Predicting ligand-binding function in families of bacterial receptors

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.

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Participation of Critical Residues from the Extreme C-Terminal End of the Human Androgen Receptor in the Ligand Binding Function†

Biochemistry ◽

10.1021/bi010146q ◽

2001 ◽

Vol 40 (29) ◽

pp. 8431-8437 ◽

Cited By ~ 14

Author(s):

Bouchra Tahiri ◽

Gilles Auzou ◽

Jean-Claude Nicolas ◽

Charles Sultan ◽

Brigitte Lupo

Keyword(s):

Androgen Receptor ◽

Ligand Binding ◽

Binding Function ◽

Critical Residues ◽

Human Androgen Receptor

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Cell-specific, activation-dependent regulation of neutrophil CD32A ligand-binding function

Blood ◽

10.1182/blood.v95.3.1069.003k14_1069_1077 ◽

2000 ◽

Vol 95 (3) ◽

pp. 1069-1077 ◽

Cited By ~ 48

Author(s):

Shanmugam Nagarajan ◽

Kala Venkiteswaran ◽

Michael Anderson ◽

Umar Sayed ◽

Cheng Zhu ◽

...

Keyword(s):

Ligand Binding ◽

Chinese Hamster Ovary Cells ◽

Biological Significance ◽

Chinese Hamster ◽

Phosphatidyl Inositol ◽

Ovary Cells ◽

Binding Function ◽

Affinity Modulation ◽

Activated Neutrophils ◽

Novel Strategy

Neutrophils express 2 low-affinity FcγR, FcγRIIIB (CD16B), and FcγRIIA (CD32A). CD16B is a glycosyl-phosphatidyl inositol-anchored molecule, whereas CD32A is a polypeptide-anchored molecule. These 2 receptors also differ in their signaling. The biological significance of coexpression of 2 FcγRs with distinct membrane anchors and signaling capacities is not clearly understood. Using neutrophils from a CD16B-deficient donor and normal neutrophils treated with anti-CD16 monoclonal antibodies, the authors demonstrated that affinity modulation of CD32A is one of the mechanisms by which neutrophils regulate their FcγR-dependent functions. Neutrophils isolated from a CD16B− donor rosetted poorly with sheep erythrocytes opsonized with rabbit IgG (EA) (12% ± 2% versus 80% ± 6% for control) and were unable to mediate immunophagocytosis. However, activation of CD16B−neutrophils with fMLP, a bacterial chemotactic peptide, increased the CD32A-dependent EA rosetting to 58%. The CD32A-dependent rosetting of fMLP-activated normal neutrophils also increased nearly 5-fold, but there was no increase in CD32A expression. The CD32A-dependent immune complex (IC) binding was also increased in activated neutrophils. This affinity regulation was not observed with CD32A expressed on Chinese hamster ovary cells. These results suggest that in resting neutrophils CD32A is in a low-affinity state and that these cells primarily engage CD16B for IC binding. However, once the neutrophils are activated, the CD32A is converted to a high-affinity state that leads to CD32A-dependent ligand binding and signaling. These results suggest that neutrophils adopt a novel strategy to engage the 2 different FcγR selectively during physiologic and pathologic conditions to carry out their functions efficiently.

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Naturally Occurring CCR5 Extracellular and Transmembrane Domain Variants Affect HIV-1 Co-receptor and Ligand Binding Function

Journal of Biological Chemistry ◽

10.1074/jbc.274.23.16228 ◽

1999 ◽

Vol 274 (23) ◽

pp. 16228-16234 ◽

Cited By ~ 53

Author(s):

O. M. Zack Howard ◽

Aiko-Konno Shirakawa ◽

Jim A. Turpin ◽

Andrew Maynard ◽

Gregory J. Tobin ◽

...

Keyword(s):

Ligand Binding ◽

Transmembrane Domain ◽

Naturally Occurring ◽

Binding Function ◽

Hiv 1

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The role of glycosylation in the biosynthesis and acquisition of ligand- binding activity of the folate-binding protein in cultured KB cells

Blood ◽

10.1182/blood.v77.6.1171.1171 ◽

1991 ◽

Vol 77 (6) ◽

pp. 1171-1180 ◽

Cited By ~ 15

Author(s):

