Tissue barrier function is directly mediated by tight junction transmembrane proteins known as claudins. Cells that form tight junctions typically express multiple claudin isoforms which suggests that heterotypic (head-to-head) binding between different claudin isoforms may play a role in regulating paracellular permeability. However, little is known about motifs that control heterotypic claudin compatibility. We found that although claudin-3 and claudin-4 were heteromerically compatible when expressed in the same cell, they did not heterotypically interact despite having extracellular loop (EL) domains that are highly conserved at the amino acid level. Claudin-1 and -5, which were heterotypically compatible with claudin-3, did not heterotypically bind to claudin-4. In contrast, claudin-4 chimeras containing either the first EL domain or the second EL domain of claudin-3 were able to heterotypically bind to claudin-1, claudin-3, and claudin-5. Moreover, a single point mutation in the first extracellular loop domain of claudin-3 to convert Asn 44 to the corresponding amino acid in claudin-4 (Thr) produced a claudin capable of heterotypic binding to claudin-4 while still retaining the ability to bind to claudin-1 and -5. Thus, control of heterotypic claudin-claudin interactions is sensitive to small changes in the EL domains.

Claudins are proteins that participate in epithelial barrier function and regulate paracellular permeability. By immunohistochemistry of adult rat lung sections, claudin-3, claudin-4, and claudin-5 were found to be co-expressed by type II alveolar epithelial cells. Claudin-3 and claudin-4 were also co-expressed by some alveolar epithelial cells adjacent to type II cells. In contrast, claudin-5 was expressed throughout the alveolus. Isolated primary rat alveolar epithelial cells in culture also expressed claudin-3, claudin-4, and claudin-5, but showed little claudin-1 and claudin-2 expression. Claudin expression by isolated cells at both the mRNA and protein level varied with time in culture. In particular, claudin-3 and claudin-5 co-localized and were distributed around the alveolar cell periphery, but claudin-4 expression was heterogeneous. We also found that paracellular permeability was increased when cultured alveolar epithelial cells were treated with a fatty acid amide, methanandamide. Methanandamide did not alter cell viability. Claudin-3, claudin-4, claudin-5, occludin, and zona occludens 1 remained localized to cell-cell contact sites at the plasma membrane in methanandamide-treated cells, suggesting that plasma membrane localization of these junction proteins is not sufficient for maintaining barrier function. However, methanandamide-treated cells showed a 12-fold increase in claudin-5 expression and a 2- to 3-fold increase in claudin-3, consistent with the notion that specific changes in claudin expression levels may correlate with changes in alveolar epithelial barrier function.

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.

Claudins are a family of nearly two dozen transmembrane proteins that are a key part of the tight junction barrier that regulates solute movement across polarized epithelia. Claudin family members interact with each other, as well as with other transmembrane tight junction proteins (such as occludin) and cytosolic scaffolding proteins (such as zonula occludens-1 (ZO-1)). Although the interplay between all of these different classes of proteins is critical for tight junction formation and function, claudin family proteins are directly responsible for forming the equivalent of paracellular ion selective channels (or pores) with specific permeability and thus are essential for barrier function. In this review, we summarize current progress in identifying structural elements of claudins that regulate their transport, assembly, and function. The effects of oxidant stress on claudins are also examined, with particular emphasis on lung epithelial barrier function and oxidant stress induced by chronic alcohol abuse.

We previously identified the 11 amino acid C1 region of the cytoplasmic domain of P-selectin as essential for an endosomal sorting event that confers rapid turnover on P-selectin. The amino acid sequence of this region has no obvious similarity to other known sorting motifs. We have analyzed the sequence requirements for endosomal sorting by measuring the effects of site-specific mutations on the turnover of P-selectin and of the chimeric protein LLP, containing the lumenal and transmembrane domains of the low density lipoprotein receptor and the cytoplasmic domain of P-selectin. Endosomal sorting activity was remarkably tolerant of alanine substitutions within the C1 region. The activity was eliminated by alanine substitution of only one amino acid residue, leucine 768, where substitution with several other large side chains, hydrophobic and polar, maintained the sorting activity. The results indicate that the endosomal sorting determinant is not structurally related to previously reported sorting determinants. Rather, the results suggest that the structure of the sorting determinant is dependent on the tertiary structure of the cytoplasmic domain. INTRODUCTIONThe function of membrane-bounded organelles requires correct targeting of resident membrane proteins to each organelle and in many cases selective cycling of membrane proteins between organelles. Selective localization and targeting of membrane proteins to their appropriate destinations depend on structural features of these proteins, termed sorting determinants. Sorting determinants are recognized by sorting machinery that functions to concentrate proteins bearing the appropriate sorting determinants into specific transport vesicles, which can then carry their cargo vectorially to the correct destination. A number of sorting determinants have been characterized, including those that mediate localization to clathrin-coated pits in the trans-Golgi network (TGN) 11 or at the cell surface, sorting to the basolateral cell surface in polarized epithelial cells, and selective transport from endosomes to lysosomes (Sandoval and Bakke, 1994;Mellman, 1996;Kirchhausen et al., 1997;Marks et al., 1997).Many sorting determinants are contained in short segments of amino acid sequence in the cytoplasmic domains of transmembrane proteins (Sandoval and Bakke, 1994;Marks et al., 1997). These short sequences have been defined by mutagenesis experiments in which systematic substitution of amino acid residues identifies only a few mutations that disrupt sorting activity. To date, the most prevalent and most extensively studied determinants that operate in post-Golgi trafficking pathways are those that require a tyrosine residue and additional residues in specific contexts * Present address: Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908. † Corresponding author: Department of Cell Biology, Box 439 HSC, University of Virginia, Charlottesville VA 22908. E-mail address: sag4y@virginia.edu. 1 Abbreviations used: CHO, Ch...

