Identification of a murine Peyer's patch--specific lymphocyte homing receptor as an integrin molecule with an alpha chain homologous to human VLA-4 alpha

Lymphocyte homing is controlled by organ-specific interactions of lymphocytes and high endothelial venules (HEV). Adhesion of lymphocytes to Peyer's patch HEV, but not to peripheral node HEV, is inhibited by an antibody recognizing the murine lymphocyte antigen LPAM-1. Lymphoma cell variants were selected on the FACS for differences in LPAM-1 expression: the binding capacity of these variants to Peyer's patch HEV directly correlates with the level of LPAM-1 expression. The anti-LPAM-1 antibody recognizes the alpha subunit of an Mr 160,000/130,000 cell surface alpha beta heterodimer. The association of LPAM-1 alpha and beta chains requires the presence of Ca2+ ions. Proteins of Mr 84,000 and Mr 62,000 present in LPAM-1 immunoprecipitates appear to be products of the proteolytic processing of alpha chains. The structure of LPAM-1 is virtually identical to that of the human integrin receptor VLA-4. The cross-reactivity of a monospecific rabbit antiserum demonstrated the similarity between the human VLA-4 alpha chain and the alpha subunit of LPAM-1.     

Discovery of very late antigen-4 (VLA-4, alpha4beta1 integrin) allosteric antagonists

Integrins are cell adhesion receptors that mediate cell-to-cell, or cell-to-extracellular matrix adhesion. They represent an attractive target for treatment of multiple diseases. Two classes of small molecule integrin inhibitors have been developed. Competitive antagonists bind directly to the integrin ligand binding pocket and thus disrupt the ligand-receptor interaction. Allosteric antagonists have been developed primarily for α(L)β(2)- integrin (LFA-1, lymphocyte function-associated antigen-1). Here we present the results of screening the Prestwick Chemical Library using a recently developed assay for the detection of α(4)β(1)-integrin allosteric antagonists. Secondary assays confirmed that the compounds identified: 1) do not behave like competitive (direct) antagonists; 2) decrease ligand binding affinity for VLA-4 ～2 orders of magnitude; 3) exhibit antagonistic properties at low temperature. In a cell based adhesion assay in vitro, the compounds rapidly disrupted cellular aggregates. In accord with reports that VLA-4 antagonists in vivo induce mobilization of hematopoietic progenitors into the peripheral blood, we found that administration of one of the compounds significantly increased the number of colony-forming units in mice. This effect was comparable to AMD3100, a well known progenitor mobilizing agent. Because all the identified compounds are structurally related, previously used, or currently marketed drugs, this result opens a range of therapeutic possibilities for VLA-4-related pathologies.     

A novel integrin (alpha E beta 4) from human epithelial cells suggests a fourth family of integrin adhesion receptors.

A new member of the integrin superfamily of adhesion receptors was isolated from human epithelial cells. Analogously to other integrins, this molecule is a heterodimer comprised of structurally unrelated subunits, both glycosylated. Unequivocal amino-acid sequence homologies were observed between these subunits and integrin alpha and beta chain sequences, indicating that this epithelial heterodimer is a novel integrin. No obvious serologic cross-reactivities were detected with other integrins. The beta chain of the epithelial integrin displayed a mol. wt significantly higher than other integrin beta chains, possibly due to a large sialic acid content. Integrin heterodimers are grouped into three families, based on which of three beta chains (beta 1, beta 2 and beta 3) they contain. Therefore, the epithelial integrin may represent the prototype of a fourth integrin family, because it contains a structurally distinct beta chain. The designation alpha E beta 4 is proposed for this novel human integrin.     

The lymphocyte metalloprotease MDC-L (ADAM 28) is a ligand for the integrin alpha4beta1.

