Post-translational modifications of alpha5beta1 integrin by glycosaminoglycan chains. The alpha5beta1 integrin is a facultative proteoglycan

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.     

Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin

In contrast to sessile cell types, lymphocytes migrate through the vasculature to become diffusely distributed in tissues or organized in lymphoid structures. A complex array of adhesion molecules including selectins, integrins and their counter-receptors mediate lymphocyte homing and migration into tissues and may be constitutively expressed or induced. However, the molecules that mediate the tissue-specific retention of lymphocytes within the parenchyma have not been identified. Along the epithelium at the basolateral surface of enterocytes, intestinal intraepithelial lymphocytes are found. These T cells of the mucosal immune system serve as a model for the tissue-specific compartmentalization of lymphocytes. We investigated whether the localization of these intestinal intraepithelial lymphocytes could be mediated by specific interactions between adhesion molecules expressed selectively on this subpopulation of T cells and tissue-restricted adhesion molecules on epithelial cells. Here we show that heterotypic adhesive interactions between epithelial cells and intraepithelial lymphocytes in vitro are mediated by E-cadherin and the alpha E beta 7 integrin.     

Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis

Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120-kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM-1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and prolonged activation of VLA-5 may mediate subsequent interactions with the basement membrane and extracellular matrix.     

Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3

SUMMARY: Cellular invasion depends on cooperation between adhesive and proteolytic mechanisms. Evidence is provided that the matrix metalloproteinase MMP-2 can be localized in a proteolytically active form on the surface of invasive cells, based on its ability to bind directly integrin alpha v beta 3. MMP-2 and alpha v beta 3 were specifically colocalized on angiogenic blood vessels and melanoma cells in vivo. Expression of alpha v beta 3 on cultured melanoma cells enabled their binding to MMP-2 in a proteolytically active form, facilitating cell-mediated collagen degradation. In vitro, these proteins formed an SDS-stable complex that depended on the noncatalytic C-terminus of MMP-2, since a truncation mutant lost the ability to bind alpha v beta 3. These findings define a single cell-surface receptor that regulates both matrix degradation and motility, thereby facilitating directed cellular invasion.     

Transforming growth factor beta 1 suppresses transformation in hepatocytes by regulating alpha 1 beta 1 integrin expression

We previously reported that (a) treatment of the ras-transformed hepatocyte cell line NR4 with transforming growth factor (TGF) beta 1 suppresses many characteristics associated with the transformed phenotype including altered morphology, actin cytoskeleton reorganization, and anchorage-independent growth such that the cells more closely resemble the immortalized CWSV1 parent cell line; (b) transformed NR4 cells expressed significantly less alpha 1 integrin RNA than the immortalized CWSV1 cells; and (c) TGF-beta 1 treatment of NR4 cells stimulated the expression of alpha 1 and beta 1 integrin RNAs. In this report, the role of the alpha 1 beta 1 integrin in TGF-beta 1-mediated suppression of the ras-transformed phenotype was investigated. We determined that (a) the cell surface integrin that increased in response to TGF-beta 1 treatment of NR4 cells was alpha 1 integrin; (b) TGF-beta 1 altered the ability of NR4 cells to attach to collagen and laminin, the extracellular matrix components that interact with the alpha 1 beta 1 integrin receptor; (c) TGF-beta 1 treatment resulted in relocalization of the alpha 1 integrin on the NR4 cell surface; and (d) TGF-beta 1-mediated inhibition of anchorage-independent growth was blocked by the presence of alpha 1 integrin antibody. A cell line that overexpresses alpha 1 integrin was derived from NR4 cells; characterization of these cells indicated that they continued to express H-ras RNA but were less transformed than the parent NR4 cells. Specifically, they had an altered morphology, an organized actin cytoskeleton, and reduced ability to demonstrate anchorage-independent growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interrelationship of alpha4 integrin and matrix metalloproteinase-2 in the pathogenesis of experimental autoimmune encephalomyelitis