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro. The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet a-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the 'lysosome-like' secretory granules of platelets and melanocytes. P-selectin, a cell adhesion protein that functions in leukocyte recruitment early in the inflammatory response, is targeted to secretory granules in platelets and endothelial cells, and delivered to the cell surface upon regulated granule exocytosis in response to inflammatory stimuli (1). Cell surface Pselectin is rapidly internalized in endothelial cells and transfected cell lines (2,3), then undergoes selective sorting from early endosomes to late endosomes, resulting in rapid recycling through late endosomes and the trans-Golgi network (TGN) (4). Rapid internalization followed by endosomal sorting provides temporal regulation for cell surface expression of P-selectin following granule exocytosis, and also results in rapid delivery of P-selectin to lysosomes when it is not packaged in secretory granules (5-7). These three sorting activities are critical to regulating the adhesive function of this protein.The 35-amino acid cytoplasmic domain of P-selectin ( Figure 1) mediates each of the three sorting activities. Rapid internalization of P-selectin occurs in clathrin-coated pits (3). Although a conventional internalization signal was not identifiable by alanine-scanning mutagenesis, the cytoplasmic domain of P-selectin has recently been shown to associate with the medium (m) subunit of the AP-2 adaptor through an extended tyrosine-containing motif (8). Endosomal sorting of P-selectin is mediated by a sorting determinant that functions independently of the rapid internalization activity, and can be abolished by the single amino acid substi...

Amyloid b protein, the major component of the senile plaques in Alzheimer's disease, is generated by secretory and endocytic processing of amyloid precursor protein. Internalized amyloid precursor protein either recycles to the plasma membrane, where a-secretase resides, or moves to acidic compartment(s) for b-secretase exposure. While the trans-Golgi network contains b-secretase activity, recent examination of the subcellular distribution of this proteinase, called BACE, has led to the suggestion that b-secretase activity might also reside at the plasma membrane and in endosomes. To examine the role of endocytic compartments in b-secretase processing of amyloid precursor protein, the wild-type and endosomal sorting mutant P-selectin cytoplasmic domains were used to control movement of amyloid precursor protein through endosomes. Amyloid precursor protein/P-selectin, which is sorted from early to late endosomes, undergoes significantly less a-secretase cleavage, and more b-secretase cleavage, than amyloid precursor protein/P-selectin768A, a mutant that recycles more efficiently to the cell surface. Our results demonstrate that endosomal sorting influences relative exposure of the amyloid precursor protein/P-selectin chimeras to a-and b-secretase activities, and suggest that, because delivery to late endocytic compartments favors b-secretase processing of amyloid precursor protein, there is likely limited b-secretase activity in early endosomes or at the cell surface. We propose that the trans-Golgi network may be involved in both secretory and endocytic generation of amyloid b protein. Alzheimer's disease (AD) is the most common form of dementia in late middle-aged and older adults, affecting an estimated 10% of all humans over age 65 (1). The sole invariant pathological trait of AD is the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles. The major protein component of these amyloid deposits is a 40-42 908 amino acid peptide, Ab, derived through endoproteolytic cleavage of a type I integral membrane protein, amyloid precursor protein (APP). APP processing is mediated by three proteinase activities designated a-, b-, and g-secretases. Recently, ADAMs 10 and 17 have been shown to be involved in a-secretase cleavage of APP (2,3), and a transmembrane aspartic protease, BACE, has been identified as b-secretase (4-8). Both a-and b-secretases cleave within the APP lumenal domain, releasing large lumenal fragments, aAPPs and bAPPs, respectively, and generating membrane-associated carboxy-terminal fragments ( Figure 1A). Cleavage of APP by b-secretase produces the amino-terminus of Ab, while cleavage by a-secretase occurs 16 residues carboxy-terminal to the b cleavage site, precluding formation of Ab (9). g-secretase cleaves both the a-and b-secretase products, producing p3 and Ab, respectively.Much work has been done to identify the subcellular locations of the secretase cleavage events. Cleavage of APP by a-secretase occurs at the cell surface (10,11) and is limited by residence time ...

Tight junction proteins known as claudins are structurally required for tissue barrier function. Epithelial cells typically express multiple claudin isoforms which, in turn, influence paracellular ion permeability. This suggests that heterologous claudin‐claudin interactions are important for formation of tight junctions, however, little is known about structural motifs which determine whether or not claudins interact. To explore this, we examined heterotypic (head‐to‐head) binding between different claudin isoforms in HeLa cells, which are claudin‐null, yet express other tight junction proteins including occludin, JAM‐A, ZO‐1, ZO‐2 and ZO‐3. We found that claudin‐3 and claudin‐4 did not heterotypically interact, despite having extracellular loop (EL) domains that are highly conserved at the amino acid level. Claudin‐1 and claudin‐5, which were heterotypically compatible with claudin‐3 did not bind to claudin‐4, suggesting recognition of a common motif. Interestingly, claudin‐4 chimeras containing either the first EL domain or the second EL domain of claudin‐3 were able to heterotypically bind to claudin‐1, claudin‐3 and claudin‐5. This suggests heterotypic binding was not determined by a simple binary interaction between individual EL domains. Instead, a more likely model is that heterotypic binding is mediated by a simultaneous interaction involving both pairs of EL domains.