The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion.

DOCK proteins are guanine nucleotide exchange factors for Rac and Cdc42 GTPases. DOCK1 is the founding member of the family and acts downstream of integrins via the canonical Crk-p130Cas complex to activate Rac GTPases in numerous contexts. In contrast, DOCK5, which possesses the greatest similarity to DOCK1, remains sparingly studied. Here we establish that DOCK5 has a non-redundant role in regulating motile and invasive capacities of epithelial cells. DOCK1 is constitutively associated with sites of integrin attachment termed focal adhesions (FAs). In contrast, we demonstrate that DOCK5 recruitment to FAs in Hela cells is restricted by GIT2, an established regulator of FA signaling. We determine that GIT2 is targeted to FAs in response to Rho-ROCK signaling and actomyosin contractility. Accordingly, inhibition of ROCK activity or MLC function promotes enrichment of DOCK5 in membrane protrusions and nascent cell-substratum adhesions. We further demonstrate that GIT2 inhibits the interaction of DOCK5 with Crk. Moreover, we show that depletion of GIT2 promotes DOCK5-dependent activation of the Crk-p130Cas signaling cascade to promote Rac1-mediated lamellipodial protrusion and FA turnover. The antagonism between GIT2 and DOCK5 extends to non-transformed MCF10A mammary epithelial cells, with DOCK5 'dialing-up' and GIT2 'dialing-down' invasiveness. Finally, we determine that DOCK5 inhibition attenuates invasion and metastasis of MDA-MB-231 cells and prolongs life span of mice injected with these cells. Collectively, our work identifies DOCK5 as a key regulator of epithelial invasion and metastasis, and demonstrates that suppression of DOCK5 by GIT2 represents a previously unappreciated mechanism for coordination of Rho and Rac GTPases.

Quantification of tissue inhibitor of metalloproteinases 2 in plasma from healthy donors and cancer patients.

Tissue inhibitor of metalloproteinases (TIMP)-2 is a highly conserved molecule, which binds both active and latent matrix metalloproteinase (MMP)-2. TIMP-2 is also involved in the activation of MMP-2 on the cell surface. A quantitative enzyme-linked immunosorbent assay (ELISA) was established and optimized for measurement of TIMP-2 in plasma. The capturing antibody in the ELISA was a monoclonal, while the detecting antibody was a chicken polyclonal antibody recognizing the native form of human TIMP-2. The levels of TIMP-2 were measured in ethylenediaminetetraacetic acid (EDTA) and citrate plasma from healthy donors. The median values were determined as 163 ng/ml (n = 186) with a range of 109-253 ng/ml for EDTA plasma and 139 ng/ml (n = 77) with a range of 95-223 ng/ml for citrate plasma. The TIMP-2 concentration in citrate plasma from 15 patients with advanced, stage IV breast cancer had a median value of 160 ng/ml, only slightly higher but statistically distinguishable from the level found in citrate plasma from the healthy donors. In addition, the TIMP-2 concentration in EDTA plasma from colorectal cancer patients revealed a significantly higher level in plasma from patients with Dukes stage A (P = 0.01) compared with patients with more advanced Dukes stages.

The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis.

The urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) is a newly discovered member of the macrophage mannose receptor family that was reported to interact with ligand-bound urokinase plasminogen activator receptor (uPAR), matrix metalloprotease-13 (MMP-13), and collagen V on the cell surface. We have determined the sites of expression of this novel receptor during murine postimplantation development. uPARAP/Endo180 was expressed in all tissues undergoing primary ossification, including the developing bones of the viscerocranium and calvarium that ossify intramembranously, and developing long bones undergoing endochondral ossification. uPARAP/Endo180 mRNA was expressed by both immature osteoblasts and by mature osteocalcin-producing osteoblasts-osteocytes, and was coexpressed with MMP-13. Interestingly, osteoblasts also expressed uPAR. Besides bone-forming tissues, uPARAP/Endo180 expression was detected only in a mesenchymal condensation of the midbrain and in the developing lungs. The data suggest a function of this novel protease receptor in bone development, possibly mediated through its interactions with uPAR, MMP-13, or collagen V.

Identification of two estrogen regulated genes associated with growth regulation of human breast cancer.

We have identified two estrogen regulated gene products in the E(2) growth inhibited human breast cancer xenograft, T61; one showing 100% homology to the human BAC clone RP11-112E16, the other 100% homology to the human CPR3/DNJ3 gene. Verification by Northern blot analyses showed an up-regulation of the BAC clone RP11-112E16 and the CPR3/DNJ3 mRNAs upon E(2) treatment. Treatment of T61 tumors with tamoxifen, leading to static tumor growth, also increased the expression of the BAC clone RP11-112E16 and the CPR3/DNJ3 mRNAs. A similar association between growth inhibition and BAC clone RP11-112E16 and CPR3/DNJ3 mRNA induction was observed in MCF-7 cells treated with ICI 182.780. In MCF-7 cells, treatment with E(2) resulted in growth stimulation concomitant with a decrease in the BAC clone RP11-112E16 and CPR3/DNJ3 mRNA expression. Treatment with a combination of E(2) and ICI 182.780 abolished the anti-estrogen induced increase in BAC clone RP11-112E16 and CPR3/DNJ3 mRNA expression, indicating that regulation of the gene products is mediated through the ER. The association between growth inhibition and BAC clone RP11-112E16 or CPR3/DNJ3 mRNA expression was supported by high expression of both gene products in brain tissue. Further investigations are ongoing to clarify the biological function of these two gene products.