Previous studies have suggested that surface expression of alpha4 integrin by autoreactive T-cell clones is necessary for the clones to induce experimental autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis. To provide direct evidence for this phenomenon, we have transfected alpha4 integrin into C19alpha4-LO, a myelin basic protein-reactive T-cell clone that does not express alpha4 integrin and does not induce EAE when adoptively transferred into a susceptible mouse strain. Transfection of alpha4 integrin converted this clone to an alpha4 integrin-expressing clone that induced EAE. We then examined potential mechanisms by which alpha4 integrin may facilitate the disease process. C19 T-cell clones adhered equally to a monolayer of microvascular endothelial cells, regardless of level of alpha4 integrin expression. However, in contrast to T-cell clones that do not express alpha4 integrin, T-cell clones that express alpha4 integrin (endogenously or by transfection) transmigrated through an endothelial cell layer and subendothelial matrix at an enhanced rate and adhered to recombinant vascular cell adhesion molecule-1 (rVCAM-1) and the CS1 fragment of fibronectin, and after adhesion to these ligands, a matrix-degrading metalloproteinase (MMP-2) was induced and activated. The clones were also shown to constitutively express the membrane-type matrix metalloproteinase (MT1-MMP), an enzyme that activates MMP-2. GM6001 and UK-221,316, inhibitors of metalloproteinases, reduced alpha4 integrin-mediated transmigration and EAE induction by C19 T-cell clones. In addition, we studied a second EAE-inducing T-cell clone, MM4, which constitutively expresses alpha4 integrin and MMP-2. Engagement of alpha4 integrin on the MM4 clone up-regulated the expression and activation of MMP-2, without changing the expression of MT1-MMP. MMP inhibitors also reduced transmigration of and EAE induction by the MM4 T-cell clone. These studies demonstrate directly that expression of alpha4 integrin by autoreactive T-cell clones is required for adoptive transfer of EAE in this model. We also define a role for alpha4 integrin in the disease process in mediating the induction and coordinate activation of a matrix metalloproteinase (MMP-2), which facilitates T-cell transmigration.     

VCAM-1 is internalized by a clathrin-related pathway in human endothelial cells but its alpha 4 beta 1 integrin counter-receptor remains associated with the plasma membrane in human T lymphocytes

Lymphocyte extravasation involves a step(s) of de-adhesion to allow trans- and subendothelial migration in response to inflammatory signals. We show here that ligated VCAM-1 was rapidly internalized (t1/2 14.5 min) in ECV 304 endothelial cells and in TNF-alpha-primed human umbilical vein-derived endothelial cells (t1/2 11.2 min). The process required energy (ATP), intracellular Ca2+, an intact cytoskeletal network and active protein kinases. The internalization of VCAM-1 involved a clathrin-dependent pathway based on the observations that 1) it was inhibited in cells treated with lysosomotropic agents or with a hypertonic concentration of sucrose, and 2) internalized VCAM-1 colocalized with clathrin. In contrast, the cross-linked alpha 4 beta 1 integrin counter-receptor of VCAM-1 remained associated with the plasma membrane of purified peripheral T and Jurkat cells. Our results suggest a model where VCAM-1 would initially participate in the retention of T cells to the endothelium by binding alpha 4 beta 1 integrin. Lymphocyte de-adhesion would be facilitated as a result of the internalization of VCAM-1. The persistent cell surface expression of alpha 4 beta 1 integrin would allow the migrating T cells to interact with and receive signal(s) from its fibronectin ligand of the extracellular matrix.     

Dual control by divalent cations and mitogenic cytokines of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity expressed by human hemopoietic cells

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function alpha 4 beta 1 and alpha 5 beta 1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by "inside-out" mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn2+, Mg2+ and Ca2+ for alpha 4 beta 1 and alpha 5 beta 1 activation by "inside-out" mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-beta 1 integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of beta 1 integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, alpha 4 beta 1 and alpha 5 beta 1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca2+, Mg2+ or Mn2+ were able to restore adhesive functions of alpha 4 beta 1 and alpha 5 beta 1 when activated by 8A2, only Mg2+ and Mn2+ were able to support activation of alpha 4 beta 1 and alpha 5 beta 1 by cytokines. Furthermore, high concentrations of Ca2+ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn2+ and cytokines but not by 8A2. On the contrary, in the presence of both Ca2+ and Mg2+, Mn2+ had an additive effect on the activation of alpha 4 beta 1 and alpha 5 beta 1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca2+, Mg2+ and Mn2+ may modulate in vivo alpha 4 beta 1 and alpha 5 beta 1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.     

