YIGSR Sequence Research Articles

One-Pot Synthesis of Bioinspired Peptide-Decorated Apatite Nanoparticles for Nanomedicine.

Hybrid organic-inorganic bio-inspired apatite nanoparticles (NPs) are attractive for biomedical applications and especially in nanomedicine. Unfortunately, their applications in nanomedicine are limited by their broad particle size distributions and uncontrolled drug loading due to their multistep synthesis process. Besides, very few attempts at exposing bioactive peptides on apatite NPs are made. In this work, an original one-pot synthesis of well-defined bioactive hybrid NPs composed of a mineral core of bioinspired apatite surrounded by an organic corona of bioactive peptides is reported. Dual stabilizing-bioactive agents, phosphonated polyethylene glycol-peptide conjugates, are prepared and directly used during apatite precipitation i) to form the organic corona during apatite precipitation, driving the size and shape of resulting hybrid NPs with colloidal stabilization and ii) to expose peptide moieties (RGD or YIGSR sequences) at the NPs periphery in view of conferring additional surface properties to enhance their interaction with cells. Here, the success of this approach is demonstrated, the functionalized NPs are fully characterized by Fourier-transform infrared, Raman, X-ray diffraction, solid and liquid state NMR, transmission electron microscopy, and dynamic light scattering, and their interaction with fibroblast cells is followed, unveiling a synergistic proliferative effect.

Functionalization of 3D printed polymeric bioresorbable stents with a dual cell-adhesive peptidic platform combining RGDS and YIGSR sequences.

Biomimetic surface modification with cell-adhesive peptides is a promising approach to improve endothelialization of current bioresorbable stents (BRS). Among them, RGDS and YIGSR sequences have been reported to mediate adhesion and migration of endothelial cells (ECs) while preventing platelet activation. This work presents the functionalization of novel 3D-printed poly-L-lactic acid (PLLA) and poly(L-lactic-co-ε-caprolactone) (PLCL) BRS with linear RGDS and YIGSR sequences, as well as a dual platform (PF) containing both motifs within a single biomolecule. Functionalized surfaces were characterized in terms of static contact angle, biomolecule distribution under confocal fluorescence microscopy and peptide quantification via detachment from the surface, showing a biomolecule density in the range of 0.5 to 3.5 nmol cm-2. Biological evaluation comprised a cell adhesion test on functionalized films with ECs and a blood perfusion assay on functionalized stents to assess ECs response and device hemocompatibility, respectively. Cell adhesion assays evidenced significantly increased cell number and spreading onto functionalized films with respect to control samples. Regarding stents' hemocompatibility, platelet adhesion onto PLCL stents was severely decreased with respect to PLLA. In addition, functionalization with RGDS, YIGSR and the PF rendered BRS stents displaying even further reduced platelet adhesion. In conclusion, the combination of intrinsically less prothrombogenic materials such as PLCL and its functionalization with EC-discriminating adhesive biomolecules paves the way for a new generation of BRS based on accelerated re-endothelialization approaches.

A functionalized self-assembling peptide containing E7 and YIGSR sequences enhances neuronal differentiation of spermatogonial stem cells on aligned PCL fibers for spinal cord injury repair.