The interaction of lymphocytes with other cells is critical for normal immune surveillance and response. MDC-L (ADAM 28), a member of the ADAM (a disintegrin and metalloprotease) protein family, is expressed on the surface of human lymphocytes. ADAMs possess a disintegrin-like domain similar in sequence to small non-enzymatic snake venom peptides that act as integrin antagonists. We report here that the disintegrin domain of MDC-L is recognized by the leukocyte integrin alpha(4)beta(1). Recombinant Fc fusion proteins possessing the disintegrin domain of MDC-L supported adhesion of the T-lymphoma cell line, Jurkat, in a concentration- and divalent cation-dependent manner. Adhesion of Jurkat cells to the disintegrin domain of MDC-L was inhibited by an anti-MDC-L monoclonal antibody (mAb), Dis1-1. The epitope for mAb Dis1-1 was localized within 59 residues of the disintegrin domain. Recombinant expression of this 59-residue fragment of the disintegrin domain also supported cell adhesion. Adhesion of Jurkat cells to the MDC-L disintegrin domain was specifically inhibited by anti-alpha(4) and anti-beta(1) function-blocking mAbs. Furthermore, adhesion of various cell lines to MDC-L correlated with expression of the integrin alpha(4)-subunit. Transfected K562 cells expressing alpha(4)beta(1) adhered to the disintegrin domain in contrast to non-transfected K562 cells. We further investigated the binding of recombinant MDC-L disintegrin domain (rDis-Fc) in solution. The rDis-Fc was found to bind to Jurkat cells in solution in a concentration-dependent and saturable manner. Both adhesion and solution binding of rDis-Fc was inhibited by the alpha(4)beta(1) ligand mimetic CS-1 peptide. Additionally, recognition of the MDC-L disintegrin domain required "activation" of lymphocyte beta(1) integrins. The interaction of MDC-L with alpha(4)beta(1) may potentially regulate metalloprotease function by targeting or sequestering the active protease on the cell surface. These results suggest a potential role for the lymphocyte ADAM, MDC-L, in the interaction of lymphocytes with alpha(4)beta(1)-expressing leukocytes.     

Structure-function analysis of the human integrin VLA-4 (alpha 4/beta 1). Correlation of proteolytic alpha 4 peptides with alpha 4 epitopes and sites of ligand interaction.

The structure-function relationship of the human integrin VLA-4 (alpha 4/beta 1; CD49d/CD29), has been studied in the human B-cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150-kDa non-proteolyzed form of the alpha 4 chain, which could be digested upon mild trypsin treatment to generate the 80- and 65-kDa proteolyzed forms, as well as alpha 4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55- and 50-kDa alpha 4 peptides. The trypsin-generated 80- and 65-kDa alpha 4 polypeptides, but not the 55- and 50-kDa fragments, were able to associate with the beta 1 chain. Distinct anti-VLA-4 mAb against four different alpha 4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150-kDa alpha 4 chain both associated or non-associated with the beta 1 chain. The alpha 4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti-alpha 4 mAb directed against the four different alpha 4 epitopes. On the other hand, the 55-kDa alpha 4 peptide was present in precipitates from anti-alpha 4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti-alpha 4 mAb specific for epitope B2. The different adhesive capacities of the VLA-4 integrin, namely the interaction with a 38-kDa fibronectin fragment containing the CS-1 region of plasma fibronectin (Fn-38), the binding to the vascular cell adhesion molecule-1 (VCAM-1), or the ability to mediate the anti-alpha 4-induced cell aggregation, were not altered on VLA-4 from cells upon mild trypsin treatment, when compared to non-treated cells. However, the 55- and 50-kDa alpha 4 forms generated by high-dose pronase cell treatment, failed to mediate cell interaction with Fn-38 or VCAM-1 ligands, and cell aggregation could not be triggered through VLA-4 under these conditions.     

Assembly and function of integrin receptors is dependent on opposing alpha and beta cytoplasmic domains.

