Invadopodia are membrane extensions of aggressive tumor cells that function in the activation of membranebound proteases occurring during tumor cell invasion. We explore a novel and provocative activity of integrins in docking proteases to sites of invasion, termed invadopodia. In the absence of collagen, ␣ 3 ␤ 1 integrin and the gelatinolytic enzyme, seprase, exist as nonassociating membrane proteins. Type I collagen substratum induces the association of ␣ 3 ␤ 1 integrin with seprase as a complex on invadopodia. The results show that ␣ 3 ␤ 1 integrin is a docking protein for seprase to form functional invadopodia. In addition, ␣ 5 ␤ 1 integrin may participate in the adhesion process necessary for invadopodial formation. Thus, ␣ 3 ␤ 1 and ␣ 5 ␤ 1 integrins play major organizational roles in the adhesion and formation of invadopodia, promoting invasive cell behavior.The integrin family of transmembrane adhesion proteins has been shown to exhibit multiple functions, including adhesion to extracellular matrix (ECM), 1 cytoskeleton organization, and signal transduction (1-4). Because integrin and integrin-associated molecules are enriched at membrane protrusions called invadopodia (5-10), we hypothesized that integrins may also be involved in recruiting proteases to these sites of cell invasion. In support of this hypothesis, the ␣ v ␤ 3 integrin has been shown to modulate ECM proteolytic activities by recruiting a major soluble protease, matrix metalloproteinase-2, to the cell surface (11). Moreover, both adhesive and signaling activities of integrins can be regulated by the interaction between integrins and the urokinase plasminogen activator/receptor (12). We have shown that in LOX melanoma cells, a 170-kDa membrane gelatinase, seprase, was localized to invadopodia and associated with the invasive phenotype (13)(14)(15)(16). Sequencing data on the 97-kDa protein subunit of seprase indicates only a short (six) amino acid sequence at the cytoplasmic amino terminus (14), suggesting that seprase localization at invadopodia may be dependent upon other membrane proteins such as integrins. Here, we show immunoprecipitation, immunofluorescence, and cell surface cross-linking experiments demonstrating that seprase and ␣ 3 ␤ 1 integrin associate at invadopodia in a collagendependent manner. EXPERIMENTAL PROCEDURESAntibodies and Immunofluorescence Labeling-Anti-seprase monoclonal antibodies (mAbs) D28 and D8 have previously been described (14, 15). Anti-␤ 1 polyclonal (number 3847) and mAb 13 antibodies were used to detect ␤ 1 integrins (17), and anti-vitronectin receptor antibodies was used to detect the ␤ 3 subunit of the vitronectin receptor (Life Technologies, Inc.).Anti-␣ 2 integrin (mouse mAb clone P1E6), anti-␣ 3 integrin (mouse mAb clone P1B5, both from Becton and Dickinson Immunocytometry Systems, San Jose, CA and Telios, San Diego, CA), and rat anti-␣ 6 (clone GoH3, Serotec Inc, Partners, Raleigh, NC) were used to perform immunoprecipitation. Anti-␣ 5 mAb 11 was used to detect ␣ 5 ␤ 1 integrin (17). Anti-placental...

The 170-kDa membrane-bound gelatinase, seprase, is a cell surface protease, the expression of which correlates with the invasive phenotype of human melanoma and carcinoma cells. We have isolated seprase from cell membranes and shed vesicles of LOX human melanoma cells. The active enzyme is a dimer of N-glycosylated 97-kDa subunits. Sequence analysis of three internal proteolytic fragments of the 97-kDa polypeptide revealed up to 87.5% identity to the 95-kDa fibroblast activation protein ␣ (FAP␣), the function of which is unknown. Thus, we used reverse transcription-polymerase chain reaction to generate a 2.4-kilobase cDNA from LOX mRNA with FAP␣ primers. COS-7 cells transfected with this cDNA expressed a 170-kDa gelatinase that is recognized by monoclonal antibodies directed against seprase. Sequence analysis also showed similarities to the 110-kDa subunit of dipeptidyl peptidase IV (DPPIV). Like DPPIV, the gelatinase activity of seprase was completely blocked by serine-protease inhibitors, including diisopropyl fluorophosphate. Seprase could be affinitylabeled by [ 3 H]diisopropyl fluorophosphate, but the proteolytically inactive 97-kDa subunit could not, confirming the existence of a serine protease active site on the dimeric form. Proteolytic activity is lost upon dissociation into its 97-kDa subunit following treatment with acid, heat, or cysteine and histidine-modifying agents. We conclude that seprase, FAP␣, and DPPIV are related serine integral membrane proteases and that seprase is similar to DPPIV, the proteolytic activities of which are dependent upon subunit association.Seprase was originally identified from a human malignant melanoma cell line LOX, which exhibited aggressive behavior in experimental metastasis (1, 2). It is a membrane glycoprotein with gelatinase activity that is expressed and localized at the invasion front during invasion into the ECM 1 by human melanoma, breast carcinoma cells, and chicken embryo fibroblasts transformed by Rous sarcoma virus (3-6). The protease is a hydrophobic glycoprotein soluble in Triton X-100 and in SDS (3). Seprase shows gelatinolytic activity as demonstrated by gelatin zymography (3,4). The enzyme maintains maximal activity at neutral pH, which can be further enhanced by SDS buffer, EDTA, the cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane and dithiothreitol. However, seprase has a peculiar protease inhibitor profile; it is inhibited by the protease inhibitors PMSF and NEM (3). A major problem in purifying seprase was the apparent low level of this protein in cultured cell lines. Because LOX cells produced a large quantity of membrane vesicles that were enriched in seprase, we isolated seprase from the cell membranes and shed vesicles in LOX conditioned medium and characterized its subunit composition and its enzymatic activity. We found that the protease had an apparent molecular mass of 170 kDa composed of proteolytically inactive 97-kDa subunits. Amino acid analysis of internal proteolytic fragments of the 97-kDa polypep...

Seprase is a homodimeric 170 kDa integral membrane gelatinase whose expression correlates with the invasiveness of the human melanoma cell line LOX. Here, we report the molecular cloning of a cDNA that encodes the 97 kDa subunit of seprase. Its deduced amino acid sequence predicts a type II integral membrane protein with a cytoplasmic tail of 6 amino acids, followed by a transmembrane domain of 20 amino acids and an extracellular domain of 734 amino acids. The carboxyl terminus contains a putative catalytic region (approximately 200 amino acids) which is homologous (68% identity) to that of the nonclassical serine protease dipeptidyl peptidase IV (DPPIV). The conserved serine protease motif G-X-S-X-G is present as G-W-S-Y-G. However, sequence analysis of seprase cDNA from LOX and other cell lines strongly suggests that seprase and human fibroblast activation protein alpha (FAP alpha) are products of the same gene. We propose that seprase/FAP alpha and DPPIV represent a new subfamily of serine integral membrane proteases (SIMP).

SUMMARYA 3H+-release method has been developed for the assay of P-hydroxylation of the adrenolytic drug mitotane. P-3H-rnitotane was synthesized by the reduction of 142-chlorophenyl)-l-(4-chlorophenyl)-2,2,2-trichloroethane by an aluminium-HgZC12 couple in the presence of 3Hz0. For P-hydroxylation of mitotane, the jH+-release assay is more efficient and sensitive than a method utilizing 14C-mitotane and chromatographic separation of metabolites by HPLC. The 3H+-release assay has been used to evaluate the ability of adrenal tumors to metabolize mitotane via the Phydroxylation route.

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