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Bmx/Etk, a member of the Tec/Btk family of nonreceptor kinases, has recently been shown to mediate cell motility in signaling pathways that become activated upon integrin-mediated cell adhesion (Chen, R., Kim, O., Li, M., Xiong, X., Guan, J. L., Kung, H. J., Chen, H., Shimizu, Y., and Qiu, Y. (2001) Nat Cell Biol. 3, 439-444). The molecular mechanisms of Bmx-induced cell motility have so far remained unknown. Previous studies by us and others have demonstrated that a complex formation between the docking protein p130Cas (Cas) and the adapter protein Crk is instrumental in connecting several stimuli to the regulation of actin cytoskeleton and cell motility. We demonstrate here that expression of Bmx leads to an interaction between Bmx and Cas at membrane ruffles, which are sites of active actin remodeling in motile cells. Expression of Bmx also enhances tyrosine phosphorylation of Cas and Cas.Crk complex formation, and coexpression of Bmx with Cas results in an enhanced membrane ruffling and haptotactic cell migration. Importantly, a mutant form of Bmx that fails to interact with Cas also fails to induce cell migration. Furthermore, expression of a dominant-negative form of Cas that is incapable of interacting with Crk inhibits Bmx-induced membrane ruffling and cell migration. These studies suggest that Bmx-Cas interaction, phosphorylation of Cas by Bmx, and subsequent Cas.Crk complex formation functionally couple Bmx to the regulation of actin cytoskeleton and cell motility.

Leukocyte infiltration during inflammation is mediated by the sequential actions of adhesion molecules and chemokines. By using a rat ureteral obstruction model, we showed previously that L-selectin plays an important role in leukocyte infiltration into the kidney. Here we report the purification, identification, and characterization of an L-selectin-binding heparan sulfate proteoglycan (HSPG) expressed in the rat kidney. Partial amino acid sequencing and Western blotting analyses showed that the L-selectin-binding HSPG is collagen XVIII, a basement membrane HSPG. The binding of L-selectin to isolated collagen XVIII was specifically inhibited by an anti-L-selectin monoclonal antibody, EDTA, treatment of the collagen XVIII with heparitinase or heparin but not by chemically desulfated heparin. A cell binding assay showed that the L-selectin-collagen XVIII interaction mediates cell adhesion. Interestingly, collagen XVIII also interacted with a chemokine, monocyte chemoattractant protein-1, and presented it to a monocytic cell line, THP-1, which enhanced the alpha(4)beta(1) integrin-mediated binding of the THP-1 cells to vascular cell adhesion molecule-1. Thus, collagen XVIII may provide a link between selectin-mediated cell adhesion and chemokine-induced cellular activation and accelerate the progression of leukocyte infiltration in renal inflammation.

The complete primary structure of the mouse type XIII collagen chain was determined by cDNA cloning. Comparison of the mouse amino acid sequences with the previously determined human sequences revealed a high identity of 90%. Surprisingly, the mouse cDNAs extended further in the 5' direction than the previously identified human clones. The 5' sequences contained a new in-frame ATG codon for translation initiation which resulted in elongation of the N-terminal noncollagenous domain by 81 residues. These N-terminal sequences lack a typical signal sequence but include a highly hydrophobic segment that clearly fulfills the criteria for a transmembrane domain. The sequence data thus unexpectedly suggested that type XIII collagen may be located on the plasma membrane, with a short cytosolic N-terminal portion and a long collagenous extracellular portion. These sequence data prompted us to generate antipeptide antibodies against type XIII collagen in order to study the protein and its subcellular location. Western blotting of human tumor HT-1080 cell extract revealed bands of over 180 kDa. These appeared to represent disulfide-bonded multimeric polypeptide forms that resolved upon reduction into 85-95-kDa bands that are likely to represent a mixture of splice forms of monomeric type XIII collagen chains. These chains were shown to contain the predicted N-terminal extension and thus also the putative transmembrane segment. Immunoprecipitation of biotinylated type XIII collagen from surface-labeled HT-1080 cells, subcellular fractionation, and immunofluorescence staining were used to demonstrate that type XIII collagen molecules are indeed located in the plasma membranes of these cells.