The membrane proximal regions of integrin alpha and beta subunits are highly conserved in evolution. In particular, all integrin alpha subunits share the KXGFFKR sequence at the beginning of their cytoplasmic domains. Previous work has shown that this domain is important in integrin receptor assembly. Using chimeric integrin alpha and beta subunits, we show that the native cytoplasmic domains of both subunits must be present for efficient assembly. Most strikingly, chimeric alpha 1 and beta 1 subunits with reciprocally swapped intracellular domains dimerize selectively into collagen IV receptors expressed at high levels on the surface. However, these receptors, which bind ligand efficiently, are deficient in a variety of post-ligand binding events, including cytoskeletal association and induction of tyrosine phosphorylation. Furthermore, deletion of the distal alpha cytoplasmic domain in the swapped heterodimers leads to ligand-independent focal contact localization, which also occurs in wild-type subunits when the distal cytoplasmic domain is deleted. These results show that proper integrin assembly requires opposed alpha and beta cytoplasmic domains, and this opposition prevents ligand-independent focal contact localization. Our working hypothesis is that these two domains may associate during receptor assembly and provide the mechanism for integrin receptor latency.     

The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites

Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.     

A novel integrin beta subunit is associated with the vitronectin receptor alpha subunit (alpha v) in a human osteosarcoma cell line and is a substrate for protein kinase C.

We demonstrate that a novel integrin beta subunit is present in association with the vitronectin receptor (VNR) alpha subunit on the surface of MG-63 human osteosarcoma cells. This beta subunit and the glycoprotein IIIa beta subunit (beta 3) were both found complexed with VNR alpha on MG-63 cells and in at least two other human cell types we examined. Tryptic peptide mapping indicated that the two beta subunits are related but distinct. The novel beta chain, referred to here as beta s, was not recognized by the monoclonal antibody AP3, which recognizes GPIIIa, nor by an antiserum raised against a peptide from the COOH-terminal cytoplasmic domain of beta 3. Both receptor complexes bound to and were specifically eluted from a column containing the cell adhesion peptide GRGDSP. The unique beta subunit became phosphorylated at high stoichiometry when MG-63 cells or AG1523 human fibroblasts were treated with the phorbol-ester tumor promoter phorbol 12-myristate 13-acetate. This phosphorylation occurred mainly on serine and probably at one major site, as determined by phosphotryptic peptide mapping. Protein kinase C phosphorylated the beta s subunit of intact receptor in vitro, at the same site phosphorylated in treated cells, indicating that protein kinase C is likely to be responsible for this phosphorylation in vivo.     

Identification of a novel integrin beta subunit expressed on cultured monocytes (macrophages). Evidence that one alpha subunit can associate with multiple beta subunits.

The vitronectin receptor (VnR) is one member of a subset of cell adhesion receptors within the integrin supergene family which shares the beta 3 subunit (IIIa). We show here that the VnR is absent from the surface of monocytes freshly isolated from blood but is expressed on these cells after a period of in vitro culture. Such cultured monocytes (macrophages) from a patient with type I Glanzmann's thrombasthenia, however, failed to express the VnR. Instead, immunoprecipitation with a monoclonal antibody directed to the VnR alpha chain (alpha v) revealed a novel integrin comprising alpha v associated noncovalently with a 100-kDa beta subunit (beta 3b), immunologically unrelated to the VnR beta subunit (beta 3a). This same novel integrin complex was also identified on 10-day-old macrophages from healthy donors, but on these cells, the beta 3b subunit was co-expressed with the classical VnR complex of alpha v beta 3a. The novel beta 3b subunit was not identified by monoclonal or polyclonal antibodies to IIIa (beta 3a) nor by a monoclonal antibody to the classical VnR complex. The beta 3b subunit could be distinguished from beta 3a by its relatively greater migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction, by its distinct isoelectric point upon two-dimensional gel electrophoresis, and by one-dimensional peptide mapping. Neither platelets nor B lymphoblasts from this patient with Glanzmann's thrombasthenia expressed any VnR on their surface, whereas control cells from a normal donor expressed the classical VnR but not the beta 3b subunit. The two beta chains, and hence also the combined receptor complexes, appeared to be differentially regulated. These findings provide the first example of an integrin alpha chain complexed with more than a single beta chain in the same cell. Furthermore, the differential regulation of expression of the different beta subunits that associate with the VnR alpha chain on cultured monocytes suggests a role for the novel receptor complex during monocyte/macrophage differentiation.     