Background: Spinal cord injury (SCI) induces neuronal death and disrupts the nerve fiber bundles, which leads to partial or complete sensorimotor function loss of the limbs. Transplantation of exogenous neurons derived from stem cells to the lesion site becomes a new neurorestorative strategy for SCI treatment. Spermatogonial stem cells (SSCs) can attain pluripotency features by converting to embryonic stem-like cells in vitro. However, differentiating SSCs into lineage-specific neurons is quite difficult and low efficiency. Methods: Immunofluorescence, immunohistochemistry, Western blotting, whole-cell patch clamp, and behavioral tests were performed to verify that self-assembled hydrogels could improve the directional differentiation efficiency of SSCs and the feasibility of SSC-derived neurons in the treatment of spinal cord injury. Results: We developed a novel self-assembled peptide Nap-FFGEPLQLKMCDPGYIGSR (Nap-E7-YIGSR) coated with aligned electrospun PCL fibers to enhance neuronal differentiation of SSCs. The Nap-E7-YIGSR peptide could evenly self-assemble on the surface of PCL fibers, enhanced the materials's hydrophilicity, and improved the SSC affinity of PCL fibers through the stem cell adhesion peptide sequence EPLQLKM domain. In addition, Nap-E7-YIGSR could effectively induce SSC neuron differentiation by activating the integrin β1/GSK3β/β-catenin signaling pathway. Moreover, implanting the induced neurons derived from SSCs into SCI lesion sites in rats resulted in the formation of new relay circuits, myelination, and synapse formation. Furthermore, SSC-derived neurons could survive and function in the spinal cord injury microenvironment, boosting the recovery of locomotion. Conclusion: The combination of the multifunctional peptide and aligned fibers can potentially trigger SSC differentiation to neurons, facilitating neuronal replacement therapy and promoting functional recovery after SCI.

Surface Modification of Multiple Bioactive Peptides to Improve Endothelialization of Vascular Grafts.

Endothelialization is an effective approach to prevent thrombus formation and enhance vascular graft survival. Surface modification of biomolecules has been proved to be effective in regulating endothelial cell behaviors. In this study, several peptides including YIGSR, RGD, and REDV sequences are covalently immobilized on the surface of electrospun silk fibroin scaffolds and the effects of combined application of these peptides on cell behaviors are studied. The results show that, compared with the scaffolds modified with single peptides, the scaffolds modified with dual peptides (YIGSR+RGD) could significantly enhance the proliferation of human umbilical vein endothelial cells (HUVECs). However, the combination of REDV+RGD or YIGSR+REDV does not promote the adhesion or proliferation of HUVECs. Notably, YIGSR-modified scaffolds improved HUVEC migration significantly in comparison to REDV- or RGD-modified groups. Moreover, its combination with either of these two peptides also presents excellent effect on cell migration. Thus, all the data suggest that the combined application of peptides might be a promising method to enhance the endothelialization of small-diameter vascular grafts.

Hyperbranched poly(ϵ-lysine) substrate presenting the laminin sequence YIGSR induces the formation of spheroids in adult bone marrow stem cells.

Unlike the fibroblast-like cells formed upon monolayer culture of human mesenchymal stem cells, the natural stem cell niche of the bone marrow and other types of tissues favours the formation of 3-dimensional (3D) cell clusters. The structuring and biological activity of these clusters are regulated by the contacts established by cells with both the basement membrane and neighbour cells and results in their asymmetric division and the consequent maintenance of both a stem population and a committed progeny. The present work demonstrates the potential of a synthetic substrate to mimic the stem cell niche in vitro. The side amino groups of a linear Poly-L-lysine were modified with hyperbranched poly-(ϵ-lysine) peptides, named as dendrons, tethered with the laminin-mimicking sequence, YIGSR. These dendrons presented the YIGSR sequence at the uppermost molecular branching ensuring a controlled spacing of the bioligand. When used to coat the surface of tissue culture plates in a serum-free in vitro cell culture system, the substrate was able to mimic the most relevant features of the basement membrane of the stem cell niche, i.e. the mesh structure of Collagen Type IV and the availability of laminin bioligands relevant to integrin biorecognition. The substrate biomimetic properties were tested for their ability to support the formation of human bone marrow mesenchymal stem cells (hMSCs) 3D spheroids similar to those observed in the natural stem cell niches and their ability to maintain stem cell pluripotency markers. These features were related to the substrate-specific expression and localisation of (i) cell adhesion receptors (i.e. β-integrin and N-cadherin), (ii) transcription factors of pluripotency markers and cytoskeleton protein and (iii) regulators of cell migration throughout cell culture passages 2 to 4. The results clearly demonstrate the formation of 3D spheroids starting from the asymmetric division of substrate-adhering spread cells, the clustering of relevant integrins and the expression of specific intracellular pathways controlling cytoskeleton formation suggesting their potential use as a substrate for the handling of stem cells prior to transplantation procedures.

