Gangliosides interact directly with plasminogen and urokinase and may mediate binding of these fibrinolytic components to cells

Receptors for the fibrinolytic molecules plasminogen and urokinase are expressed at high capacity on a wide variety of peripheral blood cells and transformed cell lines. We have considered whether gangliosides, components of the outer leaflets of cell membranes, may modulate the interactions of these fibrinolytic ligands with cells. Radiolabeled plasminogen and urokinase bound directly to insolubilized gangliosides. The interactions were saturable and were 50% inhibited by 2.2 microM unlabeled plasminogen or 12 nM unlabeled urokinase, respectively. A panel of gangliosides inhibited binding of both ligands to U937 monocytoid cells, and the order of decreasing inhibitory effectiveness was GD1a greater than GM1 greater than GT1b greater than GM2, while GM3 was minimally effective. The individual components of gangliosides, hexoses, hexosamines, sialic acid, GM1 pentasaccharide, ceramides, and glucocerebrosides were ineffective in in inhibiting the binding of plasminogen and urokinase either to cells or to insolubilized gangliosides. Binding of both ligands to endothelial cells and granulocytes and binding of plasminogen to platelets were also inhibited by gangliosides. U937 cells were cultured with gangliosides to allow incorporation of these glycolipids into the cell membranes. After 3 days of culture, both urokinase binding and plasminogen binding to the cells became enhanced. These results suggest that gangliosides can directly bind to these fibrinolytic components and may mediate or modulate the interactions of plasminogen and urokinase with a variety of cell types.     

Coronary artery disease and the thrombospondin single nucleotide polymorphisms

GeneQuest was a high throughput, large-scale analysis of single nucleotide polymorphisms (SNPs) to identify gene associated with familial, premature coronary artery disease and myocardial infarction. The three SNPs showing the highest and most significant associations with disease were all members of the thrombospondin gene family, thrombospondin-1, thrombospondin-2 and thrombospondin-4. These unanticipated associations have kindled efforts to understand how the three SNPs influence the structures and functions of the thrombospondins. The SNP in thrombospondin-1 and thrombospondin-4 reside in their coding regions and result in single amino acid changes: in thrombospondin-1, the predominant asparagine at position 700 is changed to a serine while, in thrombospondin-4, it is a change of an alanine to a proline at position 387. The SNP in thrombospondin-2 is a base change in the 3'-untranslated region of the mRNA. At this early stage of investigation, predictive analyses suggest that the substitutions in thrombospondin-2 and thrombospondin-4 should alter structure, and there is direct evidence to indicate that the thrombospondin-1 SNP alters conformational stability. In addition, profound differences in the function of the thrombospondin-4 SNP variants have been identified with respect to their capacity to support endothelial cell adhesion and proliferation. While substantial additional information is needed to understand if and how the polymorphic forms of the thrombospondins affect coronary artery disease, the data assembled to date suggest marked effects of these SNPs on the structures and functions of the thrombospondins, which are consistent with induction of a proatherogenic and prothrombotic phenotype.     

Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness

Kindlins are integrin-interacting proteins essential for integrin-mediated cell adhesiveness. In this study, we focused on the evolutionary origin and functional specialization of kindlins as a part of the evolutionary adaptation of cell adhesive machinery. Database searches revealed that many members of the integrin machinery (including talin and integrins) existed before kindlin emergence in evolution. Among the analyzed species, all metazoan lineages—but none of the premetazoans—had at least one kindlin-encoding gene, whereas talin was present in several premetazoan lineages. Kindlin appears to originate from a duplication of the sequence encoding the N-terminal fragment of talin (the talin head domain) with a subsequent insertion of the PH domain of separate origin. Sequence analysis identified a member of the actin filament–associated protein 1 (AFAP1) superfamily as the most likely origin of the kindlin PH domain. The functional divergence between kindlin paralogues was assessed using the sequence swap (chimera) approach. Comparison of kindlin 2 (K2)/kindlin 3 (K3) chimeras revealed that the F2 subdomain, in particular its C-terminal part, is crucial for the differential functional properties of K2 and K3. The presence of this segment enables K2 but not K3 to localize to focal adhesions. Sequence analysis of the C-terminal part of the F2 subdomain of K3 suggests that insertion of a variable glycine-rich sequence in vertebrates contributed to the loss of constitutive K3 targeting to focal adhesions. Thus emergence and subsequent functional specialization of kindlins allowed multicellular organisms to develop additional tissue-specific adaptations of cell adhesiveness.     