Potent cyclic monomeric and dimeric peptide inhibitors of VLA-4 (alpha4beta1 integrin)-mediated cell adhesion based on the Ile-Leu-Asp-Val tetrapeptide.

Potent monomeric and dimeric cyclic peptide very late antigen-4 (VLA-4) inhibitors have been designed based on a tetrapeptide (Ile-Leu-Asp-Val) sequence present in a 25-amino acid peptide (CS-1) reported in the literature. The peptides, synthesized by the SPPS techniques, were evaluated in the in vitro cell adhesion assays and in the in vivo inflammation models. The N- to C-terminal cyclic peptides such as cyclo(Ile-Leu-Asp-Val-NH-(CH2)2-S-(CH2)2-CO) (28) and cyclo(MeIle-Leu-Asp-Val-D-Ala-D-Ala) (31), monomeric and dimeric peptides containing piperazine (Pip) or homopiperazine (hPip) residues as linking groups, e.g. cyclo(MeIle-Leu-Asp-Val-Pip-CH2CO-NH-(CH2)2-S-CH2-CO) (49) and cyclo(MeIle-Leu-Asp-Val hPip-CH2CO-MeIle-Leu-Asp-Val-hPip-CH2CO) (58) and cyclic peptides containing an amide bond between the side chain amino group of an amino acid such as Lys and the C-terminal Val carboxyl group, e.g. Ac-cyclo(D-Lys-D-Ile-Leu-Asp-Val) (62) and beta-Ala-cyclo(D-Lys-D-Leu-Leu-Asp-Val) (68) were more potent than CS-1 in inhibiting the adhesion of the VLA-4-expressing MOLT-4 cells to fibronectin. The more potent compounds were highly selective and did not affect U937 cell adhesion to fibronectin (VLA-5), PMA-differentiated U937 cell adhesion to intercellular cell adhesion molecule- 1-expressing Chinese hamster ovary cells (LFA-1) and ADP-induced platelet aggregation (GPIIb/IIIa). A number of the more potent compounds inhibited ovalbumin-induced delayed type hypersensitivity in mice and some were 100-300 times more potent (ED50 = 0.003-0.009 mg/kg/day, s.c.) than CS-1. Two peptides, Ac-cyclo(D-Lys D-Ile-Leu-Asp-Val) (62) and cyclo(CH2CO-Ile-Leu-Asp-Val-Pip-CH2CO-Ile-Leu-Asp-Val-Pip) (55), were formulated in poly(DL-lactide-co-glycolide) depots and the release profile was investigated in vitro over a 30-day period.     

Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin alpha8beta1 in the embryonic kidney

The epithelial-mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin alpha8beta1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin alpha8beta1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, alpha8beta1-AP detects a novel ligand of 70-90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin alpha8beta1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas alpha8beta1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by alpha8beta1-AP and forms a complex with alpha8beta1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating alpha8beta1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.     

Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes

In human keratinocytes cultured in conditions which allow differentiation and stratification and are suitable to reconstitute a fully functional epidermis, alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were respectively polarized to the basal and lateral domains of the plasmamembrane both in growing colonies and in the reconstituted epidermis. Conversely, the alpha v integrin subunit, presumably in association with beta 5, was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4: (a) was organized in typical patches which often showed a "leopard skin" pattern where spots corresponded to microfilament-free areas; (b) was not associated with focal contacts containing vinculin and talin but rather corresponded to relatively removed contact areas of the basal membrane as shown by interference reflection microscopy; and (c) was coherent to patches of laminin secreted and deposited underneath the ventral membrane of individual cells. The two beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1), both endowed with laminin receptor properties, were not associated with focal adhesions under experimental conditions allowing full epidermal maturation but matched the lateral position of vinculin (but not talin), cingulin, and desmoplakin, all makers of intercellular junctions. Often thin strips of laminin were observed in between the lateral aspects of individual basal keratinocytes. The integrin complex alpha v beta 5 had a topography similar to that of talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The discrete topography of beta 1 and beta 4 integrins has a functional role in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion (De Luca, M., R. N. Tamura, S. Kajiji, S. Bondanza, P. Rossino, R. Cancedda, P. C. Marchisio, and V. Quaranta. Proc. Natl. Acad. Sci. USA. 87:6888-6892). Moreover, the surface polarization of integrins followed attachment and depended both on the presence of Ca2+ in the medium and on the integrity of the cytoskeleton. We conclude that our in vitro functional tests and structural data suggest a correlation between the pattern of integrin expression on defined plasmamembrane domains and the mechanism of epidermal assembly.     

Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin.

The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor alpha 4 beta 1. Using assays measuring the spreading of A375-SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH-terminal heparin-binding domain (HepII), but lacking CS1 and CS5, is completely sensitive to anti-alpha 4 and anti-beta 1 antibodies, suggesting that HepII contains a third alpha 4 beta 1-binding sequence. Examination of the primary structure of HepII revealed a sequence with homology to CS1. A 19mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment. H1-dependent adhesion was completely sensitive to anti-alpha 4 and anti-beta 1 antibodies. When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of H1 was found to be completely inhibitory. The active region of H1 was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CS1. Taken together, these results identify a novel peptide sequence in the HepII region of fibronectin that supports alpha 4 beta 1-dependent cell adhesion.     

Streptococcus plasmid pAM alpha 1 is a composite of two separable replicons, one of which is closely related to Bacillus plasmid pBC16.

A tetracycline resistance plasmid of Streptococcus faecalis, pAM alpha 1, is shown to contain two independent sets of replication functions, separated from each other on either side by short (300- to 400-base-pair) sequences of homology. The homologous sequences are oriented as direct repeats and therefore permit the dissociation of pAM alpha 1 into its component replicons, referred to here as pAM alpha 1 delta 1 and pAM alpha 1 delta 2, as the reciprocal products of a simple intramolecular recombination. pAM alpha 1 delta 1 is a 4.6-kilobase plasmid which carries the tet gene, and pAM alpha 1 delta 2 is a 5.1-kilobase plasmid which carries no known selectable marker. pAM alpha 1 delta 1 is shown to replicate efficiently in Bacillus subtilis and to confer tetracycline resistance on Bacillus hosts. We demonstrate by restriction mapping analysis that pAM alpha 1 delta 1 is virtually identical to a 4.6-kilobase tetracycline resistance plasmid of Bacillus cereus, pBC16, which is known to show extensive homology to plasmid isolates from Staphylococcus species (such as pUB110), as well as from other Bacillus species. The pAM alpha 1 delta 1-pBC16-pUB110 replicon thus exists naturally in at least three different gram-positive genera, indicating that these plasmids have a high degree of interspecific functional adaptability and supporting the view that plasmid DNA is commonly exchanged among many species of gram-positive bacteria in their natural environments.     

The major laminin receptor of mouse embryonic stem cells is a novel isoform of the alpha 6 beta 1 integrin