The urokinase receptor associated protein (uPARAP/endo180): a novel internalization receptor connected to the plasminogen activation system.

The urokinase-mediated plasminogen activation system plays a central role in the extracellular proteolytic degradation reactions in cancer invasion. In this review article we discuss a number of recent findings identifying a new cellular receptor protein, uPARAP, that interacts with components of this proteolytic system. uPARAP is a high molecular weight type-1 membrane protein, belonging to the macrophage mannose receptor protein family. On the surface of certain cells, uPARAP forms a ternary complex with the pro-form of the urokinase-type plasminogen activator (uPA) and its primary receptor (uPAR). While the biological consequences of this reaction have not yet been verified experimentally, a likely event is ligand internalization because uPARAP is a constitutively recycling internalization receptor. uPARAP also binds at least one component, collagen type V, in the extracellular matrix meshwork, pointing to a potential role in proteolytic substrate presentation. Additional ligands have been proposed, including collagenase-3 and glycoproteins capable of interacting with one of the multiple carbohydrate recognition-type domains of uPARAP. In various adult tissues uPARAP is present on fibroblasts, macrophages and a subset of endothelial cells. In fetal tissues the protein has also been demonstrated in certain bone forming regions. Hypotheses on the physiological function of uPARAP include regulatory roles in extracellular proteolysis. This type of function would be likely to direct the local turnover of proteases and their substrate degradation products and thus may add to the complicated interplay between several cell types in governing restricted tissue degradation.

Differential binding of urokinase and peptide antagonists to the urokinase receptor: evidence from characterization of the receptor in four primate species.

The urokinase plasminogen activator receptor (uPAR) is a membrane protein active in localizing the plasminogen activation cascade system on the cell surface. The resulting pericellular proteolytic activity is responsible for degradation reactions in the extracellular matrix that are needed for the invasion of cancer cells, thus making uPAR a potential target for anti-invasive therapy based on binding antagonists. A remarkable property of the uPA-uPAR system is a pronounced species specificity in ligand recognition. We have now cloned and studied uPAR from four primate species and show that even though these sequences contain very few substitutions relative to the human uPAR, the receptor protein products differ markedly in terms of ligand selectivity. Thus, a well described competitive peptide antagonist directed against the human uPAR reacts with only one of the monkey receptors (chimpanzee uPAR), in spite of the fact that uPAR from all of the four species cross-reacts with human uPA. Notably, uPAR from African green monkey, which is completely devoid of reactivity with the peptide, contains only three substitutions relative to chimpanzee uPAR in the molecular regions critical for binding. These findings aid the elucidation of the structure/function relationship of uPAR and, unexpectedly, identify a structural distinction governing the binding of uPA and a very similar peptide antagonist.

A urokinase receptor-associated protein with specific collagen binding properties.

The plasminogen activation cascade system, directed by urokinase and the urokinase receptor, plays a key role in extracellular proteolysis during tissue remodeling. To identify molecular interaction partners of these trigger proteins on the cell, we combined covalent protein cross-linking with mass spectrometry based methods for peptide mapping and primary structure analysis of electrophoretically isolated protein conjugates. A specific tri-molecular complex was observed upon addition of pro-urokinase to human U937 cells. This complex included the urokinase receptor, pro-urokinase, and an unknown, high molecular weight urokinase receptor-associated protein. The tryptic peptide mixture derived from a cross-linked complex of pro-urokinase and the latter protein was analyzed by nanoelectrospray tandem mass spectrometric sequencing. This analysis identified the novel protein as the human homologue of a murine membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices.

Differential gene expression in human mammary carcinoma cells: identification of a new member of a receptor family.

Differential gene expression in mammary carcinoma cell lines MCF-7 and MCF-7ADR was investigated by Differential Display Technique. These two cell lines represent a model system for progression of mammary carcinoma: MCF-7 is estradiol-dependent for growth, estrogen-receptor positive, tamoxifen responsive, vimentin negative, Adriamycin sensitive and not invasive in vitro or in vivo while MCF-7ADR is estradiol-independent for growth, estrogen-receptor negative, tamoxifen resistant, vimentin positive, Adriamycin resistant and invasive in vitro and in vivo. Here we describe the identification of a new receptor expressed exclusively in MCF-7ADR. The receptor covers 157 amino acids with a predicted topology of four transmembrane and two extracellular domains. Additional members of the gene family are: peripheral membrane protein PMP22, epithelial membrane protein EMP-1 and squamous cell differentiation marker CL-20.