Identification of Functionally Important Amino Acid Residues within the C2-Domain of Human Factor V Using Alanine-Scanning Mutagenesis

Sw, Kim; Ma, Quinn-Allen; Jt, Camp; Macedo-Ribeiro, Sandra; Fuentes‐Prior, Pablo; Bode, Wolfram; Kane, WH

doi:10.1021/bi992256r

Cited by 72 publications

(127 citation statements)

References 38 publications

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“…The suggested ␤-barrel structure has been recently confirmed by x-ray crystallography for the second discoidin domain of factor V and VIII (39,40). Based on these structural data, a surface epitope involved in binding of factor V to membrane-anchored phosphatidylserine has been suggested (41,42). Such structural and functional information should help for future definition and analysis of regions of functional importance in DDR1.…”

Section: Discussionmentioning

confidence: 69%

Mapping of Epitopes in Discoidin Domain Receptor 1 Critical for Collagen Binding

Curat¹,

Eck²,

Dervillez³

et al. 2001

Journal of Biological Chemistry

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The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.Discoidin domain receptors 1 and 2 (DDR1 and DDR2) 1 have been recognized as a distinct tyrosine kinase receptor subfamily because of structural and functional homologies. In their extracellular region, both receptors show a domain homologous to the Dictyostelium discoideum protein discoidin I. DDR1 and DDR2 are also functionally related by the observation that collagen acts as cognate ligand for both receptors. Whereas DDR1 activation is achieved by all collagens so far tested (types I-VI and VIII), DDR2 is only activated by fibrillar collagens, in particular by collagen type I and type III. In contrast to most other tyrosine kinase receptors, activation of DDRs can take several hours (1, 2).The cDNA coding for human DDR1 has been cloned from several tissues or carcinoma cells (3-7). Gene orthologs to human DDR1 have been identified in mice, rats, and Caenorhabditis elegans (8 -10). Expression of human DDR1 is predominantly seen in epithelial cells, particularly from kidney, lung, gastrointestinal tract, and brain, but also in corneal and dermal fibroblasts (7, 9, 11-13). DDR1 seems to be also involved in the differentiation of cerebellar granular neurons (14). Upregulated DDR1 expression has been reported from breast, ovarian, esophagus and brain tumors (5,(15)(16)(17)(18)(19). Human DDR1 is located on chromosome 6p21.3 in close proximity to HLA genes, which belong to the telomeric region (class I) of the major histocompatibility complex (20). The juxtamembrane regions in DDR1 and DDR2 are much longer than in other receptor tyrosine kinases (176 and 147 amino acids, respectively). Furthermore, the extracellular domain of DDR1 is shed by an unidentified protease, res...

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Section: Discussionmentioning

confidence: 69%

Mapping of Epitopes in Discoidin Domain Receptor 1 Critical for Collagen Binding

Curat¹,

Eck²,

Dervillez³

et al. 2001

Journal of Biological Chemistry

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“…The crucial role of this triad common to all discoidin domain family members is con¢rmed by the observation that mutations inside the triplet motif of FaVIII are known to cause hemophilia A [13] and to destabilize the fold of the C2 domain of FaV [32]. In the case of retinoschisis single point mutations to cysteine of each amino acid of the triad cause the pathology.…”

Section: Resultsmentioning

confidence: 99%

Effects of pathological mutations on the stability of a conserved amino acid triad in retinoschisin

Fraternali¹,

Cavallo

Musco

2003

FEBS Letters

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A three-dimensional model has been calculated for the discoidin domain of retinoschisin (RS1), the protein involved in the X-linked juvenile retinoschisis. The model allows for a mapping of the pathological retinoschisis missense mutations and a rationale for the structural e¡ects of an evolutionary conserved surface exposed triad (W122^R200^W163). Molecular dynamics simulations of the triad mutants models, together with ab initio energy calculations of the complexes corresponding to the triad show that the observed pathological mutations sensibly destabilize local interactions and the entire fold. Moreover the presented model reveals evidence of a putative site for membrane association. ß

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“…Deletion of the entire C2 domain results in a complete loss of phosphatidylserine-specific membrane binding (20). Alaninescanning mutagenesis within the C2 identified several key polar and hydrophobic amino acids as necessary for achieving maximal cofactor function (21,22).…”

mentioning

confidence: 99%

The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function

Adams

Hockin

Mann

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

142

157

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In vertebrate hemostasis, factor Va serves as the cofactor in the prothrombinase complex that results in a 300,000-fold increase in the rate of thrombin generation compared with factor Xa alone. Structurally, little is known about the mechanism by which factor Va alters catalysis within this complex. Here, we report a crystal structure of protein C inactivated factor Va (A1⅐A3-C1-C2) that depicts a previously uncharacterized domain arrangement. This orientation has implications for binding to membranes essential for function. A high-affinity calcium-binding site and a copperbinding site have both been identified. Surprisingly, neither shows a direct involvement in chain association. This structure represents the largest physiologically relevant fragment of factor Va solved to date and provides a new scaffold for the future generation of models of coagulation cofactors.

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Identification of Functionally Important Amino Acid Residues within the C2-Domain of Human Factor V Using Alanine-Scanning Mutagenesis

Cited by 72 publications

References 38 publications

Mapping of Epitopes in Discoidin Domain Receptor 1 Critical for Collagen Binding

Mapping of Epitopes in Discoidin Domain Receptor 1 Critical for Collagen Binding

Effects of pathological mutations on the stability of a conserved amino acid triad in retinoschisin

The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function

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