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.

This phase 2 study suggests that a subgroup of patients, when treated with fampridine, experiences a clinically relevant improvement in walking ability, which is sustained for at least 14 weeks.

Suspension of cultured human foreskin keratinocytes (HKs) with trypsin phosphorylates tyrosine residues on an 80-kDa membrane glycoprotein, p80 (Xia, Y., Gil, S. G., and Carter, W. G. (1996) J. Cell Biol. 132, 727-740). Readhesion dephosphorylates p80. Sequencing of a p80 cDNA established identity to CUB domain-containing protein 1 (CDCP1), a gene elevated in carcinomas. CDCP1/p80 cDNA encodes three extracellular CUB domains, a transmembrane domain, and two putative cytoplasmic Tyr phosphorylation sites. Treatment of adherent HKs with suramin, a heparin analogue, or inhibitors of phosphotyrosine phosphatases (PTPs; vanadate or calpeptin) increases phosphorylation of p80 and a novel 140-kDa membrane glycoprotein, gp140. Phosphorylated gp140 was identified as a trypsin-sensitive precursor to p80. Identity was confirmed by digestion and phosphorylation studies with recombinant gp140-GFP. Plasmin, a serum protease, also converts gp140 to p80, providing biological significance to the cleavage in wounds. Phosphorylation of gp140 and p80 are mediated by Src family kinases at multiple Tyr residues including Tyr 734 . Dephosphorylation is mediated by PTP(s). Conversion of gp140 to p80 prolongs phosphorylation of p80 in response to suramin and changes in adhesion. This distinguishes gp140 and p80 and explains the relative abundance of phosphorylated p80 in trypsinized HKs. We conclude that phosphorylation of gp140 is dynamic and balanced by Src family kinase and PTPs yielding low equilibrium phosphorylation. We suggest that the balance is altered by conversion of gp140 to p80 and by adhesion, providing a novel transmembrane phosphorylation signal in epithelial wounds.Wounding of quiescent epidermis activates changes in adhesion and cell signaling resulting in keratinocyte migration, basement membrane (BM) 1 repair, and wound closure (1-3).Based on an in vitro model for wound activation, we reported that trypsin detachment of cultured human foreskin keratinocytes (HKs) promotes phosphorylation of tyrosine residues on an 80-kDa membrane glycoprotein (p80) (4). Phosphorylated p80 (P-p80) in suspended HKs is dephosphorylated upon readhesion to laminin 5 via integrins ␣ 6 ␤ 4 and ␣ 3 ␤ 1 . In work here, we purified and characterized p80. We wished to understand whether phosphorylated p80 identified in an in vitro deadhesion/readhesion screen (4) might be involved in physiologically significant wound activation. Quiescent epidermis adheres to laminin 5 in the BM via integrin ␣ 6 ␤ 4 in hemidesmosome (HD) cell junctions. Wounding epidermis generates leading and following subpopulations of keratinocytes at the wound margin (5). Leading keratinocytes migrate over exposed dermal collagen via integrin ␣ 2 ␤ 1 and fibronectin via integrin ␣ 5 ␤ 1 . However, leading cells also deposit laminin 5 as a provisional BM and interact with these deposits via integrin ␣ 3 ␤ 1 (5-7). The interaction of leading cells with deposited laminin 5 generates distinct transmembrane signals when compared with interaction with dermal ligands. For example,...

Epidermolysis bullosa with pyloric atresia (EB-PA), an autosomal recessive genodermatosis, manifests with neonatal cutaneous blistering associated with congenital pyloric atresia. The disease is frequently lethal, but nonlethal cases have also been reported. Expression of the alpha6 beta4 integrin is altered at the dermal-epidermal basement-membrane zone; recently, mutations in the corresponding genes (ITGA6 and ITGB4) have been disclosed in a limited number of patients, premature termination codons in both alleles being characteristic of lethal variants. In this study, we have examined the molecular basis of EB-PA in five families, two of them with lethal and three of them with nonlethal variants of the disease. Mutation analysis disclosed novel lesions in both ITGB4 alleles of each proband. One of the patients with lethal EB-PA was a compound heterozygote for premature termination-codon mutations (C738X/4791delCA), whereas the other patient with a lethal variant was homozygous for a missense mutation involving a cysteine residue (C61Y). The three nonlethal cases had missense mutations in both alleles (C562R/C562R, R1281W/R252C, and R1281W/R1281W). Immunofluorescence staining of skin in two of the nonlethal patients and in one of the lethal cases was positive, yet attenuated, for alpha6 and beta4 integrins. These results confirm that ITGB4 mutations underlie EB-PA and show that missense mutations may lead to nonlethal phenotypes.