A naturally occurring Tyr143HisαIIb mutation abolishes αIIbβ3 function for soluble ligands but retains its ability for mediating cell adhesion and clot retraction: comparison with other mutations causing ligand-binding defects

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3485-3491 ◽  
Author(s):  
Teruo Kiyoi ◽  
Yoshiaki Tomiyama ◽  
Shigenori Honda ◽  
Seiji Tadokoro ◽  
Morio Arai ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Binding Sites ◽  
Clot Retraction ◽  
Naturally Occurring ◽  
Binding Function ◽  
293 Cells ◽  
Ligand Binding Sites ◽  
Functional Defect ◽  
And Function

The molecular basis for the interaction between a prototypic non–I-domain integrin, αIIbβ3, and its ligands remains to be determined. In this study, we have characterized a novel missense mutation (Tyr143His) in αIIb associated with a variant of Glanzmann thrombasthenia. Osaka-12 platelets expressed a substantial amount of αIIbβ3(36%-41% of control) but failed to bind soluble ligands, including a high-affinity αIIbβ3-specific peptidomimetic antagonist. Sequence analysis revealed that Osaka-12 is a compound heterozygote for a single 521T>C substitution leading to a Tyr143His substitution in αIIb and for the null expression of αIIb mRNA from the maternal allele. Given that Tyr143 is located in the W3 4-1 loop of the β-propeller domain of αIIb, we examined the effects of Tyr143His or Tyr143Ala substitution on the expression and function of αIIbβ3 and compared them with KO (Arg-Thr insertion between 160 and 161 residues of αIIb) and with the Asp163Ala mutation located in the same loop by using 293 cells. Each of them abolished the binding function of αIIbβ3 for soluble ligands without disturbing αIIbβ3 expression. Because immobilized fibrinogen and fibrin are higher affinity/avidity ligands for αIIbβ3, we performed cell adhesion and clot retraction assays. In sharp contrast to KO mutation and Asp163AlaαIIbβ3, Tyr143HisαIIbβ3-expressing cells still had some ability for cell adhesion and clot retraction. Thus, the functional defect induced by Tyr143HisαIIb is likely caused by its allosteric effect rather than by a defect in the ligand-binding site itself. These detailed structure–function analyses provide better understanding of the ligand-binding sites in integrins.

scholarly journals Identification of putative ligand-binding sites of the integrin α4β1 (VLA-4, CD49d/CD29)

1995 ◽  
Vol 305 (3) ◽  
pp. 945-951 ◽  
Author(s):  
T Kamata ◽  
W Puzon ◽  
Y Takada
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Adhesion Molecule ◽  
Cho Cells ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Cell Adhesion Molecule ◽  
Chinese Hamster ◽  
Ligand Binding Sites ◽  
Cell Adhesion Molecule 1

Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected].

Ligand binding to integrin αvβ3requires tyrosine 178 in the αv subunit

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 175-182 ◽  
Author(s):  
Shigenori Honda ◽  
Yoshiaki Tomiyama ◽  
Nisar Pampori ◽  
Hirokazu Kashiwagi ◽  
Teruo Kiyoi ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Integrin Αvβ3 ◽  
Alanine Scanning Mutagenesis ◽  
Α Subunit ◽  
Fibrinogen Binding ◽  
293 Cells ◽  
Ligand Binding Sites ◽  
Arginine Glycine Aspartic Acid

Abstract Integrin αvβ3 has been implicated in angiogenesis and other biological processes. However, the ligand-binding sites in αv, a non–I-domain α subunit, remain to be identified. Recently in αIIb, the other partner of the β3 subunit, several discontinuous residues important for ligand binding were identified in the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alanine-scanning mutagenesis in 293 cells was used to investigate the role of these loops (cysteine [C]142-C155 and glycine [G]172-G181) of αv in ligand binding. Wild-type αvβ3 was able to bind soluble fibrinogen following integrin activation either by 0.5 mM manganese dichloride (MnCl2) or a mutation of β3 threonine (T)562 to asparagine. However, mutation of tyrosine (Y)178 to alanine in the predicted G172-G181 loop of αv abolished fibrinogen binding, and alanine (A) substitutions at adjacent residues phenylalanine (F)177 and tryptophan (W)179 had a similar effect. Cells expressing Y178Aαvalso failed to bind to immobilized fibrinogen. Moreover, the Y178A mutation abolished the binding of WOW-1 Fab, a monovalent ligand-mimetic anti-αvβ3 antibody, and the expression of β3 ligand–induced binding sites (LIBS) induced by arginine-glycine-aspartic acid-tryptophan (RGDW). In sharp contrast to the data obtained with αIIb, none of the mutations in the predicted W3 4-1 loop in αv impaired ligand binding. These results implicate αv Y178 in ligand binding to αvβ3, and they suggest that there are key structural differences in the adhesive ligand-binding sites of αvβ3 and αIIbβ3.