Adhesion-activating phorbol ester increases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes

Lymphocytes activate adhesion to intracellular adhesion mlecule 1 (ICAM-1) via leukocyte function associated antigen 1 (LFA-1), their major beta 2 integrin, in response to PMA (phorbol 12-myristate 13-acetate) without an increase in the number of receptors expressed. The molecular details of the mechanism are unknown. To determine the effect of PMA activation on LFA-1 movement within the plasma membrane, we used the single particle tracking technique to measure the diffusion rate of LFA-1 molecules on EBV-transformed B cells before and after PMA activation. Diffusion of LFA-1 on unactivated cells was restricted compared to CR1 (CD35), another transmembrane protein of equivalent size. PMA caused a 10-fold increase in the diffusion rate of LFA-1 without any effect on CD35. The increased LFA-1 motion induced by PMA was random, not directed, indicating that it was due to a release of constraints rather than the application of forces. The diffusion rates of LFA-1 are consistent with cytoskeletal attachment before and free diffusion after PMA. Cytochalasin D led to an equivalent increase in mobility and, at low doses, stimulated adhesion, implying that the nonadhesive state of LFA-1 is actively maintained by the lymphocyte cytoskeleton.     

Specific regulation of VLA-4 and alpha 4 beta 7 integrin expression on human activated T lymphocytes

Modulation of VLA integrins was studied in several human T cell clones upon specific and nonspecific cellular activation. Human activated T lymphocytes down-regulated both alpha 4 beta 1 and alpha 4 beta 7 integrins upon specific recognition of alloantigens (cytotoxic T cells) or in the presence of Staphylococcus enterotoxin B (superantigen recognizing noncytotoxic T cells). In contrast, the expression of other membrane integrins, such as VLA-1 and VLA-5 integrins, was not modified. Down-regulation of alpha 4 beta 1 and alpha 4 beta 7 integrins was observed as early as 3 h after stimulation, lasted later than 72 h and was partially inhibited by cytochalasin D. Interestingly, neither target cells nor NK cells modulated CD49d expression after interaction with T cells of K562, respectively, suggesting that CD49d expression was linked to specific T cell activation. The down-regulation of the CD49d chain in T cell clones stimulated with immobilized anti-CD3 mAbs confirmed the role of TCR-mediated activation in CD49d regulation. However, the CD3-independent cellular aggregation induced by soluble anti-CD43 mAb was also able to strongly down-regulate alpha 4 beta 1 and alpha 4 beta 7. The present work shows the first evidence that CD49d subunit-bearing integrin expression is distinctly regulated from other integrins after Ag or superantigen recognition by human activated T cells. CD49d modulation may be relevant for the traffic and tissue localization of locally activated T cells during immune responses.     

Intracerebral interleukin-1beta impairs response to tumor invasion: involvement of adrenal catecholamines

