrin in tissue homogenates also has an apparent molecular mass of > 400 kD. While the amino acid sequence encoded by the 350-bp extension has no homology to published rat agrin, it includes a stretch of 15 amino acids that is 80% identical to a previously identified bovine HSPG. The extension is required for binding of agrin to ECM. AChR aggregates induced by recombinant agrin that includes the extension are considerably smaller than those induced by agrin fragments, suggesting that binding of agrin to ECM modulates the size of receptor clusters. In addition, we found a site encoding seven amino acids at the NH2-terminal end of agrin that is alternatively spliced. While motor neurons express the splice variant with the seven amino acid long insert, muscle cells mainly synthesize isoforms that lack this insert. In conclusion, the cDNAs described here code for chick agrin that has all the characteristics previously allocated to endogenous agrin.

Agrin binds to the nerve-muscle basal lamina via laminin.

Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

Agrin can mediate acetylcholine receptor gene expression in muscle by aggregation of muscle-derived neuregulins.

The neural isoforms of agrin can stimulate transcription of the acetylcholine receptor (AChR) epsilon subunit gene in electrically active muscle fibers, as does the motor neuron upon the formation of a neuromuscular junction. It is not clear, however, whether this induction involves neuregulins (NRGs), which stimulate AChR subunit gene transcription in vitro by activating ErbB receptors. In this study, we show that agrin- induced induction of AChR epsilon subunit gene transcription is inhibited in cultured myotubes overexpressing an inactive mutant of the ErbB2 receptor, demonstrating involvement of the NRG/ErbB pathway in agrin- induced AChR expression. Furthermore, salt extracts from the surface of cultured myotubes induce tyrosine phosphorylation of ErbB2 receptors, indicating that muscle cells express biological NRG-like activity on their surface. We further demonstrate by RT-PCR analysis that muscle NRGs have Ig-like domains required for their immobilization at heparan sulfate proteoglycans (HSPGs) of the extracellular matrix. In extrasynaptic regions of innervated muscle fibers in vivo, ectopically expressed neural agrin induces the colocalized accumulation of AChRs, muscle-derived NRGs, and HSPGs. By using overlay and radioligand-binding assays we show that the Ig domain of NRGs bind to the HSPGs agrin and perlecan. These findings show that neural agrin can induce AChR subunit gene transcription by aggregating muscle HSPGs on the muscle fiber surface that then serve as a local sink for focal binding of muscle-derived NRGs to regulate AChR gene expression at the neuromuscular junction.

Alternative splicing of agrin alters its binding to heparin, dystroglycan, and the putative agrin receptor.

Agrin is a heparan sulfate proteoglycan that induces aggregation of acetylcholine receptors (AChRs) at the neuromuscular synapse. This aggregating activity is modulated by alternative splicing. Here, we compared binding of agrin isoforms to heparin, alpha-dystroglycan, and cultured myotubes. We find that the alternatively spliced 4 amino acids insert (KSRK) is required for heparin binding. The binding affinity of agrin isoforms to alpha-dystroglycan correlates neither with binding to heparin nor with their AChR-aggregating activities. Moreover, the minimal fragment sufficient to induce AChR aggregation does not bind to alpha-dystroglycan. Nevertheless, this fragment still binds to cultured muscle cells. Its binding is completed only by agrin isoforms that are active in AChR aggregation, and therefore this binding site is likely to represent the receptor that initiates AChR clustering.

Time-dependent morphology and adhesion of osteoblastic cells on titanium model surfaces featuring scale-resolved topography.

The role of micrometer and submicrometer surface roughness on the interaction of cells with titanium model surfaces of well-defined topography was investigated using human bone-derived cells (MG63 cells). The early phase of interactions was studied using a kinetic morphological analysis of adhesion, spreading and proliferation of the cells. By SEM and double immunofluorescent labeling of vinculin and actin, it was found that the cells responded to nanoscale roughness by a higher cell thickness and a delayed apparition of the focal contacts. A singular behavior was observed on nanoporous oxide surfaces, where the cells were more spread and displayed longer and more numerous filopods. On electrochemically microstructured surfaces with hemispherical cavities, arranged in a hexagonal pattern, the MG63 cells were able to go inside, adhere and proliferate in cavities of 30 or 100 microm in diameter, whereas they did not recognize the 10 microm diameter cavities. Cells adopted a 3D shape when attaching inside the 30 microm diameter cavities. Condensation of actin cytoskeleton correlated with vinculin-positive focal contacts on cavity edges were observed on all microstructured surfaces. Nanotopography on surfaces with 30 microm diameter cavities had little effect on cell morphology compared to flat surfaces with same nanostructure, but cell proliferation exhibited a marked synergistic effect of microscale and nanoscale topography.