ID2015 Preparation of graphene oxide/oligopeptides composite for promoting mesenchymal stem cell proliferation

Human mesenchymal stem cells (hMSCs) treatments are being tested clinically for a range of disorders. However, ex vivo expansion of hMSCs is still depending on human or animal source extracellular matrix (ECM) molecules. These ECM molecules are expensive to manufacture, have limited scalability and high batch-to-batch variability. Surface modification techniques have been instrumental in the development of scaffolds that promote cell-surface interactions. In this study, the surface of graphene oxide (GO) was modified in order to promote the attachment and proliferation of mesenchymal stem cells (hMSCs). YIGSR, WYQNMIR, IKVAV and QHREDGS peptide sequences derived from ECM adhesion peptides were combined alternatively to form oligopeptides and each oligopeptide was attached to GO through π-π and electrostatic interactions. The presence of peptide bond in the GO/peptide composites were confirmed by using modified Lowry method. The GO/Peptide-IKVAV composite showed increase proliferation rate and better attachment of hMSCs at day 1 as compared to GO substrate. In addition, the GO/Peptide-IKVAV composite proved to be a suitable environment for the time-dependent viability of hMSCs. The data obtained here collectively demonstrates that the GO/Peptide-IKVAV composite is a potential substrate for the adhesion and proliferation of hMSCs

Tunable, Injectable Hydrogels Based on Peptide-Cross-Linked, Cyclized Polymer Nanoparticles for Neural Progenitor Cell Delivery.

A PEG-based cyclized vinyl polymer was synthesized via one-step RAFT polymerization and used as a precursor of injectable hydrogels. Dithiol linkers including laminin-derived peptides containing IKVAV and YIGSR sequences and DTT were used for gelation. Fast and adjustable gelation rate was achieved through nucleophile-initiated thiol-Michael reaction under physiological conditions. Low swelling ratio and moderate degradation rate of the formed hydrogels were observed. 3D encapsulation of neural progenitor cells in the synthetic hydrogel showed good cell viability over 8 days. The long-term cell survival and proliferation were promoted by the introduction of laminin-derived peptides. This hydrogel platform based on peptide-cross-linked, cyclized vinyl polymers can be used as a universal hydrogel template for 3D cell encapsulation.

The surface modification of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymers to improve the attachment of urothelial cells

Biocompatible and biodegradable poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] substrates were modified to improve the attachment of porcine urothelial cell culture. The pristine copolymer exhibits excellent mechanical properties to replace the bladder tissue, but its surface lacks chemical functionalities to interact with cells. Thus, wet chemical treatments based on NaOH and ethylenediamine in aqueous [ED(aq)] and isopropanol [ED(isoOH)] media to functionalize the P(HB-co-HHx) films surfaces were compared. Among these treatments, short ED(aq) treatment was able to decrease the hydrophobicity, rendering a surface with amino groups and without a significant alteration of the mechanical properties. Furthermore, to enhance the interaction with urothelial cells, laminin derived YIGSR sequence was covalently bound to these amino functionalized substrates. The focal attachment was clearly improved with this last treatment, comparing with those results found with the unmodified and first-step functionalized P(HB-co-HHx).

Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration

To control specific endothelial cell (EC) functions, cell adhesive RGDS, EC specific REDV and YIGSR peptides, and angiogenic SVVYGLR sequences were covalently immobilized onto polyethylene terephthalate (PET) surfaces for the purpose of cell culture. X-ray photoelectron spectroscopy, atomic force microscopy, fluorescence microscopy and contact angle measurement were employed for characterization of surface modifications. The peptide density on PET surfaces was evaluated by fluorescence microscopy. The surfaces immobilized with peptides were exposed to human umbilical vein endothelial cells to study their specific effects onto EC functions. The results showed that the surface functionalized by these peptides enhanced the EC adhesion, spreading and migration as compared with native PET surfaces. Specifically, the RGDS peptides induced more cell adhesion than other peptides. The YIGSR and SVVYGLR sequences induced more cell spreading and cell migration, represented by intense focal adhesion at the leading edges of cell spreading and migration. The bi-functionalization of RGDS and SVVYGLR peptides (MIX) combined the advantages of both peptides and induced significant EC adhesion, spreading and migration. Our study indicates that the surface functionalization by peptides specific for ECs, especially the combination of RGDS with SVVYGLR or YIGSR peptides, has potential applications in promoting endothelialization of vascular prostheses and for construction of vascularized tissues in tissue engineering.

