The Membrane Anchor Influences Ligand Binding Two-dimensional Kinetic Rates and Three-dimensional Affinity of FcγRIII (CD16)

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Binding of pathogen-bound immunoglobulin G (IgGFc ␥ receptors (Fc␥Rs) 7 are a family of cell surface glycoproteins with varying affinities for the Fc region of immunoglobulins G (IgG). Three classes of human Fc␥Rs have been described: Fc␥RI (CD64), Fc␥RII (CD32), and Fc␥RIII (CD16), which are widely distributed in hematopoietic cell lineages. These include at least 12 different isoforms many of which are polymorphic. For example, CD16 has two isoforms, a and b, that differ by six amino acids in the extracellular domain and the presence (CD16a) or absence (CD16b) of the transmembrane and cytoplasmic domains (1).The IgG-Fc␥R interaction-mediated binding of antibody-opsonized pathogens to leukocytes is a key event by which antibody effector functions are initiated. Kinetic rates and binding affinity are critical determinants of an IgG-Fc␥R interaction, as they control how likely and how rapidly such binding will occur, how many bonds will be formed, and how long the bonds last. In addition, it has been hypothesized that these parameters are related to the signaling events following the initial binding (2-4). Using a micropipette method, we have measured the kinetic rates and binding affinities of several IgG-Fc␥R interactions when both interacting molecules are anchored on apposing surfaces. We found that in this assay the half-lives of these IgG-Fc␥R bonds are in the order of seconds (5-9).In addition to interaction parameters of surface-linked molecules, the kinetic rates and affinities of Fc␥Rs for soluble IgG are also of interest, because in vivo interactions of immobilized IgG to leukocyte Fc␥Rs are subject to competitive binding of soluble antibodies in sera. Of the three Fc␥Rs, CD64 binds to monomeric IgG with high affinity (K d ϳ tens of nM) (10), CD32 and CD16b are of low affinities (K d , several and several tens of M, respectively) (6, 11, 12), and CD16a is considered as an intermediate affinity receptor (K d ϳ hundreds of M) for monomeric IgG (6,10,12). In addition to the above results, which were obtained by conventional assays using Fc␥R-expressing cells, there were two studies using the surface plasmon resonance (SPR) technology and soluble Fc␥Rs. Galon et al. (13) reported half-lives of the order of 10 min and equilibrium dissociation constants of several micromolar for sCD16b NA2 binding to human (h) IgG1 and IgG3. By comparison, Maenaka et al. (14) found a much faster off-rate (half-lives of the order of seconds) but similar K d for IgG-sCD16b NA2 binding. Here, we report kinetic and affinity measurements of a soluble dimeric human Fc␥RIIIa (sCD16a) interacting with various IgG ligands in both monomeric and multimeric forms using SPR. We found that sCD16a interacted with monomeric rabbit (Rb) IgG, hIgG, hIgG1, and hIgG3 with on-rates of 1.8 ϫ 10 4 , 6.5 ϫ 10 3 , 8.2 ϫ 10 3 , and 1.1 ϫ 10 4 M Ϫ1 s Ϫ1, off-rates of 1.9 ϫ 10 Ϫ2 , 4.7 ϫ 10 Ϫ3 , 5.7 ϫ 10 Ϫ3 , and 5.9 ϫ 10 Ϫ3 s Ϫ1 , and equilibrium dissociation constants of 1.1, 0.72, 0.71, and 0.56 M, respectively. sCD16a bound with aggregated li...

Section: ؊1 Smentioning

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: ؊1 Smentioning

confidence: 99%

See 1 more Smart Citation

Affinity and Kinetic Analysis of Fcγ Receptor IIIa (CD16a) Binding to IgG Ligands

Jiang²,

Nagarajan

et al. 2007

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“…The F/G loop also hosts critical epitopes for IgG binding (10). The CD16b-CLBFcgran1 dissociation constant, K d (ϭ 1/K a ), was found to be 8 Ϯ 2 nM with reconstituted RBCs and 12 Ϯ 5 nM with transfected CHO cells (11), indicating the lack of any significant binding enhancement for RBCs with an antibody in solution. It is also further evidence of the equivalency of the two expression modes.…”

Section: Enhanced Binding To Rbcs Is Not Evident In the Threedimensiomentioning

confidence: 99%

Quantifying the Impact of Membrane Microtopology on Effective Two-dimensional Affinity

