We have previously described peptide-binding proteins of 72 and 74 kDa (PBP72/74), which have been implicated as playing a role in antigen processing and are serologically related to the 70-kDa heat shock protein (hsp7O) family. Here we report the cloning and sequencing of the cDNA encoding PBP74 in mice and in humans, accomplished by using amino acid sequence information obtained from the purified protein. We show that PBP74 is highly homologous to members of the hsp7O family but, significantly, is not identical to any known member of this family. Inspection of the cDNA nucleotide sequence indicates that it encodes a 46-residue N-terminal peptide which is not present in the mature protein. Transcription and translation in vitro of the PBP74 cDNA verified that it encodes a form of PBP74 which is larger than the mature protein. The presequence does not conform to known motifs for organelle-targeting sequences, and at present, its function is not known. By confocal microscopy, PBP74 was localized to cytoplasmic vesicles but not to the nucleus, mitochondria, or plasma membrane by using antibodies specific for the N-terminal 16 residues of PBP74. By RNA filter hybridization analysis, PBP74 mRNAs are detected in all cell types tested. Exposure of cells to heat shock does not result in an increase in the mRNA levels of PBP74, unlike the dramatic increase observed for the stress-inducible hsp7O mRNA. Thus, PBP74 appears to be a constitutive, new member of the hsp7O family.

We examined the role of Laminin-5 (Ln-5) an extracellular matrix component of breast gland basement membrane, in supporting migration of normal (HUMEC), immortalized (MCF-10A), and malignant breast epithelial cells that exhibit different degrees of metastatic potential (MDA-MB-435>MDA-MB-231>MCF-7). HUMEC, MCF-10A, and MCF-7 cells all adhered to purified Ln-5 through the alpha3beta1 integrin receptor in adhesion assays. However, HUMEC and MCF-10A cells remained statically adherent, while MCF-7 cells migrated on Ln-5 in Transwell and colloidal gold displacement assays. Anti-alpha3 integrin antibodies blocked migration of MCF-7 cells on Ln-5. MDA-MB-231 and MDA-MB-435 cells bound and migrated on Ln-5 through a beta1 integrin receptor that is insensitive to antibodies that block the function of alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphaV integrin subunits. Migration of all cell types tested was blocked by CM6, a monoclonal antibody directed to a cell adhesion site on the alpha3 chain of Ln-5. Thus, Ln-5 may play an important role in regulating adhesion and migration in normal and transformed breast epithelium. Our results indicate that the type of integrin utilized by breast cells to interact with Ln-5, as well as its functional state, may determine whether cells will be statically adherent or migratory on Ln-5.

It is well established that integrins and extracellular matrix (ECM) play key roles in cell migration, but the underlying mechanisms are poorly defined. We describe a novel mechanism whereby the integrin ␣6␤1, a laminin receptor, can affect cell motility and induce migration onto ECM substrates with which it is not engaged. By using DNAmediated gene transfer, we expressed the human integrin subunit ␣6A in murine embryonic stem (ES) cells. ES cells expressing ␣6A (ES6A) at the surface dimerized with endogenous ␤1, extended numerous filopodia and lamellipodia, and were intensely migratory in haptotactic assays on laminin (LN)-1. Transfected ␣6A was responsible for these effects, because cells transfected with control vector or ␣6B, a cytoplasmic domain ␣6 isoform, displayed compact morphology and no migration, like wild-type ES cells. The ES6A migratory phenotype persisted on fibronectin (Fn) and Ln-5. Adhesion inhibition assays indicated that ␣6␤1 did not contribute detectably to adhesion to these substrates in ES cells. However, anti-␣6 antibodies completely blocked migration of ES6A cells on Fn or Ln-5. Control experiments with monensin and anti-ECM antibodies indicated that this inhibition could not be explained by deposition of an ␣6␤1 ligand (e.g., Ln-1) by ES cells. Cross-linking with secondary antibody overcame the inhibitory effect of anti-␣6 antibodies, restoring migration or filopodia extension on Fn and Ln-5. Thus, to induce migration in ES cells, ␣6A␤1 did not have to engage with an ECM ligand but likely participated in molecular interactions sensitive to anti-␣6␤1 antibody and mimicked by cross-linking. Antibodies to the tetraspanin CD81 inhibited ␣6A␤1-induced migration but had no effect on ES cell adhesion. It is known that CD81 is physically associated with ␣6␤1, therefore our results suggest a mechanism by which interactions between ␣6A␤1 and CD81 may up-regulate cell motility, affecting migration mediated by other integrins. INTRODUCTIONCell migration is crucial to embryonic development, tissue remodeling, and cancer invasion. To migrate properly, cells must integrate multiple incoming signals. Once committed to migration, they coordinately regulate, both spatially and temporally, surface receptors and cytoskeleton to generate traction and movement (Huttenlocher et al., 1996;Lauffenburger and Horwitz, 1996). Migration usually occurs over extracellular matrix (ECM) 1 and is accompanied by characteristic morphological changes. Cell protrusions (e.g., filopodia or lamellipodia) are sites where adherence ‡ Corresponding author: 10550 North Torrey Pines Road, Mail drop SBR12, La Jolla, CA 92037. 1 Abbreviations used: ECM, extracellular matrix; ES cell, embryonic stem cell; Fn, fibronectin; LIF, leukemic inhibitory factor; Ln, laminin.© 1997 by The American Society for Cell Biology 2253 contacts for traction generally are formed. To accomplish forward movement, there must be a balance between the establishment of plasma membrane-ECM adherence contacts at the cell leading edge and their coordinated as...

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Helper T cells recognize fragments of antigen bound to the class II molecules on the surface of antigen-presenting cells. Naturally processed antigenic fragments have been isolated from the class II molecules and shown to be heterogeneous in length, ranging from 13 to 25 residues, and to vary at both the N and C termini. A 15-residue peptide in an extended conformation is predicted to fit in an open peptide-binding cleft of the class II molecules. Thus, the longer peptides observed bound to class II presumably have regions which reside outside the cleft. It is not known if the additional length contributes significantly to T cell activation. We have carried out a systematic analysis of the antigenicity of peptides of increasing length beyond the minimally defined T cell antigenic peptide. Here we show that the full functional activities of peptides representing the major antigenic determinant of the protein antigen, cytochrome c, minimally require that the peptides be 23 amino acids long. The long peptides do not require processing and are presented by purified class II molecules incorporated into synthetic membranes, indicating that such peptides associate directly with class II and require no additional cellular machinery for presentation. We also show that a hybrid peptide, 51 residues in length, containing a 29-residue cytochrome c peptide and a "promiscuous" peptide of tetanus toxoid, is more antigenic than the 23-residue peptide alone and significantly, does not require processing. Thus, the additional peptide length, although not predicted to bind in the peptide-binding groove of the MHC class II molecule, has a significant impact on the ability of the peptides to stimulate T cell responses maximally.