Surface modification of poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) copolymer films for promoting interaction with bladder urothelial cells

Often bladder dysfunction and diseases lead to therapeutic interventions that require partial or complete replacement of damaged tissue. For this reason, the development of biomaterials to repair the bladder by promoting the adhesion and growth of urothelial cells is of interest. With this aim, a modified copolyester of biocompatible and biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] was used as scaffold for porcine urothelial cell culture. In addition to good biocompatibility, the surface of P(HB-co-HV) substrates was modified to provide both, higher hydrophilicity and a better interaction with urothelial cells. Chemical treatments with ethylenediamine (ED) and sodium hydroxide (NaOH) led to substrate surfaces with decreasing hydrophobicity and provided functional groups that enable the grafting of bioactive molecules, such as a laminin derived YIGSR sequence. Physico-chemical properties of modified substrates were studied and compared with those of the pristine P(HB-co-HV). Urothelial cell morphology on treated substrates was studied. The results showed that focal attachment and cell-related properties were improved for peptide grafted polymer compared with both, the unmodified and functionalized copolyester.

Enhancement of gene transfer using YIGSR analog of Tat‐derived peptide

Cell penetrating peptide based gene carriers are notably known for low level of gene transfer. To remedy this, as laminin receptor (LR) has been previously linked to tumor metastasis, the LR-binding domain (YIGSR) as well as a scrambled sequence (SGIYR) were added to Tat-derived peptide sequence (YIGSR-Tat and SGIYR-Tat respectively). Peptides cellular uptake was assessed with high-LR (HT1080) and low-LR (HT29) cell lines by flow cytometry. Their ability to form complexes with DNA was examined using YOPRO-1 fluorescence assay and their transfection efficiencies evaluated using a luciferase reporter gene assay. DNA complexes were formed at (+/-) charge ratios as low as 2:1. While no conclusion could be drawn on the effect of YIGSR sequence on peptides uptake in both cell lines, a significant improvement in gene transfection in HT1080 cells was achieved using YIGSR-Tat compared to Tat and SGIYR-Tat. Additionally this increased efficiency was inhibited by excess free YIGSR. No significant difference in transfection efficiency was observed between Tat, SGIYR-Tat and YIGSR-Tat based complexes in HT29 cells. These studies demonstrate that attachment of receptor-binding ligand (YIGSR) to Tat-derived peptide can improve the efficiency of gene transfer in LR-positive cells (HT1080).

Peptide-surface modification of poly(caprolactone) with laminin-derived sequences for adipose-derived stem cell applications

Human adipose tissue has been recognized as a source of adult stem cells for tissue engineering applications such as bone, cartilage, and soft tissue repair. For the success of these tissue-engineering approaches, a cell delivery vehicle such as a hydrogel or scaffold is required to position the stem cells at the site of need. Surface modification techniques have been instrumental in the development of scaffolds that promote cell-surface interactions. In this study, poly(caprolactone) (PCL), surfaces were modified in order to promote the attachment and proliferation of adipose-derived stem cells (ASCs). RGD, YIGSR, and IKVAV peptide sequences derived from the extracellular matrix protein laminin were each covalently attached to an aminated polymer surface using carbodiimide chemistry. The surface was characterized using scanning electron microscopy (SEM), goniometry and X-ray photoelectron spectroscopy (XPS). The attachment and proliferation of ASCs was assessed on the different peptide-treated surfaces. XPS analysis confirmed the presence of the peptide sequences on the surface of the polymer as indicated by the increase in the nitrogen/carbon ratio on the surface of the polymer. Among all peptide sequences tested, IKVAV-treated surfaces had a significantly greater number of ASCs bound 2 and 3 days after cell seeding. SEM confirmed differences in the morphology of the cells attached to the three peptide-treated surfaces. These results indicate that IKVAV is a suitable peptide sequence for use in surface modification techniques aimed at improving the attachment of ASCs to a tissue-engineered scaffold.

