Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions , such as adhesion , proliferation , polarization , and motility of epidermal cells. Here , we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with ␤1 integrin, ␣-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS. (Am J

Collagen XVII, a hemidesmosomal component, mediates the adhesion of epidermal keratinocytes to the underlying basement membrane. It exists as a full-length transmembrane protein and a soluble ectodomain that is proteolytically released from the cell surface by sheddases of a disintegrin and metalloproteinase (ADAM) family; TACE, the tumor necrosis factor-␣-converting enzyme, is the major physiological proteinase. Because both collagen XVII and the ADAMs are transmembrane proteins, their plasma membrane microenvironment can influence shedding. Lipid rafts, assemblies of sphingolipids and cholesterol within the plasma membrane, are responsible for the separation of membrane proteins and are thought to regulate shedding of cell surface proteins. In this study we analyzed the influence of the cholesterol-depleting agent methyl-␤-cyclodextrin (M␤CD), which disintegrates lipid rafts, on the shedding of collagen XVII in HaCaT keratinocytes and in transfected COS-7 cells. Increasing concentrations of M␤CD led to a dose-dependent decrease of membrane cholesterol levels and to stimulation of collagen XVII shedding. The stimulation was completely inhibited by sheddase inhibitors, and experiments with COS-7 cells co-transfected with TACE and collagen XVII demonstrated that TACE mediated the low cholesterol-dependent shedding. Co-patching analysis by double immunofluorescence staining revealed co-localization of collagen XVII with the raft resident phosphatidylinositol-linked placental alkaline phosphatase and segregation from the non-raft protein human transferrin receptor, indicating that a majority of collagen XVII molecules was incorporated into lipid rafts. These data deliver the first evidence for the role of plasma membrane lipid organization in the regulation of collagen XVII shedding and, therefore, in the regulation of keratinocyte migration and differentiation.Collagen XVII, previously called BP180, is a hemidesmosomal transmembrane protein (1) that plays a critical role in maintaining the linkage between the intracellular and the extracellular structural elements involved in epidermal adhesion. This function is indirectly demonstrated by blistering skin diseases. Mutations in the collagen XVII gene, COL17A1, lead to junctional epidermolysis bullosa (2), a human blistering skin disorder. Furthermore, in bullous autoimmune skin diseases, autoantibodies to collagen XVII perturb cell adhesion and lead to epidermal-dermal separation and skin blistering (3). As a type II transmembrane protein, collagen XVII contains three 180-kDa ␣-chains that fold to form a homotrimer with collagenous triple helical segments built up by the typical Gly-X-Tyr repeat sequences. Each ␣-chain consists of an intracellular N-terminal domain of 466 amino acids, a short transmembrane stretch, and a large extracellular C terminus of 1008 amino acids (4). The ectodomain contains 15 collagenous subdomains, intervened by 16 short non-collagenous sequences.Collagen XVII belongs to the novel group of the collagenous transmembrane proteins (5) and w...

