Mark D. Hulett

Multiple regions of human Fc gamma RII (CD32) contribute to the binding of IgG.

The low affinity receptor for IgG, FcγRII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. FcγRII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human FcγRII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by FcγRII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between FcγRII and the structurally related FcεRI α chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116 and Ser130-Thr135 resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116 and Ser130-Thr135 domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131 profoundly decreased the binding of hIgG1, whereas substitution of Asp133 and Pro134 increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by FcγRII but, importantly, that the domain 2 regions Ser109-Val116 and Phe129-Thr135 also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of FcγRII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.

The second and third extracellular domains of FcγRI (CD64) confer the unique high affinity binding of IgG2a

FcgammaRI (CD64) is functionally unique as it is the only FcgammaR able to bind monomeric IgG with high affinity. FcgammaRI is also structurally distinct, containing an extracellular Ig-interactive region of three Ig-like domains in contrast to the two domains of the low affinity receptors FcgammaRII and FcgammaRIII. Previous studies have demonstrated that the third domain of FcgammaRI plays a crucial role in high affinity IgG binding of the receptor, with the first and second domains together forming a low affinity IgG binding motif. In this study the individual functional contributions of the first and second domains of FcgammaRI to IgG binding have been investigated. Chimeric FcgammaR were generated by exchanging extracellular domains between mouse FcgammaRI and the structurally related yet distinct low affinity receptor for IgG, mouse FcgammaRII. The replacement of both domains 1 and 2 of FcgammaRI with domains 1 and 2 of FcgammaRII results in a dramatic change in IgG binding characteristics, as this receptor loses the capacity to bind monomeric IgG with high affinity and also demonstrates a broader specificity (binding not only IgG2a but also IgG1 and 2b. IgG3 was not tested). However, the substitution of FcgammaRII domain 2 of this chimeric receptor with domain 2 of FcgammaRI (generating a chimeric receptor with domain 1 of FcgammaRII linked to domains 2 and 3 of FcgammaRI) was found to reconstitute the specific high affinity monomeric IgG2a binding of wild-type FcgammaRI, albeit with a slightly reduced affinity (1.8-fold lower than wild-type FcgammaRI). These findings suggest that it is the specific interaction between domains 2 and 3 of FcgammaRI, with domain 1 playing a supporting role in maintaining the conformational stability of the receptor, that is the major structural requirement to confer the unique Ig binding characteristics of FcgammaRI.

Identification of the Immunoglobulin Binding Regions (IBR) of FcγRII and FcɛRI

Fc receptors (FcR) are cell surface glycoproteins that bind the Fc region of immunoglobulin (Ig) and are found on the cell surface of the vast majority of leukocytes, FcR have been defined for all classes of Ig and play important roles in the protection of an organism against infection by providing the host with a link between immunological function (antibody production) and paraimmunological functions such as immune complex removal by macrophages and neutrophils or the sensitization of mast cells with IgE. The biological roles of FcR, their diversity, function and early biochemical characterization as well as their recently understood molecular genetics have been extensively reviewed elsewhere and will not be covered here (Spiegelberg 1984, Unkeless et al. 1988, Van de Winkel & Anderson 1991, Ravetch & Kinet 1991). This manuscript will review our attempts to define the regions of IgG and IgE FcR that are involved in Ig binding and develop a model of the structure of Fc receptors. Three distinct classes of human and murine FcR for IgG (FcyR) have been defined on the basis of their distinct structures, specificities and affinities for IgG. Thus FcyRI has a high affinity for monomeric IgG (10^M) and shows preferential binding of certain IgG subclasses, whereas FcyRII and FcyRIII have a very low affinity for IgG monomer (< 10*M) but avidly bind IgG complexes (Unkeless et al. 1988, Van de Winkel & Anderson 1991, Ravetch & Kinet 1991). A single high-affinity receptor for IgE (FceRI) is present on mast cells and binds IgE with affinity of 10M and is distinct from an unrelated low-affinity IgE receptor FccRII or CD23 (Metzger et al. 1986, Ravetch & Kinet 1991).

Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1.

Met-ase-1 is a 30000Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3− large granular lymphocytes. We screened a mouse genomic library with ratMet-ase-1 cDNA, and obtained bacteriophage clones that contained the mouseMet-ase-1 gene. The mouseMet-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. MouseMet-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3− GM1+ large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1+ cell line 4–16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouseMet-ase-1 mRNA. The 5′ flanking region of the mouseMet-ase-1 gene also shares considerable regions of identity with the 5′ flanking region of the ratMet-ase-1 gene. A 3.3 kb segment of 5′ sequence flanking the mouseMet-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouseMet-ase-1 5′ flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4–16 mouse NK1.1+ cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouseMet-ase-1. The predicted hexapropeptide of mouseMet-ase-1 (Asn−6 to Gln−1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouseMet-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouseMet-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.

Fcγ receptors: Gene structure and receptor function

Molecular studies of murine FcγR have revealed much exciting new information about the structure and regulation of FcγRI and FcγRII genes and of the FcγRI protein. The FcγRI gene is composed of six exons, whereas the FcγRII gene is composed of ten. The extracellular domains are encoded by individual exons in both genes (three in FcγRI and two in FcγRII); however, the FcγRII gene shows greatest complexity in the region encoding the cytoplasmic tail and membrane spanning region, which is encoded by four exons compared to only one in the FcγRI gene. Expression of FcγRII is controlled by elements within the first 641 bases upstream of the transcription initiation site. The function of the domains of FcγRI has been defined with the surprising finding that in the absence of the third domain the first two extracellular domains function as a broadly specific low affinity FcγRII-like receptor.

Cloning and characterization of a novel NK cell-specific serine protease gene and its functional 5′-flanking sequences

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30 000 Mr serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5′-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5′-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5′-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5′-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines.

The Structural Basis of the Interaction of IgE and FcεRI

FceRI is capable of inducing one of the most powerful and violent pharmacological responses known. Indeed the association of IgE with FceRI, and subsequent aggregation is a most important interaction in the induction of human disease, and causes more chronic misery (in the West at least) than the engagement of any other immunological receptor. As approximately one in five people are afflicted with IgE dependent allergies—most notably allergic rhinitis or ‘hay fever’ and asthma—there has been a large effort made by many groups in studying this receptor, its ligand and the consequences of its activation. The impetus to study this receptor probably stems from its pathological role rather than its physiological one, which is still somewhat undefined, but with evidence pointing to an anti-parasite role.1

Immunoglobulin Fc receptors: Diversity, structure, and function

Publisher Summary This chapter discusses the diversity, structure, and function of immunoglobulin (Ig) Fc receptors. Receptors for immunoglobulins, Fc receptors (FcR), play a fundamentally important role in immunity and resistance to infection by providing the means of linking humoral immunity to cellular effector mechanisms. Indeed, as will be seen below, these widespread and abundant receptors participate in the removal and destruction of antibody-coated particles; activate and induce cells to kill antibody-coated targets; are induced by, and, after cross linking, induce the production and release of cytokines and inflammatory mediators; transport immunoglobulins; and are also involved in the regulation of immunity. Fc receptors are cell membrane molecules that specifically bind the Fc portion of an immunoglobulin heavy chain. These receptors have been defined on leukocytes for all Ig classes—IgM, IgD, IgG, IgE, and IgA for which they are named, that is, the IgG receptors are Fc γR, IgE receptors are FcɛR, and so forth. Where more than one class of receptor is defined for an Ig isotype, the receptors are designated thus: FcγRI, FcγRII, or FcɛRI, FcɛRII, and so forth. The exception to these rules are the receptors expressed on non-leukocytes—that is, the receptor for polymeric Ig called the poly Ig receptor, or a unique receptor in the gut of neonatal rats, the FcRn. In addition to their specificity for Ig classes, Fc receptors are also classified based on reactivity with monoclonal antibodies and their affinity for Ig.