Transcripts for the alpha 1 chain of mouse type XVIII collagen were found to be heterogeneous at their 5'-ends and to encode three variant N-terminal sequences of the ensuing 1315-, 1527-, or 1774-residue collagen chains. The variant mRNAs appeared to originate from the use of two alternate promoters of the alpha 1(XVIII) chain gene, resulting in the synthesis of either short or long N-terminal non-collagenous NC1 domains, the latter being further subject to modification due to alternative splicing of the transcripts. As a result, the 1527- and 1774-residue polypeptides share the same signal peptide, and the lengths of their NC1 domains are 517 or 764 amino acid residues, respectively, while the 1315-residue polypeptide has a different signal peptide and a 301-residue NC1 domain. The longest NC1 domain was strikingly characterized by a 110-residue sequence with 10 cysteines, which was found to be homologous with the previously identified frizzled proteins belonging to the family of G-protein-coupled membrane receptors. Thus, it is proposed that the cysteine-rich motif, termed fz, represents a new sequence motif that can be found in otherwise unrelated proteins. Tissues containing mainly one or two NC1 domain mRNA variants or all three NC1 domains were identified, indicating that there is tissue-specific utilization of two alternate promoters and alternative splicing of alpha 1(XVIII) transcripts.

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, plays a central role in collagen synthesis as it catalyzes the formation of 4-hydroxyproline residues by the hydroxylation of proline in X-Pro-Gly sequences. We report here that the human gene for the catalytically important alpha subunit is more than 69 kilobase pairs and consists of 16 exons. The exons that encode solely protein sequences vary from 54 to 240 base pairs (bp), and the introns vary from 750 to more than 16,000 bp. The 133 bp of 5'-untranslated sequences of the mRNA are coded by two exons, and these sequences contain inverted repeats with a potential for stem-loop formation, which may be involved in translational control of the expression of this gene. The 5'-flanking region contains a TATa motif at -29 relative to the major transcription site but no CCAAT motif. The 5'-flanking region and the downstream sequences contain several motifs that may act as binding sites for various transcription factors. Evidence has previously been reported for a mutually exclusive alternative splicing of RNA transcripts of this gene. The present data indicate that the mutually exclusive sequences found in the mRNAs are coded by two consecutive, homologous 71-bp exons 9 and 10. These exons are identical in their first 5 bp and the overall identity between them is 61% at the nucleotide level and 58% at the level of the coded amino acids. Both types of mRNA were found to be expressed in all of the tissues studied, but in some tissues the type coding for exon 9 or 10 sequences was more abundant than the other type.

We have isolated cDNAs that complete the elucidation of the primary structure of the mouse alpha 1(XVIII) collagen chain, a polypeptide homologous to the alpha 1(XV) collagen chain. The 1315-residue alpha 1(XVIII) chain includes a 25-residue signal peptide, a 301-residue NH2-terminal non-collagenous domain (NC1), a 674-residue collagenous sequence with nine interruptions of 10-24 residues, and a 315-residue COOH-terminal noncollagenous domain (NC11). Seven of the collagenous domains and both flanking noncollagenous domains share homology with the alpha 1(XV) chain. The COOH-terminal noncollagenous domains are unique to the alpha 1(XVIII) and alpha 1(XV) chains, and they contain a homologous beginning, a variable portion, and a highly homologous COOH-terminal half with 4 conserved cysteines. The differences in the collagenous sequences probably preclude the existence of the two chains in the same molecule, however. A 12.5-kilobase pair genomic sequence was found to contain the 12 extreme 3'-exons of the alpha 1(XVIII) gene, covering 40% of the coding sequences. Exons start with either a complete codon or a split codon for the glycines of Gly-Xaa-Yaa repeats, and seven exons completely cover the NC11 domain. Comparison of the sequences encoded by these seven exons with the corresponding region of the alpha 1(XV) gene indicated conserved exon-intron organization, suggesting that the two genes derive from a common ancestor.

Types XV and XVIII collagens form a new subgroup within the diverse family of collagens, both being characterized by extensive interruptions in their collagenous sequences. We isolated human alpha 1(XV) chain cDNAs that extend beyond the sequences previously derived from a 2127-nucleotide clone. The cDNAs cover 5172 nucleotides of the 5300-4400-nucleotide mRNAs and encode a polypeptide of 1388 residues, including a 25-residue signal peptide, a 530-residue NH2-terminal noncollagenous domain (NC1), a 577-residue collagenous sequence with eight interruptions of 7-45 residues, and a 256-residue COOH-terminal noncollagenous domain (NC10). A thrombospondin sequence motif found previously in certain other collagen types was identified in the NC1 domain of type XV collagen, whereas the NC10 domain was characterized by the presence of four cysteine residues. The exon-intron organization of the human gene was determined, corresponding to the extreme COOH-terminal 2/3 + 275 residues. NC10 is encoded by seven exons, six of which encode solely protein sequences and vary in size between 60 and 186 base pairs, whereas the last one corresponds to the end of the polypeptide and the 3'-untranslated sequences. The extreme 5' exon analyzed was a junction exon encoding both collagenous and noncollagenous sequences. This initial characterization is indicative of a multiexon arrangement for the alpha 1(XV) gene.