scholarly journals Amino Acids in Nine Ligand-Prefer Ramachandran Regions

2015 ◽  
Vol 2015 ◽  
pp. 1-10
Author(s):  
Chen Cao ◽  
Lincong Wang ◽  
Xiaoyang Chen ◽  
Shuxue Zou ◽  
Guishen Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hydrogen Bonds ◽  
Ligand Binding ◽  
Binding Sites ◽  
Van Der Waals ◽  
Dihedral Angles ◽  
Ligand Binding Sites ◽  
And Function ◽  
The Relationship ◽  
Van Der Waals Contacts

Several secondary structures, such asπ-helix and left-handed helix, have been frequently identified at protein ligand-binding sites. A secondary structure is considered to be constrained to a specific region of dihedral angles. However, a comprehensive analysis of the correlation between main chain dihedral angles and ligand-binding sites has not been performed. We undertook an extensive analysis of the relationship between dihedral angles in proteins and their distance to ligand-binding sites, frequency of occurrence, molecular potential energy, amino acid composition, van der Waals contacts, and hydrogen bonds with ligands. The results showed that the values of dihedral angles have a strong preference for ligand-binding sites at certain regions in the Ramachandran plot. We discovered that amino acids preceding the ligand-preferϕ/ψbox residues are exposed more to solvents, whereas amino acids following ligand-preferϕ/ψbox residues form more hydrogen bonds and van der Waals contacts with ligands. Our method exhibited a similar performance compared with the program Ligsite-csc for both ligand-bound structures and ligand-free structures when just one ligand-binding site was predicted. These results should be useful for the prediction of protein ligand-binding sites and for analysing the relationship between structure and function.

Missense mutations in the β3 subunit have a different impact on the expression and function between αIIbβ3 and αvβ3

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 931-938 ◽  
Author(s):  
Seiji Tadokoro ◽  
Yoshiaki Tomiyama ◽  
Shigenori Honda ◽  
Hirokazu Kashiwagi ◽  
Satoru Kosugi ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Regulatory Mechanism ◽  
Japanese Patients ◽  
Enzyme Linked Immunosorbent Assay ◽  
Missense Mutations ◽  
Glanzmann Thrombasthenia ◽  
Binding Function ◽  
Expression Studies ◽  
293 Cells ◽  
And Function

Abstract αIIbβ3 and αvβ3 belong to the β3integrin subfamily. Although the β3 subunit is a key regulator for the biosynthesis of β3 integrins, it remains obscure whether missense mutations in β3 may induce the same defects in both αIIbβ3 and αvβ3. In this study, it is revealed that thrombasthenic platelets with a His280Pro mutation in β3, which is prevalent in Japanese patients with Glanzmann thrombasthenia, did contain significant amounts of αvβ3 (about 50% of control) using sensitive enzyme-linked immunosorbent assay. Expression studies showed that the His280Proβ3 mutation impaired αIIbβ3 expression but not αvβ3 expression in 293 cells. To extend these findings, the effects of several β3 missense mutations leading to an impaired αIIbβ3expression on αvβ3 function as well as expression was examined: Leu117Trp, Ser162Leu, Arg216Gln, Cys374Tyr, and a newly created Arg216Gln/Leu292Ser mutation. Leu117Trp and Cys374Tyr β3 mutations did impair αvβ3 expression, while Ser162Leu, Arg216Gln, and Arg216Gln/Leu292Ser mutations did not. With regard to ligand binding function, Ser162Leu mutation induced especially distinct effects between 2 β3 integrins: it markedly impaired ligand binding to αIIbβ3 but not to αvβ3 at all. These data clearly demonstrate that the biosynthesis and the ligand binding function of αIIbβ3 and those of αvβ3 are regulated in part by different mechanisms. Present data would be a clue to elucidate the regulatory mechanism of expression and function of β3 integrins.