CA Luhrs

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Binding Protein ◽

Binding Property ◽

Affinity Matrix ◽

Kb Cells ◽

Folate Binding Protein ◽

Binding Function ◽

High Mannose ◽

Endoglycosidase H

Abstract The biosynthesis, processing, and ligand-binding function of the membrane-associated and soluble forms of the folate-binding protein (FBP) in KB cells, a cultured human cell line, were studied using pulse- chase labeling with [35S] methionine. The intermediary and mature forms of the protein were isolated by immunoprecipitation and affinity chromatography and analyzed by sodium dodecyl sulfate electrophoresis and autoradiography. The earliest species identified had an Mr of 32 Kd and disappeared over 5 hours concomitant with the appearance of a 38-Kd cellular FBP. As the 38-Kd species disappeared, a 40-Kd form appeared in the medium. When tunicamycin was added to the culture medium to inhibit core glycosylation, a 26-Kd aglycosylated species and minor 28- Kd and 30-Kd forms appeared. Endoglycosidase H, which cleaves high mannose but not complex oligosaccharides, reduced the 32-Kd species to 26-Kd but the enzyme had no effect on the 38-Kd form, indicating that this species is complex glycosylated. Monensin, which blocks complex glycosylation, also inhibited synthesis of the 38-Kd species. Although both the 32-Kd and 38-Kd forms had ligand-binding sites (as demonstrated by binding to a folate-Sepharose matrix), the 26-Kd aglycosylated species, labeled in the presence of tunicamycin, lacked similar binding sites because it did not bind to the affinity matrix. In contrast, the aglycosylated 26-Kd form, which was obtained by treatment of the 32-Kd species with endoglycosidase H, did bind to the folate affinity matrix, indicating that it retained ligand-binding function. Thus, the high mannose oligosaccharide moiety is not required for the folate-binding property of the FBP, but its addition to the polypeptide chain precedes a later step that is necessary for the mature protein to have ligand-binding function.

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A Mutation in the  Subunit of the Platelet Integrin IIbβ3 Identifies a Novel Region Important for Ligand Binding

Blood ◽

10.1182/blood.v93.3.918 ◽

1999 ◽

Vol 93 (3) ◽

pp. 918-924 ◽

Cited By ~ 19

Author(s):

Eileen Collins Tozer ◽

Elizabeth K. Baker ◽

Mark H. Ginsberg ◽

Joseph C. Loftus

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Single Amino Acid ◽

Chimeric Receptor ◽

Amino Acid Residues ◽

Genetic Approach ◽

Specific Amino Acid ◽

Transfected Cells ◽

Binding Function ◽

A Cell

Abstract An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIbβ. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (D→V) was identified. Although well expressed on the surface of transfected cells, IIbD224Vβ3 as well as IIbD224Aβ3 did not bind IIbβ3-specific ligands or a RGD peptide, a ligand shared in common with vβ3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIbβ3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin  subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed β-propeller model for integrin  subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIbβ3 receptor.

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A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques.

The Journal of Cell Biology ◽

10.1083/jcb.119.4.913 ◽

1992 ◽

Vol 119 (4) ◽

pp. 913-921 ◽

Cited By ~ 83

Author(s):

Y Takada ◽

J Ylänne ◽

D Mandelman ◽

W Puzon ◽

M H Ginsberg

Keyword(s):

Ligand Binding ◽

Point Mutation ◽

Dominant Negative ◽

Rgd Sequence ◽

Binding Function ◽

Beta 1 Integrin ◽

Conserved Region ◽

Adhesion Plaques

A point mutation in a highly conserved region of the beta 1 subunit, Asp130 to Ala (D130A) substitution, abrogates the Arg-Gly-Asp (RGD)-dependent binding of alpha 5 beta 1 to fibronectin (FN) without disrupting gross structure or heterodimer assembly. The D130A mutation also interferes with binding to invasin, a ligand that lacks RGD sequence. In spite of the lack of detectable FN binding by alpha 5 beta 1(D130A), it was recruited to adhesion plaques formed on FN by endogenous hamster receptors. Thus, intact ligand binding function is not required for recruitment of alpha 5 beta 1 to adhesion plaques. Overexpression of beta 1(D130A) partially interfered with endogenous alpha 5 beta 1 function, thus defining a dominant negative beta 1 integrin mutation.

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Platelet Surface PDI Controls the Ligand-Binding Function of Glycoprotein Ibalpha and Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions

Blood ◽

10.1182/blood.v126.23.235.235 ◽

2015 ◽

Vol 126 (23) ◽

pp. 235-235

Author(s):

Kyungho Kim ◽

Jing Li ◽

Robert K Andrews ◽

Joyce Chiu ◽

Philip Hogg ◽

...

Keyword(s):