Ecto-phosphorylation is emerging as an important mechanism to regulate cellular ligand interactions and signal transduction. Here we show that extracellular phosphorylation of the cell surface receptor collagen XVII regulates shedding of its ectodomain. Collagen XVII, a member of the novel family of collagenous transmembrane proteins and component of the hemidesmosomes, mediates adhesion of the epidermis to the dermis in the skin. The ectodomain is constitutively shed from the cell surface by metalloproteinases of the ADAM (a disintegrin and metalloproteinase) family, mainly by tumor necrosis factor-␣ converting enzyme (TACE). We used biochemical, mutagenesis, and structural modeling approaches to delineate mechanisms controlling ectodomain cleavage. A standard assay for extracellular phosphorylation, incubation of intact keratinocytes with cell-impermeable [␥-32 P]ATP, led to collagen XVII labeling. This was significantly diminished by both broad-spectrum extracellular kinase inhibitor K252b and a specific casein kinase 2 (CK2) inhibitor. Collagen XVII peptides containing a putative CK2 recognition site were phosphorylated by CK2 in vitro, disclosing Ser 542 and Ser 544 in the ectodomain as phosphate group acceptors. Phosphorylation of Ser 544 in vivo and in vitro was confirmed by immunoblotting of epidermis and HaCaT keratinocyte extracts with phosphoepitopespecific antibodies. Functionally, inhibition of CK2 kinase activity or mutation of the phosphorylation acceptor Ser 544 to Ala significantly increased ectodomain shedding, whereas overexpression of CK2␣ inhibited cleavage of collagen XVII. Structural modeling suggested that the phosphorylation of serine residues prevents binding of TACE to its substrate. Thus, extracellular phosphorylation of collagen XVII by ecto-CK2 inhibits its shedding by TACE and represents novel mechanism to regulate adhesion and motility of epithelial cells.Cells need a versatile, fast mechanism to respond to changes in their microenvironment during development, growth, and regeneration. Regulated proteolysis on the cell surface offers a post-translational mechanism to remove or structurally modify surface associated proteins at any time (1). For a multitude of type I and type II transmembrane proteins, such as receptors, cell adhesion molecules, and growth factors, soluble forms have been identified that have functions distinct from their membrane-bound counterparts. These are often generated through ectodomain shedding, a general mechanism that influences interactions of different cell types with the environment in a broad spectrum of biological and pathological processes (1, 2). However, regulation of the ectodomain shedding remains poorly understood, namely, targeting of the sheddase to particular substrate depending on biological context.One prime example in this context is collagen XVII, a prototype of the novel family of collagenous transmembrane proteins (3). It is a structural component of hemidesmosomes that mediate adhesion of the epidermal keratinocytes to the underlying...

Collagen XXIII belongs to the class of type II orientated transmembrane collagens. A common feature of these proteins is the presence of two forms of the molecule: a membrane-bound form and a shed form. Here we demonstrate that, in mouse lung, collagen XXIII is found predominantly as the full-length form, whereas in brain, it is present mostly as the shed form, suggesting that shedding is tissue-specific and tissue-regulated. To analyze the shedding process of collagen XXIII, a cell culture model was established. Mutations introduced into two putative proprotein convertase cleavage sites showed that altering the second cleavage site inactivated much of the shedding. This supports the idea that furin, a major physiological protease, is predominantly responsible for shedding. Furthermore, our studies indicate that collagen XXIII is localized in lipid rafts in the plasma membrane and that ectodomain shedding is altered by a cholesterol-dependent mechanism. Moreover, newly synthesized collagen XXIII either is cleaved inside the Golgi/trans-Golgi network or reaches the cell surface, where it becomes protected from processing by being localized in lipid rafts. These mechanisms allow the cell to regulate the amounts of cell surface-bound and secreted collagen XXIII.The group of collagenous transmembrane proteins consists of type XIII, XVII, XXIII, and XXV collagens and several related proteins such as ectodysplasin A, the class A macrophage scavenger receptors, the MARCO1 receptor, and the group of colmedins. These are type II transmembrane proteins that contain at least one collagenous triple helical domain (summarized in Ref. 1). Collagens XIII, XXIII, and XXV are of unknown function and consist of three collagenous domains that are flanked and separated by noncollagenous domains. Whereas collagen XVII is more distantly related, types XIII, XVII, XXIII, and XXV all exist in two forms: a transmembrane form and a shed ectodomain form. Whereas collagen XVII is shed from the surface by TACE (tumor necrosis factor-␣-converting enzyme), a member of the ADAM (a disintegrin and metalloproteinase) family (2), mutation analysis of collagens XIII and XXV demonstrated that the protease furin produces the shed forms (3, 4). Initial cell culture studies suggested an involvement of furin either directly or indirectly in the cleavage of collagen XXIII as well (5). Furthermore, the co-existence in tissues of both the transmembrane and shed forms of collagen XXIII was suggested from immunoblot analyses (6).The shedding of an ectodomain amplifies the possible functional role of a protein because different forms, i.e. full-length cell surface-bound or soluble, likely have different biological activity. The "sheddase" furin is a member of a proprotein convertase family. Among other functions, furin participates in the maturation and activation of proteins at the cell surface, endowing the cell with the ability to change its functional behavior (7-9). Moreover, conversion by proteolytic cleavage can be tightly regulated (10, 11). Furin-de...

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