Interleukin-1beta (IL-1beta) is released within the brain following stress, trauma, infection, and in specific brain disorders. This centrally acting IL-1beta has recently been shown to impair peripheral immunity. Central administration of IL-1beta suppresses natural killer (NK) cell activity impairs lung clearance of tumor cells and enhances tumor colonization. Using an in vivo model of tumor colonization (lung clearance of NK-sensitive MADB106 adenocarcinoma cells), this study examined the role of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS) in mediating these effects. We demonstrate that adrenalectomy significantly attenuated the impaired lung clearance of MADB106 tumor cells induced by intracerebroventricular (i.c.v.) administration of IL-1beta (20 ng). Supplementing adrenalectomized animals with corticosterone did not reinstate the effect. The effect of IL-1beta on lung clearance was blocked by pretreatment with the beta-adrenergic antagonist, nadolol (0.5 mg/kg), but not by the alpha-antagonist phentolamine (5 mg/kg). Peripheral noradrenergic pathways are not implicated given that systemic administration of the noradrenergic neurotoxin, 6-hydroxydopamine, did not block the effect of IL-1beta. Taken together, these findings indicate that IL-1beta impairs lung clearance of MADB106 tumor cells via the actions of adrenal catecholamines, most likely epinephrine, acting at beta-adrenergic receptors in the periphery.     

Cross-talk between insulin receptor and integrin alpha5 beta1 signaling pathways

The ligation and clustering of cell surface alphabeta heterodimeric integrins enhances cell adhesion and initiates signaling pathways that regulate such processes as cell spreading, migration, differentiation, proliferation and apoptosis. Here we show that insulin treatment of Chinese hamster ovary cells expressing insulin receptors (CHO-T) markedly promotes cell adhesion onto a fibronectin matrix, but not onto bovine serum albumin or poly-lysine. Incubation of cells with a GRGDSP peptide that specifically binds integrins (but not the nonspecific GRADSP peptide) abolishes this insulin effect, as does the potent phosphoinositide 3-kinase (PI 3-kinase) inhibitor wortmannin. Moreover, a specific blocking monoclonal anti-alpha5beta1 integrin antibody, PB-1, blocks insulin-stimulated cell adhesion onto fibronectin. Conversely, activating alpha5beta1 integrins on CHO-T cells by adherence onto fibronectin markedly potentiates the action of insulin to enhance insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation. Activation of alpha5beta1 integrin also markedly potentiates the recruitment of p85-associated PI 3-kinase activity to IRS-1 in response to submaximal levels of insulin in CHO-T cells. These data indicate that insulin potently activates integrin alpha5beta1 mediated CHO-T cell adhesion, while integrin alpha5beta1 signaling in turn enhances insulin receptor kinase activity and formation of complexes containing IRS-1 and PI 3-kinase. These findings raise the hypothesis that insulin receptor and alpha5beta1 integrin signaling act synergistically to enhance cell adhesion.     

Two-stage activation for alpha5beta1 integrin binding to surface-adsorbed fibronectin

By analyzing the functional binding of alpha5beta1 integrin to adsorbed fibronectin in intact cells, we demonstrate that integrin activation results in linear increases in adhesion strength as a function of ligand density, suggesting that modulation of the receptor-ligand interaction is the dominant mechanism for adhesion during the initial stages of adhesion and that cooperative binding contributes little to initial adhesion strength. Using this experimental framework, we show the existence of three distinct activation states for alpha5beta1 integrin binding to adsorbed fibronectin for both passive, antibody-induced and active, cell-controlled activation. During the initial phase of adhesion, alpha5beta1 integrin is activated in an energy-dependent process from the nonbinding ground state to an intermediate state in which the receptor binds fibronectin and provides significant mechanical coupling. In later stages of adhesion maturation, alpha5beta1 integrin is activated to a higher binding state, which provides significant increases in adhesion strength compared with the intermediate state. These multiple binding states most likely result from different integrin conformations and reflect distinct interactions between alpha5beta1 and sites on adsorbed fibronectin. Multiple activation states for alpha5beta1 suggest the existence of distinct stages in adhesion signaling and strengthening and can provide a versatile mechanism for the regulation of adhesive interactions.     