Synthetic collagen fibers coated with a synthetic peptide containing the YIGSR sequence of laminin to promote peripheral nerve regeneration in vivo.

The usefulness of collagen fibers and the YIGSR sequence (Tyr-lle-Gly-Ser-Arg) of laminin for nerve regeneration were examined in vivo. Type I collagen gel (G-group), Type I collagen fibers (F-group), Type I collagen fibers coated with laminin (L-group) or the YIGSR sequence (Y-group) were packed into silicone tubes, 15 mm long, and transplanted to the sciatic nerves of Wistar rats. Empty silicone tubes were used as the control. The animals were sacrificed 8 weeks after transplantation. Bridging of the nerve was confirmed in the F-(7/12), Y-(7/10) and L-group (6/10), but no bridging was observed in any of the animals of the G- and control group. Nerve regeneration among the space of collagen fibers was observed, and it was suggested that fibroblasts infiltrated the gap in the substance of the degenerated collagen fibers were followed by Schwann cells on the basis of immunocytochemistry. The number of myelinated axons per regenerated tissue in the tube (density), and total area of myelinated axons per measured regenerated tissue in the tube (% axon area) in each the L- and Y-group were significantly higher than that in the F-group (P < 0.05). These results suggest the possibility of obtaining adequate nerve regeneration with new artificial materials only.

Binding of the Cysteine Proteinases Papain and Cathepsin B-like to Coated Laminin: Use of Synthetic Peptides from Laminin and from the Laminin Binding Region of the β1Integrin Subunit to Characterize the Binding Site

Cysteine proteinases of the papain superfamily, i.e., papain and cathepsin B-like proteinase, were found to be able to bind to laminin-coated wells. When papain and cathepsin B-like proteinase were used, saturable binding curves were found. The characterization of the binding site was carried out using synthetic peptides which corresponded to the most relevant functional sites of laminin and an octapeptide from the laminin binding region of the β1integrin subunit. In binding experiments, the decapeptide RNIAEIIKDI and the pentapeptide YIGSR were able to displace papain and cathepsin B-like proteinase from coated laminin. Nevertheless, the integrin β1peptide DLYYLMDL was the most powerful in the same experimental system. From these results, the C-terminal region of this cross-shaped protein, i.e., the end of the long arm, and the region including the YIGSR sequence of the short arm of the β chain would be the cysteine proteinase binding site. This binding site is probably the result of the network organization of laminin which brings two regions, separated on a single laminin molecule, into proximity. In previous work, digestion of basement membranes has been found to be associated with the binding of cysteine proteinases to these supramolecular structures [N. Guinec, V. Dalet-Fumeron, and M. Pagano (1992)FEBS Lett.308, 305–308]. The present report demonstrates that laminin is the cysteine proteinase binding protein of basement membranes. This property of laminin could be associated with tumor invasion and other tissue remodeling processes linked to proteolysis of basement membranes and extracellular matrices.

Involvement of the YIGSR sequence of laminin in protein tyrosine phosphorylation

We have examined the mechanism of signaling by the 67 kDa YIGSR binding protein of laminin and its properties in neuroblastoma cells. Ligand displacement analysis showed that the interaction with the C(YIGSR) 3-NH 2 peptide amide is of intermediate affinity (1.5×10 −7 M). Cross-linking experiments with sulfo-MBS detected an additional protein with a molecular mass of 116 kDa that binds the YIGSR sequence. Incubation of neuroblastoma cells with C(YIGSR) 3-NH 2 peptide amide or antibody directed against the 67 kDa laminin binding protein induces tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa and another heterogeneous protein group of 32 kDa.

