Abstract. We have examined cultures of neonatal human foreskin keratinocytes (HFKs) to determine the ligands and functions of integrins 0~2/31, and ot3/31 in normal epidermal stratification and adhesion to the basement membrane zone (BMZ) in skin. We used three assay systems, HFK adhesion to purified extracellular matrix (ECM) ligands and endogenous secreted ECM, localization of integrins in focal adhesions (FAs), and inhibition of HFK adhesion with mAbs to conclude: (a) A new anti-ot3fll mAb, P1F2, localized ot3/~l in FAs on purified laminin > fibronectin/collagen, indicating that laminin was the best exogenous ligand for ct3~l. However, in long term culture, ot3/~l preferentially codistributed in and around FAs with secreted laminin-containing ECM, in preference to exogenous laminin. Anti-a3/~l, mAb P1B5, detached prolonged cultures of HFKs from culture plates or from partially purified HFK ECM indicating that interaction of a3/~l with the secreted laminincontaining ECM was primarily responsible for HFK adhesion in long term culture. (b), In FA assays, a2/~l localized in FAs coincident with initial HFK adhesion to exogenous collagen, but not laminin or fibronectin. However, in inhibition assays, anti-&2/31 inhibited initial HFK adhesion to both laminin and collagen. Thus, ot2/31 contributes to initial HFK adhesion to laminin but tx3/31 is primarily responsible for longterm HFK adhesion to secreted laminin-containing ECM. (c) Serum or Ca2+-induced aggregation of HFKs resulted in relocation of 0t2/31 and ot3/31 from FAs to cell-cell contacts. Further, cell-cell adhesion was inhibited by anti-c~3/31 (P1B5) and a new anti-/31 mAb (P4C10). Thus, interaction of c~3/31 with either ECM or membrane coreceptors at cell-cell contacts may facilitate Ca2+-induced HFK aggregation. (d) It is suggested that interaction of ot3/31 with a secreted, laminin-containing ECM in cultured HFKs, duplicates the role of ot3/31 in basal cell adhesion to the BMZ in skin. Further, relocation of o~2B1 and c~3/31 to cell-cell contacts may result in detachment of cells from the BMZ and increased cell-cell adhesion in the suprabasal cells contributing to stratification of the skin.

Abstract. We have purified the platelet membrane glycoprotein Ia-IIa complex by detergent solubilization and sequential affinity chromatography on Concanavalin A-Sepharose and collagen-Sepharose. The complex, which is identical to the VLA-2 complex of lymphocytes and other cells and contains subunits of 160 and 130 kD on SDS-PAGE, was labeled with ~25I and incorporated into phosphatidyl choline liposomes. The liposomes, like intact platelets, adhered to collagenous substrates in an Mg~-dependent manner with a K'tM~ of 3.5 mM. Little adhesion of the liposomes to collagen occurred when Mg +÷ was replaced by Ca ++ or EDTA. Calcium ions inhibited the Mg +÷-dependent adhesion with a K'tca.+~ of 5.5 mM. Liposomes containing the Ia-IIa complex adhered to substrates cornposed of types I, II, III, and IV collagen, but did not effectively adhere to substrates composed of type V collagen or gelatin. Adhesion to collagen was specific. The liposomes did not adhere to fibronectin, vitronectin, laminin, thrombospondin, fibrinogen, or von WiUebrand factor substrates. The monoclonal antibody P1H5, which specifically immunoprecipitated the Ia-IIa complex, also specifically inhibited the Mg ++-dependent adhesion of both platelets and Ia-IIacontaining liposomes to collagen substrates. These findings provide additional evidence that the platelet membrane Ia-IIa complex is the mediator of Mg ++-dependent platelet adhesion to collagen and suggest that the VLA-2 complex may also function as an Mg++-dependent collagen receptor in other cells.

Abstract. We previously identified a 90-kD (GP90), collagen-binding, membrane glycoprotein, termed extracellular matrix receptor 1II (ECMR HI), that is homologous to the lymphocyte homing receptor and CD44 antigen (Gallatin, W. M., E. A. Wayner, P. A. Hoffman, T. St. John, E. C. Butcher, and W. G. Carter. 1989. Proc. Natl. Acad. Sci. USA. 86:4654-4658). CD44 is abundantly expressed in many epithelial tissues, and is localized predominantly to filopodia in cultured keratinocytes. Here we establish CD44 as a polymorphic family of related membrane proteoglycans and glycoproteins possessing extensive diversity in both glycosylation and core protein sequence. Human neonatal foreskin keratinocytes (HFKs) and QG56 lung squamous carcinoma cells express an alternatively spliced form of the CD44 core protein (termed CD44E) that contains an additional 132 amino acids in the carbohydrate attachment region of the extracellular domain. HFKs, HTI080 fibrosarcoma and QG56 cells, as well as many other human cells, contain varying ratios of GP90 and structurally related, higher molecular mass forms of CD44 that express the following characteristics: (a) each form reacted with anti-CD44 (mAbs) P1G12, P3H9, and P3H5. Each of these mAbs recognized a distinct, nonovedapping epitope present on each CD44 form. (b) Differences in mass were due primarily to variation in carbohydrate moieties, including sulfated aspargine-linked glycopeptides (GP), chondroitin sulfate (CS), and heparan sulfate (HS) glycosaminoglycans, as well as O-linked mucin and polylactosamine structure(s). The major polymorphic forms were designated HT1080 GP90 and CS180, QG56 GP230, and HFK HS/CS250, based on dominant carbohydrate moieties and relative mass. (c) The polymorphic forms use CD44 and CD44E core proteins, each containing a unique set of potential attachment sites for O-and N-glycosides and glycosaminoglycans. (d) Immunofluorescence microscopy, differential extraction with Triton-X-114 detergent, and incorporation into liposomes indicated that all the forms were membrane bound glycoconjugates. These results define CD44 as a structurally diverse, but immunologically related, set of intrinsic membrane macromolecules, and suggests that these structurally varied forms might be expected to manifest multiple functions.

Abstract. FG human pancreatic carcinoma cells use integrin otv/35 as their primary vitronectin receptor since they fail to express integrin o~v/33. These cells are unable to form focal contacts, spread, or migrate on vitronectin but readily do so on collagen in a/31 integrin-dependent manner. Transfection of FG cells with a eDNA encoding the integrin t53 subunit results in the surface expression of a functional integrin otv/33 heterodimer providing these cells with novel adhesive and biological properties. Specifically, FG cells expressing 133 acquire the capacity to attach and spread on vitronectin as well as fibrinogen with/33 localization to focal contacts. Moreover, these cells gain the capacity to migrate through a porous membrane in response to either vitronectin or fibrinogen. These results demonstrate that the/33 and/35 integrin subunits when associated with oev, promote distinct cellular responses to a vitronectin extracellular environment.

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