Re-expression of the alpha 2 beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells

To assess the role of altered alpha 2 beta 1 integrin expression in breast cancer, we expressed the alpha 2 beta 1 integrin de novo in a poorly differentiated mammary carcinoma that expressed no detectable alpha 2-integrin subunit. Expression of the alpha 2 beta 1 integrin resulted in a dramatic phenotypic alteration from a fibroblastoid, spindle-shaped, non-contact-inhibited, motile, and invasive cell to an epithelioid, polygonal-shaped, contact-inhibited, less motile, and less invasive cell. Although expression of the alpha 2 subunit did not alter adhesion to collagen, it profoundly altered cell spreading. Re-expression of the alpha 2 beta 1 integrin restored the ability to differentiate into gland-like structures in three-dimensional matrices and markedly reduced the in vivo tumorigenicity of the cells. These results indicate that the consequences of diminished alpha 2 beta 1-integrin expression in the development of breast cancer and, presumably, of other epithelial malignancies are increased tumorigenicity and loss of the differentiated epithelial phenotype.     

Neural crest cell interactions with laminin: structural requirements and localization of the binding site for alpha 1 beta 1 integrin

We have identified the sites of neural crest cell interaction with laminin in vitro by examining their ability to attach to and migrate on proteolytic fragments of the molecule and the ability of fragment-specific antibodies to inhibit these interactions. The binding site on laminin was localized to the E8 domain on the long arm of laminin, as well as the T8' fragment within this domain, but not the E1', E3, or E4 fragments. Only subfragments containing the carboxy-terminal rod-like portion of the A chain plus the corresponding B1 and B2 chains retained the attachment-promoting activity of the parent E8 fragment. In addition, interactions required maintenance of the triple-stranded and alpha-helical coiled-coil structure of this domain. Reduction and alkylation of laminin and the E8 and T8 fragments significantly reduced neural crest cell attachment and migration. An antiserum against chick alpha 1 integrin reduced migration and adhesion of neural crest cells on an intact laminin-nidogen complex, the E8 fragment, and all its active subfragments. Furthermore, we observed that neural crest cells modified laminin substrata prepared in the absence of divalent cations. Early stable attachment to these substrata was mediated by an integrin other than alpha 1, whereas later attachment and migration were mediated by alpha 1 integrins. Our results suggest that neural crest cells selectively bind to the B1-A-B2 mid-portion (T8') of the E8 domain of laminin, requiring structural integrity of this region and that they modify laminin substrata as a result of prolonged cell-matrix interactions.     

The serpin PAI-1 inhibits cell migration by blocking integrin alpha V beta 3 binding to vitronectin

During wound healing, migrating cells increase expression of both the vitronectin receptor (VNR) integrins and plasminogen activators. Here we report that vitronectin significantly enhances the migration of smooth muscle cells (SMCs), and that the specific VNR alpha V beta 3 is required for cell motility. We also show that the alpha V beta 3 attachment site on vitronectin overlaps with the binding site for plasminogen activator inhibitor (PAI)-1, and that the active conformation of PAI-1 blocks SMC migration. This effect requires high-affinity binding to vitronectin, and is not dependent on the ability of PAI-1 to inhibit plasminogen activators. Formation of a complex between PAI-1 and plasminogen activators results in loss of PAI-1 affinity for vitronectin and restores cell migration. These data demonstrate a direct link between plasminogen activators and integrin-mediated cell migration, and show that PAI-1 can control cell-matrix interactions by regulating the accessibility of specific cell-attachment sites. This indicates that the localization of plasminogen activators at sites of focal contact does not initiate a proteolytic cascade leading to generalized matrix destruction, but instead is required to expose cryptic cell-attachment sites necessary for SMC migration.     