Typel collagen fibers coated with a synthetic laminin peptide containing the YIGSR sequence promote peripheral nerve regeneration in vivo

Typel collagen fibers coated with a synthetic laminin peptide containing the YIGSR sequence promote peripheral nerve regeneration in vivo

Adhesion polypeptides are useful for the prevention of peritoneal dissemination of gastric cancer.

We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both alpha2beta1 and alpha3beta1 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequence intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhesion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients.

1248 Type1 collagen fibers coated with a synthetic laminin peptide containing the YIGSR sequence promote peripheral nerve regeneration in vivo

1248 Type1 collagen fibers coated with a synthetic laminin peptide containing the YIGSR sequence promote peripheral nerve regeneration in vivo

Inhibition of angiogenesis, tumour growth and experimental metastasis of human fibrosarcoma cells HT1080 by a multimeric form of the laminin sequence Tyr-lle-Gly-Ser-Arg (YIGSR)

A multimeric peptide, Ac-Y16, consisting of 16 YIGSR sequences from laminin was evaluated for its effect on experimental metastasis, angiogenesis and tumour growth of HT1080 human fibrosarcoma cells. Co-injection of 0.5 mg per mouse of Ac-Y16 i.v. with HT 1080 cells inhibited lung colonisation by 100%, whereas 0.5 mg per mouse of monomeric Ac-YIGSR-NH2(AcY1) inhibited by 94%. Ac-Y16 did not show any direct cytotoxicity in tumour cells in vivo. The effect of the peptides on angiogenesis and tumour growth respectively were evaluated by counting areas of neovessels and weighing tumours after the s.c. implantation of HT1080 cells with basement membrane extracts and the peptide into nude mice. Co-injection of 0.5 mg per mouse of AC-Y16 s.c. with HT1080 cells inhibited angiogenesis and tumour growth by 92% (P<0.05) and 76% (P<0.05) respectively, whereas 0.5 mg per mouse of monomeric Ac-YIGSR-NH2(Ac-Y1) inhibited angiogenesis and tumour growth by 40% (P<0.05) and 9% (P>0.05) respectively. It can be inferred from these data that anti-tumour effects of Ac-Y16 are likely to result from anti-angiogenesis. Intraperitoneal administration of Ac-Y16 was also effective in inhibiting angiogenesis, tumour growth and lung colonisation of HT1080 cells. It was concluded that the multimeric YIGSR-containing peptide, Ac-Y16, inhibits angiogenesis, tumour growth and experimental metastasis more than the monomeric form and that it is active when administered i.p., iv. and s.c.

Down-regulation of a 67-kDa YIGSR-binding Protein upon Differentiation of Human Neuroblastoma Cells

Differentiated human neuroblastoma LA-N1 cells that were exposed to dibutyryl adenosine 3',5'-cyclic monophosphate for 5 days (primed cells) showed increased adhesion to laminin-, fibronectin-, and collagen type I-coated plates as compared to unprimed cells. Moreover, primed cells seemed to adhere best to laminin. The binding site in laminin, mediating cell attachment, was identified as containing the YIGSR sequence, a known cell binding motif, located in the short arm of the B1 chain of laminin. The synthetic peptide amide, C(YIGSR)3-NH2, containing a repeat of this binding motif, inhibited the attachment of neuroblastoma cells to laminin in a competitive manner, and its inhibitory activity was inversely dependent on laminin concentrations. Affinity chromatography of membrane-extracted proteins over an Affi-Gel 10 column conjugated to C(YIGSR)3-NH2, revealed a major YIGSR-binding protein with an apparent molecular mass of 67 kDa. The 67-kDa surface membrane protein was specifically eluted from the column with the soluble C(YIGSR)3-NH2 peptide, but not with an unrelated peptide. Furthermore, no 67-kDa laminin-binding protein was recovered from an unrelated peptide matrix with the free C(YIGSR)3-NH2 peptide. Ligand blot overlay assays with biotin-labeled C(YIGSR)3-NH2 peptide demonstrated that the 67-kDa receptor is indeed a YIGSR-binding protein. This 67-kDa laminin-binding protein appeared to be down-regulated upon differentiation of LA-N1 cells, as indicated by the level of this protein and its mRNA.