Enhanced bone apposition to a chemically modified SLA titanium surface.

Increased surface roughness of dental implants has demonstrated greater bone apposition; however, the effect of modifying surface chemistry remains unknown. In the present study, we evaluated bone apposition to a modified sandblasted/acid-etched (modSLA) titanium surface, as compared with a standard SLA surface, during early stages of bone regeneration. Experimental implants were placed in miniature pigs, creating 2 circular bone defects. Test and control implants had the same topography, but differed in surface chemistry. We created the test surface by submerging the implant in an isotonic NaCl solution following acid-etching to avoid contamination with molecules from the atmosphere. Test implants demonstrated a significantly greater mean percentage of bone-implant contact as compared with controls at 2 (49.30 vs. 29.42%; p = 0.017) and 4 wks (81.91 vs. 66.57%; p = 0.011) of healing. At 8 wks, similar results were observed. It is concluded that the modSLA surface promoted enhanced bone apposition during early stages of bone regeneration.

RGD-containing peptide GCRGYGRGDSPG reduces enhancement of osteoblast differentiation by poly(L-lysine)-graft-poly(ethylene glycol)-coated titanium surfaces.

Osteoblasts exhibit a more differentiated morphology on surfaces with rough microtopographies. Surface effects are often mediated through integrins that bind the RGD motif in cell attachment proteins. Here, we tested the hypothesis that modulating access to RGD binding sites can modify the response of osteoblasts to surface microtopography. MG63 immature osteoblast-like cells were cultured on smooth (Ti sputter-coated Si wafers) and rough (grit blasted/acid etched) Ti surfaces that were modified with adsorbed monomolecular layers of a comb-like graft copolymer, poly-(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), to limit nonspecific protein adsorption. PLL-g-PEG coatings were functionalized with varying amounts of an integrin-receptor-binding RGD peptide GCRGYGRGDSPG (PLL-g-PEG/PEG-RGD) or a nonbinding RDG control sequence GCRGYGRDGSPG (PLL-g-PEG/PEG-RDG). Response to PLL-g-PEG alone was compared with response to surfaces on which 2-18% of the polymer sidechains were functionalized with the RGD peptide or the RDG peptide. To examine RGD dose-response, peptide surface concentration was varied between 0 and 6.4 pmol/cm(2). In addition, cells were cultured on uncoated Ti or Ti coated with PLL-g-PEG or PLL-g-PEG/PEG-RGD at an RGD surface concentration of 0.7 pmol/cm(2), and free RGDS was added to the media to block integrin binding. Analyses were performed 24 h after cultures had achieved confluence on the tissue culture plastic surface. Cell number was reduced on smooth Ti compared to plastic or glass and further decreased on surfaces coated with PLL-g-PEG or PLL-g-PEG/PEG-RDG, but was restored to control levels when PLL-g-PEG/PEG-RGD was present. Alkaline phosphatase specific activity and osteocalcin levels were increased on PLL-g-PEG alone or PLL-g-PEG/PEG-RDG, but PLL-g-PEG/PEG-RGD reduced the parameters to control levels. On rough Ti surfaces, cell number was reduced to a greater extent than on smooth Ti. PLL-g-PEG coatings reduced alkaline phosphatase and increased osteocalcin in a manner that was synergistic with surface roughness. The RDG peptide did not alter the PLL-g-PEG effect but the RGD peptide restored these markers to their control levels. PLL-g-PEG coatings also increased TGF-beta1 and PGE(2) in conditioned media of cells cultured on smooth or rough Ti; there was a 20x increase on rough Ti coated with PLL-g-PEG. PLL-g-PEG effects were inhibited dose dependently by addition of the RGD peptide to the surface. Free RGDS did not decrease the effect elicited by PLL-g-PEG surfaces. These unexpected results suggest that PLL-g-PEG may have osteogenic properties, perhaps correlated with effects that alter cell attachment and spreading, and promote a more differentiated morphology.

Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N-terminal domain.

Upon insertion of a signal-anchor protein into the endoplasmic reticulum membrane, either the C-terminal or the N-terminal domain is translocated across the membrane. Charged residues flanking the transmembrane domain are important determinants for this decision, but are not necessarily sufficient to generate a unique topology. Using a model protein that is inserted into the membrane to an equal extent in either orientation, we have tested the influence of the size and the folding state of the N-terminal domain on the insertion process. A small zinc finger domain or the full coding sequence of dihydrofolate reductase were fused to the N-terminus. These stably folding domains hindered or even prevented their translocation. Disruption of their structure by destabilizing mutations largely restored transport across the membrane. Translocation efficiency, however, did not depend on the size of the N-terminal domain within a range of 40-237 amino acids. The folding behavior of the N-terminal domain is thus an important factor in the topogenesis of signal-anchor proteins.

Synaptic differentiation: the role of agrin in the formation and maintenance of the neuromuscular junction.

Upon arrival of a motor axon at the muscle fiber, signals released from its growth cone initiate the formation of a synapse. This process consists of two stages: arrest of axon growth at the target area and differentiation of pre- and postsynaptic cells at the site of nerve-muscle contact. Studies of regenerating neuromuscular junctions in vertebrates have revealed that important signals for the formation of this synapse are located in the synaptic basal lamina, and attempts to identify these signals have led to the isolation of agrin and other components. In this review, we discuss the evidence for the involvement of these molecules and their potential functional role in the formation and maintenance of the neuromuscular junction, with emphasis on agrin.

Electron microscopic structure of agrin and mapping of its binding site in laminin-1.

Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with basement membranes of several tissues. Particular splice variants of agrin are essential for the formation of synaptic structures at the neuromuscular junction. The binding of agrin to laminin appears to be required for its localization to synaptic basal lamina and other basement membranes. Here, electron microscopy was used to determine the structure of agrin and to localize its binding site in laminin-1. Agrin appears as an approximately 95 nm long particle that consists of a globular, N-terminal laminin-binding domain, a central rod predominantly formed by the follistatin-like domains and three globular, C-terminal laminin G-like domains. In a few cases, heparan sulfate glycosaminoglycan chains were seen emerging from the central portion of the core protein. Moreover, we show that agrin binds to the central region of the three-stranded, coiled-coil oligomerization domain in the long arm of laminin-1, which mediates subunit assembly of the native laminin molecule. In summary, our data show for the first time a protein-protein interaction of the extracellular matrix that involves a coiled-coil domain, and they assign a novel role to this domain of laminin-1. Based on this, we propose that agrin associates with basal lamina in a polarized way.

Dystroglycan is a dual receptor for agrin and laminin-2 in Schwann cell membrane.

We have shown previously that alpha-dystroglycan with a molecular mass of 120 kDa is a Schwann cell receptor of laminin-2, the endoneurial isoform of laminin comprised of the alpha2, beta1, and gamma1 chains. In this paper, we show that Schwann cell alpha-dystroglycan is also a receptor of agrin, an acetylcholine receptor-aggregating molecule having partial homology to laminin alpha chains in the C terminus. Immunochemical analysis demonstrates that the peripheral nerve isoform of agrin is a 400-kDa component of the endoneurial basal lamina and is co-localized with alpha-dystroglycan surrounding the outermost layer of myelin sheath of peripheral nerve fibers. Blot overlay analysis demonstrates that both endogenous peripheral nerve agrin and laminin-2 bind to Schwann cell alpha-dystroglycan. Recombinant C-terminal fragment of the peripheral nerve isoform of agrin also binds to Schwann cell alpha-dystroglycan, confirming that the binding site for Schwann cell alpha-dystroglycan resides in the C terminus of agrin molecule. Furthermore, the binding of recombinant agrin C-terminal fragment to Schwann cell alpha-dystroglycan competes with that of laminin-2. All together, these results indicate that alpha-dystroglycan is a dual receptor for agrin and laminin-2 in the Schwann cell membrane.

Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma1 chain.

Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the gamma1 subunit of laminin-1, whereas no binding to alpha1 and beta1 chains was detected. By using recombinant gamma1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant gamma1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding. Interestingly, no binding to a chimeric gamma2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.