Modeling the alpha IIb beta 3 integrin solution conformation

The alpha IIb beta 3 platelet integrin is the prototypical member of a widely distributed class of transmembrane receptors formed by the noncovalent association of alpha and beta subunits. Electron microscopic (EM) images of the alpha IIb beta 3 complex show an asymmetric particle with a globular domain from which two extended regions protrude to contact the lipid bilayer. Distance constraints provided by disulfide bond patterns, epitope mapping, and ligand mimetic cross-linking studies rather suggest a somewhat more compact conformation for the alpha IIb beta 3 complex. We have studied the shape of detergent-solubilized alpha IIb beta 3 by employing a low-resolution modeling procedure in which each polypeptide has been represented as an array of interconnected, nonoverlapping spheres (beads) of various sizes. The number, size, and three-dimensional relationships among the beads were defined either solely by dimensions obtained from published EM images of integrin receptors (EM models, 21 beads), or solely by interdomain constraints derived from published biochemical data (biochemical model, 37 beads). Interestingly, although no EM data were employed in its construction, the resulting overall shape of the biochemical model was still compatible with the EM data. Both kinds of models were then evaluated for their calculated solution properties. The more elongated EM models have diffusion and sedimentation coefficients that differ, at best, by +2% and -18% from the experimental values, determined, respectively, in octyl glucoside and Triton X-100. On the other hand, the parameters calculated for the more compact biochemical model showed a more consistent agreement with experimental values, differing by -7% (octyl glucoside) to -6% (Triton X-100). Thus, it appears that using the biochemical constraints as a starting point has resulted in not only a more detailed model of the detergent-solubilized alpha IIb beta 3 complex, where the relative spatial location of specific domains the size of 5-10 kDa can be tentatively mapped, but in a model that can also reconcile the electron microscopy with the biochemical and the solution data.     

In situ distribution of integrin alpha 2 beta 1 and alpha-actinin in melanocytic proliferations

Integrin alpha 2 beta 1 is a transmembrane protein receptor for collagen and laminin previously reported as a melanoma tumor progression antigen. alpha-Actinin is an actin-binding protein reported to interact with the cytoplasmic domain of the beta 1-integrin chain of alpha 2 beta 1. In vitro, both alpha 2 beta 1 and alpha-actinin play a role in melanoma cell motility. In turn, increased melanoma cell line motility (measured as mean migration rates), correlates with metastasis. To determine the in situ distribution of these proteins, we used monoclonal antibodies directed against the alpha 2-integrin subunit of alpha 2 beta 1 and alpha-actinin on frozen sections of 33 melanocytic proliferations, which included dermal nevi, primary melanomas, and metastatic melanomas. We found that the superficial portion of all of the melanocytic proliferations tested stained for alpha-actinin. In benign nevi and superficial spreading melanoma, there was a notable loss of staining for alpha-actinin in the cells in the deep reticular dermis. In contrast, alpha-actinin was present on almost all of the tumor cells in the nodular melanomas and the melanoma metastases. Tumors stained either uniformly positive or uniformly negative for alpha 2 beta 1; the expression of this protein correlated with the later stages of melanoma progression. Our findings suggest that alpha-actinin protein levels initially decrease and then increase during melanocytic tumor progression, whereas the alpha 2 subunit protein appears in the later stages of melanoma progression. The variable distribution of these proteins is evidence for the differential adhesive and motile properties of subpopulations of cells in melanocytic proliferations.     

Identification of ligand binding sites on integrin alpha4beta1 through chemical cross-linking

We have used chemical cross-linking to identify sequences in integrin alpha4beta1 that are involved in its interactions with ligands. A recently described leucine-aspartic acid-valine (LDV)-based small molecule inhibitor of alpha4beta1 (BIO-1494), that contained a single reactive amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ability to block the interaction with a competitive inhibitor lacking the reactive group, (ii) the absolute requirement of divalent cations for cross-linking, and (iii) the lack of cross-linking to the functionally related integrin alpha4beta7. With ANB-NOS as the cross-linker, only the beta1 chain was labeled with BIO-1494, while with the more flexible cross-linker DSS both the alpha4 and beta1 chains were modified. Similar results were obtained when cross-linking was performed on K562 cells expressing alpha4beta1 but not on K562 cells expressing alpha2beta1. The site of cross-linking on the beta1 chain was localized by CNBr peptide mapping within residues 130-146, a region that contains the putative metal binding site DXSXS and for which analogous data had been generated with RGD binding to integrin alphaIIbbeta3. The striking similarity between the data we generated for an LDV ligand and published data for the RGD family supports the notion of a common ligand binding pocket formed by both integrin chains. The cross-linking strategy developed here should serve as a useful tool for studying alpha4beta1 function.