scholarly journals A novel region of the α4 integrin subunit with a modulatory role in VLA-4-mediated cell adhesion to fibronectin

1997 ◽  
Vol 327 (3) ◽  
pp. 727-733 ◽  
Author(s):  
Marisa MUÑOZ ◽  
Juan SERRADOR ◽  
Marta NIETO ◽  
Alfonso LUQUE ◽  
Francisco SÁNCHEZ-MADRID ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Binding Sites ◽  
Close Association ◽  
Cell Aggregation ◽  
Integrin Subunit ◽  
Vascular Cell Adhesion ◽  
Ligand Binding Sites ◽  
Potential Ligand ◽  
Cell Adhesion Molecule 1

The integrin VLA-4 (α4β1) is a receptor for fibronectin and vascular cell-adhesion molecule 1 (VCAM-1). Four functionally different epitopes, designated A, B1, B2 and C, have previously been defined on the α4 subunit. Using K562 α4 mutant transfectants we found that α4 amino acids Tyr151, Gln152, Asp153, Tyr154 and Val155 are important for the structure of the epitope B2. Mutations at α4 Gln152 substantially impaired the transfectant adhesion to a CS-1-containing fragment of fibronectin (FN-H89), whereas this adhesion was not affected on the other α4 mutant transfectants. None of the α4 mutations significantly altered the adhesion of the different α4 transfectants to VCAM-1. In addition, we have identified residues Gln152, Asp153 and Tyr154 as part of the α4 epitope B2 involved in homotypic cell aggregation. The decrease in adhesion to FN-H89 shown by Gln152 α4 mutant transfectants was the result of an inefficient binding of FN-H89 by VLA-4 mutated at this residue. Also, mutant VLA-4 displayed an altered reactivity with HUTS-21, an anti-β1 monoclonal antibody that reacts with functionally active VLA integrins. Adhesion to FN-H89 was not restored unless stimuli that increase the ligand-binding affinity of VLA heterodimers were added, suggesting that cell adhesion was affected in the initial phases. These results indicate that α4 Gln152 modulates cell adhesion to FN-H89 by playing important roles in the maintenance and/or the acquisition of an active state of VLA-4, an integrin that is normally expressed on the cell surface in a range of multiple activation states. The location of the α4 Gln152 residue on a loop of the upper surface of the proposed β-propeller structure suggests a close association with potential ligand-binding sites.

scholarly journals Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus

2015 ◽  
Vol 471 (3) ◽  
pp. 403-414 ◽  
Author(s):  
M. Florencia Rey-Burusco ◽  
Marina Ibáñez-Shimabukuro ◽  
Mads Gabrielsen ◽  
Gisela R. Franchini ◽  
Andrew J. Roe ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Ligand Binding ◽  
Tissue Distribution ◽  
Binding Sites ◽  
Binding Proteins ◽  
Retinol Binding Protein ◽  
Internal Cavity ◽  
Binding Characteristics ◽  
Necator Americanus ◽  
Ligand Binding Sites

Necator americanus fatty acid and retinol-binding protein-1 (Na-FAR-1) is an abundantly expressed FAR from a parasitic hookworm. The present work describes its tissue distribution, structure and ligand-binding characteristics and shows that Na-FAR-1 expands to transport multiple FA molecules in its internal cavity.

scholarly journals Adenosine Receptors of Cerebral Microvessels and Choroid Plexus

1986 ◽  
Vol 6 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Rajesh N. Kalaria ◽  
Sami I. Harik
Keyword(s):  
Cerebral Cortex ◽  
Ligand Binding ◽  
Choroid Plexus ◽  
Adenosine Receptors ◽  
Binding Sites ◽  
Specific Binding ◽  
Cerebral Microvessels ◽  
High Affinity ◽  
Receptor Sites ◽  
Ligand Binding Sites

We studied, by ligand binding methods, the two adenosine receptors, A, and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]l-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'- N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity ( Kd ∼ 250 n M) and low-affinity ( Kd ∼ 1–2 μ M) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these microvessels.

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