Ligand Binding ◽

Disulfide Bond ◽

Conflicts Of Interest ◽

Ischemia Reperfusion ◽

Critical Role ◽

Conditional Knockout ◽

Spectrometric Analysis ◽

Platelet Surface ◽

Isomerase Activity ◽

Binding Function

Abstract We previously showed that the isomerase activity of platelet surface protein disulfide isomerase (PDI), a prototypic thiol isomerase, regulates the full activation of αIIbβ3 integrin and is important for platelet accumulation, but not hemostasis, following vascular injury (Kim et al. Blood 2013). However, it remains unclear whether platelet PDI regulates the function of other platelet surface receptors. Since it was reported that platelet PDI and glycoprotein Ibα (GPIbα) were physically close on the platelet surface (Burgess et al. JBC 2000), we investigated whether platelet PDI regulates GPIbα function using megakaryocyte-specific PDI conditional knockout (CKO) mice developed by us. We found that PDI-null platelets showed a significant defect in GPIbα-mediated agglutination and von Willebrand factor (vWF) binding. Those defects were completely rescued by exogenously-added recombinant wild-type PDI (wtPDI), but not mutant PDI (dmPDI) lacking the isomerase activity. Consistently, inhibition of platelet PDI with blocking antibodies impaired GPIbα-mediated agglutination and vWF binding in human and mouse platelets, suggesting that the isomerase activity of platelet surface PDI controls the ligand-binding function of GPIbα. Studies using surface plasmon resonance revealed that wtPDI and dmPDI bound to immobilized GPIbα with a dissociation constant of 0.9 and 1.2 µM, respectively. Mass spectrometric analysis indicated that the Cys4-Cys17 disulfide bond in GPIbα, which has an allosteric -RHStaple configuration, was preferentially reduced by wtPDI, but not dmPDI. Consistent with previous studies showing that platelet GPIbα is important for platelet-neutrophil interactions, we observed that deletion of platelet PDI significantly reduced platelet-neutrophil interactions under shear conditions. Importantly, using real-time fluorescence intravital microscopy with megakaryocyte-specific PDI CKO mice, we demonstrated that platelet-specific PDI deletion did not influence neutrophil adhesion but markedly reduced platelet adhesion and accumulation on the adherent neutrophils on TNF-α-inflamed cremaster muscle venules. Infusion of wtPDI, but not dmPDI, restored reduced platelet-neutrophil interactions in the PDI CKO mice, implying the importance of platelet surface PDI activity in regulating GPIbα-mediated platelet-neutrophil interactions. Using a thromboinflammation model of hepatic ischemia/reperfusion injury, we found that platelet-specific PDI CKO mice exhibited a significant reduction in the infarct size and the serum levels of aspartate aminotransferase, a marker of liver damage. Our results suggest that the isomerase activity of platelet surface PDI regulates the redox state of the Cys4-Cys17 disulfide bond in GPIbα and is required for the ligand-binding function of GPIbα, thereby playing a critical role during platelet-neutrophil interactions under thromboinflammatory conditions. Disclosures No relevant conflicts of interest to declare.

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Differential effects of cholesterol and desmosterol on the ligand binding function of the hippocampal serotonin1A receptor: Implications in desmosterolosis

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2009.07.004 ◽

2009 ◽

Vol 1788 (10) ◽

pp. 2169-2173 ◽

Cited By ~ 27

Author(s):

Pushpendra Singh ◽

Roopali Saxena ◽

Yamuna Devi Paila ◽

Md. Jafurulla ◽

Amitabha Chattopadhyay

Keyword(s):

Ligand Binding ◽

Differential Effects ◽

Serotonin1a Receptor ◽

Binding Function

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Targeted inactivation of endothelial lipase attenuates lung allergic inflammation through raising plasma HDL level and inhibiting eosinophil infiltration

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90530.2008 ◽

2009 ◽

Vol 296 (4) ◽

pp. L594-L602 ◽

Cited By ~ 16

Author(s):

Hiroshi Otera ◽

Tatsuro Ishida ◽

Teruaki Nishiuma ◽

Kazuyuki Kobayashi ◽

Yoshikazu Kotani ◽

...

Keyword(s):

Endothelial Cells ◽

Ligand Binding ◽

Smooth Muscles ◽

Allergic Inflammation ◽

Eosinophil Infiltration ◽

Endothelial Lipase ◽

Alveolar Type ◽

Protective Effects ◽

Binding Function ◽

Targeted Inactivation

Endothelial lipase (EL) is a novel phospholipase that determines plasma high-density lipoprotein cholesterol (HDL-C) levels. We have investigated the role of HDL-C in lung allergic inflammation by using EL knockout (EL-KO) mice that are high in HDL-C. EL-KO and wild-type control mice were sensitized and challenged with ovalbumin to evoke eosinophilic inflammation in the lung. EL was expressed in epithelial cells, alveolar type II cells, and endothelial cells in the lung, and its expression was upregulated during inflammation. Concomitant with attenuated hyperresponsiveness of the airway smooth muscles, the number of eosinophils in bronchoalveolar lavage and the expression of VCAM-1 were lower in EL-KO mice than in control mice. HDL reduced cytokine-induced VCAM-1 expression in cultured endothelial cells. When plasma HDL levels were decreased to similar levels in both mouse groups by adenovirus-mediated overexpression of EL, however, eosinophil infiltration was still lower in EL-KO mice. In vitro adhesion assays revealed that EL expression on the cell surface promoted the interaction of eosinophils through the ligand-binding function of EL. In summary, targeted inactivation of EL attenuated allergic inflammation in the lung, and the protective effects in EL-KO mice were associated with high plasma HDL levels, downregulation of VCAM-1, and loss of the direct ligand-binding function of EL. Thus EL is a novel modulator of the progression of allergic asthma.

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