Migfilin, a Molecular Switch in Regulation of Integrin Activation

The linkage of heterodimeric (α/β) integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to integrin β cytoplasmic tails (CTs) induces high affinity ligand binding (integrin activation), whereas binding of another actin-linking protein, filamin, to the integrin β CTs negatively regulates this process by blocking the talin-integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the integrin adhesion sites, strongly interacts with the same region in filamin where integrin β CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from integrin and promotes the talin/integrin binding and integrin activation. Migfilin thus acts as a molecular switch to disconnect filamin from integrin for regulating integrin activation and dynamics of extracellular matrix-actin linkage.Cells reside in a protein network, the extracellular matrix (ECM).⁴ Cell-ECM contact is crucial for many physiological and pathophysiological processes and is primarily mediated by heterodimeric (α/β) transmembrane receptors, the integrins (1). Integrins engage a variety of ECM proteins via their extracellular domains while connecting to the actin cytoskeleton via their small cytoplasmic tails (CTs). The ability of integrins to bind to their ligands is uniquely controlled by the integrin CTs via a process called “inside-out signaling,” i.e. upon cellular stimulation, an integrin, typically expressed in a latent state, can receive intracellular signal(s) at its CT, which transmits through the transmembrane domain to the extracellular domain thereby converting the receptor from a low to a high affinity state (integrin activation). How such long range information transfer is initiated and regulated has been the central topic of integrin/cell adhesion research over the decades (for reviews see Refs. 2-5). Structural/biochemical studies have indicated that the inside-out signaling involves the unclasping of the integrin α/β CT complex (6-9), followed by extensive rearrangement of transmembrane domain and extracellular domain (10-13). Talin, a large actin-linking protein, was found to play a key role in the unclasping process by binding to the integrin β CTs (7-8, 14). Talin activity appears to be controlled by multiple factors or pathways (15-20).Relevant to this study is the role of filamin, another major actin cross-linking protein (21-22), in integrin activation. Filamin was found to share an overlapping binding site on integrin β CTs with talin and thus suppress the talin-integrin interaction (16). Gene silencing of filamin in various cell lines to remove the filamin-integrin connection enhances integrin activation (16, 23), whereas increased filamin-integrin interaction inhibits cell migration (24), a process critically dependent on integrin activation. Together these observations support the notion that filamin binding to integrin serves as a cellular brake to control the dynamics of the integrin activation by inhibiting talin function and ECM-cytoskeleton communication. The mechanism as to how the filamin brake is turned off to promote integrin activation and cell migration is not understood.Filamin is known to contain an N-terminal actin binding domain (ABD) and a long rod-like domain of 24 immunoglobulin-like repeats, of which repeat 21 (IgFLN21) was shown to play a key role in binding to integrin β CTs and blocking the talin-integrin β CT interaction (16). Interestingly, IgFLN21 also recognizes another intracellular protein called migfilin, which has been shown to be an important regulator of integrin-mediated cytoskeletal rearrangements, cell shape change (25), and cell migration (26).In an effort to dissect the complex intermolecular interactions between migfilin, filamin, and integrin, we have undertaken a detailed structural/functional analysis. Using NMR spectroscopy, we have mapped the precise IgFLN21 binding region in migfilin, which is located at the extreme N terminus (residues 1-24) of migfilin (migfilin-N), and we solved solution structure of the IgFLN21-migfilin-N complex. To our surprise, despite little sequence homology, migfilin binds to the same region in IgFLN21 where integrin β CT binds. Detailed NMR and biochemical analyses demonstrate that the migfilin-filamin interaction is an order of magnitude higher than the integrin-filamin interaction and that the migfilin binding to filamin can competitively dissociate filamin from integrin and thus promote the talin-integrin interaction. Using multiple functional approaches, we further show that migfilin, but not its filamin binding defective mutant, significantly enhances integrin activation. These data suggest a novel regulatory pathway in which the binding of filamin to its downstream target migfilin switches off the integrin-filamin connection, thereby promoting talin binding to and activation of integrins.     

An LRP8 variant is associated with familial and premature coronary artery disease and myocardial infarction

Our previous genomewide linkage scan of 428 nuclear families (GeneQuest) identified a significant genetic susceptibility locus for premature myocardial infarction (MI) on chromosome 1p34-36. We analyzed candidate genes in the locus with a population-based association study involving probands with premature coronary artery disease (CAD) and/or MI from the GeneQuest families (381 cases) and 560 controls without stenosis detectable by coronary angiography. A nonconservative substitution, R952Q, in LRP8 was significantly associated with susceptibility to premature CAD and/or MI by use of both population-based and family-based designs. Three additional white populations were used for follow-up replication studies: another independent cohort of CAD- and/or MI-affected families (GeneQuest II: 441 individuals from 22 pedigrees), an Italian cohort with familial MI (248 cases) and 308 Italian controls, and a separate Cleveland GeneBank cohort with sporadic MI (1,231 cases) and 560 controls. The association was significantly replicated in two independent populations with a family history of CAD and/or MI, the GeneQuest II family-based replication cohort and the Italian cohort, but not in the population with sporadic disease. The R952Q variant of LRP8 increased activation of p38 mitogen-activated protein kinase by oxidized low-density lipoprotein. This extensive study, involving multiple independent populations, provides the first evidence that genetic variants in LRP8 may contribute to the development of premature and familial CAD and MI.     

Identity of the amino acid residues involved in C3bi binding to the I-domain supports a mosaic model to explain the broad ligand repertoire of integrin alpha M beta 2

Interactions between the complement degradation product C3bi and leukocyte integrin alpha(M)beta(2) are critical for host defense against foreign pathogens and in tumor cell surveillance. To gain insight into the mechanism by which the alpha(M)I-domain of the integrin interacts with C3bi, detailed mapping of the C3bi binding site was undertaken. Previous mutagenesis studies had implicated five small structural segments within the alpha(M)I-domain in recognition of this ligand. Sets of three amino acids within the five implicated segments were mutated to the corresponding alpha(L)I-domain residues. Then, within the affected mutants, single point mutations were introduced to precisely define the requisite residues. Ultimately, H148, F150, Q204, L205, R208, T211, T213, I256, P257 were identified as being critical for C3bi binding. A synthetic peptide approach confirmed the involvement of the specified residues with the complex midsegment, Q204-I215, in C3bi recognition. Furthermore, the alpha(D)I-domain, which has a low intrinsic affinity for C3bi, acquired high affinity for the ligand when the implicated residues were inserted. The residues necessary to engage C3bi reside on or adjacent to the cation binding MIDAS site of the alpha(M)I-domain. The amino acids involved in C3bi binding are distinct from those involved in interaction of previously mapped ligands with the alpha(M)I-domain. This divergence supports a mosaic model, in which different ligands engage different amino acids to bind to alpha(M)I-domain, accounting for the broad recognition capacity of integrin alpha(M)beta(2).