Williams¹,

Nagarajan²,

Selvaraj³

et al. 2001

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Just as interactions of soluble proteins are affected by the solvent, membrane protein binding is influenced by the surface environment. This is particularly true for adhesion receptors because their function requires tightly apposed membranes. We sought to demonstrate, and further, to quantify the possible scale of this phenomenon by comparing the effective affinity and kinetic rates of an adhesion receptor (CD16b) placed in three distinct environments: red blood cells (RBCs), detached Chinese hamster ovary (CHO) cells, and K562 cells. Effective affinity reflects both the intrinsic receptor-ligand kinetics and the effectiveness of their presentation by the host membranes. Expression of CD16b, a low affinity Fc␥ receptor, was established by either transfection or spontaneous insertion via its glycosylphosphatidylinositol anchor. Binding to IgG-coated RBCs, measured using a micropipette method, indicated a 50-fold increase in effective affinity for receptors on RBCs over CHO and K562 cells, whereas the off rates were similar for all three. Electron microscopy confirmed that specific tight contacts were broad in RBC-RBC conjugates but sparse in CHO-RBC conjugates. We suggest that through modulation of surface roughness the cytoskeleton can greatly impact the effectiveness of adhesion molecules, even those with no cytoplasmic structures. Implications for locomotion and static adhesion are discussed.Cell-cell bond formation must be preceded by the creation of a contact zone, a region in which surface-bound receptors and ligands are able to bridge narrow gaps, properly aligning by lateral and rotational diffusion (1). Rough cells initially may form isolated point contacts that over time might be broadened and connected through active membrane processes (2). Alternatively, the cells may simply possess inherently smooth surfaces capable of broad initial contacts. A smooth membrane will not enhance binding to a soluble ligand because the soluble protein can diffuse freely into the membrane folds of rough cells. However, in adhering to immobilized ligand, the ability to form an expansive tight contact area is a distinct advantage. Therefore, cell-cell adhesion depends not only on the intrinsic kinetic rates of the receptor-ligand interaction but also on how effectively the host membranes present these molecules (3).In this study we quantified the effects of various microtopological presentations on cell adhesion by comparing the effective affinity of the same receptor (CD16b) in three distinct host environments: erythrocytes (RBCs), 1 Chinese hamster ovary (CHO) cells, and erythroleukemic K562 cells. Although all other Fc receptors have intracellular domains, CD16b (Fc␥RIIIb) terminates at the lipid bilayer with a glycosylphosphatidylinositol (GPI) moiety and so has no direct cytoskeletal interface. Currently there are no data to suggest the existence of multiple activation states in CD16b. These simplifications permit a more focused investigation into the role of the membrane environment in the functionality of this rec...

“…Therefore, it is possible that the cleavage and processing of RAET1G could be altered on receipt of cellular stress signals. There are other reports of two isoforms of some proteins regulated post-transcriptionally (21). Expression of both transmembrane and GPI-linked forms of NKG2D ligands could be a host strategy to evade subversion by viruses as two alternative coupling mechanisms would be more difficult to target.…”

Section: Discussionmentioning

confidence: 99%

Post-translational Modification of the NKG2D Ligand RAET1G Leads to Cell Surface Expression of a Glycosylphosphatidylinositol-linked Isoform

Ohashi

Trowsdale

2010

NKG2D is an important activating receptor on lymphocytes. In human, it interacts with two groups of ligands: the major histocompatibility complex class I chain-related A/B (MICA/B) family and the UL-16 binding protein (ULBP) family, also known as retinoic acid early transcript (RAET1). MIC proteins are membrane-anchored, but all of the ULBP/RAET1 proteins, except for RAET1E and RAET1G, are glycosylphosphatidylinositol (GPI)-anchored. To address the reason for these differences we studied the association of RAET1G with the membrane. Using epitope-tagged RAET1G protein in conjunction with antibodies to different parts of the molecule and in pulsechase experiments, we showed that the C terminus of the protein was cleaved soon after protein synthesis. Endoglycosidase H and peptide N-glycosidase treatment and cell surface immunoprecipitation indicated that most of the protein stayed in the endoplasmic reticulum, but some of the cleaved form was modified in the Golgi and transported to the cell surface. We examined the possibility of GPI anchoring of the protein in three ways: (i) Phosphatidylinositol (PI)-specific phospholipase C released the PI-linked form of the protein.(ii) The surface expression pattern of RAET1G decreased in cells defective in GPI anchoring through mutant GPI-amidase. (iii) Site-directed mutagenesis, to disrupt residues predicted to facilitate GPI-anchoring, resulted in diminished surface expression of RAET1G. Thus, a form of RAET1G is GPI-anchored, in line with most other ULBP/RAET1 family proteins. The cytoplasmic tail and transmembrane domains appear to result from gene duplication and frameshift mutation. Together with our previous results, our data suggest that RAET1G is regulated post-translationally to produce a GPI-anchored isoform. NKG2D3 is a C-type lectin-like activating receptor, expressed on natural killer (NK) cell and T cells, that plays an important role in triggering cytotoxic activity. Remarkably, the receptor interacts with multiple different ligands. In humans, the ligands fall into two groups, namely the MHC class I chainrelated A/B (MICA/B) family and the UL-16-binding protein (ULBP) or retinoic acid expressed transcript (RAET1) family (1-4). In mice, there are at least six NKG2D ligands; some of which (Rae1␣-⑀, H60c) are GPI-anchored, and the other three, H60a, H60b, and Mult1 (murine UL-16-binding protein-like transcript 1) are transmembrane proteins (5, 6).MICA/B and ULBP/RAET1 family proteins differ in domain structure and share low amino acid sequence similarity. Both MIC family proteins, MICA and MICB, consist of three ␣ domains, transmembrane domains, and cytoplasmic tail. In contrast, ULBP/RAET1 proteins have two ␣ domains and, of the six molecules, four (ULBP1, ULBP2, ULBP3, and ULBP6 (RAET1L)), are GPI-anchored proteins (4, 7). The other two, ULBP4 (RAET1E) and ULBP5 (RAET1G) have been considered to have transmembrane domains (4,8).ULBP/RAET1 family proteins share different degrees of sequence similarity and may have arisen by a series of gene duplications (4). So...