Laminin is the first extracellular matrix protein expressed in the developing mouse embryo. It is known to influence morphogenesis and affect cell migration and polarization. Several laminin receptors are included in the integrin family of extracellular matrix receptors. Ligand binding by integrin heterodimers results in signal transduction events controlling cell motility. We report that the major laminin receptor on murine embryonic stem (ES) cells is the integrin heterodimer alpha 6 beta 1, an important receptor for laminin in neurons, lymphocytes, macrophages, fibroblasts, platelets and other cell types. However, the cytoplasmic domain of the ES cell alpha 6 (alpha 6 B) differs totally from the reported cytoplasmic domain amino acid sequence of alpha 6 (alpha 6 A). Comparisons of alpha 6 cDNAs from ES cells and other cells suggest that the alpha 6 A and alpha 6 B cytoplasmic domains derive from alternative mRNA splicing. Anti-peptide antibodies to alpha 6 A are unreactive with ES cells, but react with mouse melanoma cells and embryonic fibroblasts. When ES cells are cultured under conditions that permit their differentiation, they become positive for alpha 6 A, concurrent with the morphologic appearance of differentiated cell types. Thus, expression of the alpha 6 B beta 1 laminin receptor may be favored in undifferentiated, totipotent cells, while the expression of alpha 6 A beta 1 receptor occurs in committed lineages. While the functions of integrin alpha chain cytoplasmic domains are not understood, it is possible that they contribute to transferring signals to the cell interior, e.g., by delivering cytoskeleton organizing signals in response to integrin engagement with extracellular matrix ligands. It is therefore reasonable to propose that the cellular responses to laminin may vary, according to what alpha subunit isoform (alpha 6 A or alpha 6 B) is expressed as part of the alpha 6 beta 1 laminin receptor. The switch from alpha 6 B to alpha 6 A, if confirmed in early embryos, could then be of striking potential relevance to the developmental role of laminin.     

Characterization of the integrin alpha 8 subunit: a new integrin beta 1-associated subunit, which is prominently expressed on axons and on cells in contact with basal laminae in chick embryos.

In this article, we describe the primary structure, the biochemical characterization and the tissue distribution of a novel integrin alpha subunit, named alpha 8. This subunit associates with the integrin beta 1 subunit to form alpha 8 beta 1 heterodimers. By sequence analysis, alpha 8 is more closely related to the alpha 5 and alpha v subunits than to other characterized integrin alpha subunits, but is clearly distinct from each of these. The alpha 8 subunit is expressed at moderate levels in several epithelial cells where its localization adjacent to basal laminae suggests that alpha 8-containing heterodimers interact with at least one extracellular matrix constituent. In embryos, the highest levels of alpha 8 protein expression are seen in the nervous system where alpha 8 is strongly expressed by several classes of projection neurons. The alpha 8 subunit is concentrated in axon tracts, including major projections in the spinal cord, optic system and auditory system. This tissue specific expression and cellular localization suggest that alpha 8-containing integrin receptors might promote axon outgrowth in the embryonic nervous system.     

Cloning, primary structure and properties of a novel human integrin beta subunit.

The originally described integrin beta subunits that define the three subfamilies of integrin heterodimers are beta 1, beta 2 and beta 3. In this paper, we describe the isolation of a cDNA coding for a novel human integrin beta subunit, designated as beta 5. The beta 5 cDNA was isolated from a human thymic epithelial cell library, using oligonucleotide probes that were designed from a region highly conserved among the known beta 1, beta 2 and beta 3 sequences. The beta 5 cDNA codes for 799 (or 796) amino acids, including a 23 amino acid leader sequence. There are 776 (or 773) amino acids in the mature protein, which includes a long extracellular domain of 696 amino acids, a transmembrane domain and an intracellular C-terminal domain of 57 amino acids. The beta 5 sequence resembled the known beta 3, beta 1 and beta 2 sequences by 55, 43 and 38%, respectively, including conservation of 56/56 cysteines. Rabbit antiserum was prepared against a 20 amino acid synthetic peptide predicted from the beta 5 C-terminal sequence. This serum immunoprecipitated a beta 5 protein that was 100,000 Mr (reduced) and 95,000 Mr (nonreduced). Only a single alpha subunit was detected in association with beta 5, and that alpha subunit was immunochemically indistinguishable from the alpha v subunit previously found as part of the vitronectin receptor complex. By immunoprecipitation, beta 5 was most prevalent on carcinoma cell lines, was also present on hepatoma and fibroblast cell lines, and was absent from lymphoblastoid cells and platelets.