Zena K. Indik

Sequence Variation of Bovine Elastin mRNA Due to Alternative Splicing

Poly A+ RNA, isolated from a single 210 day fetal bovine nuchal ligament, was used to synthesize cDNA by the RNase H method, using AMV reverse transcriptase for first strand synthesis and DNA polymerase I for the second strand. The cDNA was inserted into lambda gt10 using EcoRI linkers, and recombinant phage containing elastin sequences were identified by hybridization with a 1.3 kb sheep elastin cDNA clone, pcSELI (Yoon, K. et al., Biochem. Biophys. Res. Comm. 118: 261-265, 1984). Three clones containing the largest inserts of 2.9, 2.8, and 2.6 kb were selected for further study. The complete sequence analysis of the 3 clones was correlated with the sequence of 10.2 kb of the bovine elastin gene. The analyses: (i) showed that the cDNA encompassed the great majority of the translated sequence, (ii) ordered the tryptic peptides of porcine tropoelastin, (iii) determined new amino acid sequences not previously found in the porcine peptides and (iv) demonstrated that alternative splicing of the primary transcript leads to significant variation in the sequence of the translated portion of the mRNA.

Production of recombinant human tropoelastin characterization and demonstration of immunologic and chemotactic activity

Tropoelastin cannot readily be prepared in quantity from natural sources and this has limited research in several important areas including structure/function relationships and fiber assembly. In order to eliminate this limitation, human tropoelastin has been expressed in a recombinant bacterial system and the protein has been highly purified. The size, amino acid composition, and sequence of the amino terminus of the recombinant tropoelastin (rTE) all agree with values predicted by the nucleotide sequence of the cDNA used in the expression vector. The rTE exhibits cross-reactivity with antibodies directed against a mixture of peptides derived from human elastin as well as antibody against a defined peptide located at the carboxy terminus of the protein. In addition, the rTE is chemotactic for fetal calf ligament fibroblasts. These results suggest that rTE could be a useful reagent for many types of studies.

Molecular dissection of Fcγ receptor-mediated phagocytosis

Abstract Using an experimental model in COS-1 cells, we have examined the structural requirements for phagocytosis of IgG-sensitized cells by Fcγ receptors. We have established that isoforms of each of the 3 classes of the Fcγ receptors, FcγRI, FcγRII and FcγRIII, are able to transmit a phagocytic signal in the absence of the other receptor class. FcγI and RcγRIIIA require a γ-subunit for this signaling event, but FcγRIIA does not. FcγRIIA and the γ-subunit associated with FcγRI and FcγRIIIA contain 2 copies of a conserved tyrosine-containing cytoplasmic sequence, YXXL. This sequence is important for phagocytosis and is phosphorylated on tyrosine after receptor ligation. The Fcγ receptors FcγRIIB1 and FcγRIIB2 which contain only 1 copy of the YXXL cytoplasmic sequence do not include the phagocytosis of IgG-coated cells. Thus, the Fcγ receptor isoforms differ in their ability to transmit a phagocytic signal. Structure/function studies also indicate that the Fcγ receptors which induce phagocytosis differ in their requirements for phagocytic signaling.

Differential kinase requirements in human and mouse Fc‐gamma receptor phagocytosis and endocytosis

Fc gamma receptors (FcγRs) contribute to the internalization of large and small immune complexes through phagocytosis and endocytosis, respectively. The molecular processes underlying these internalization mechanisms differ dramatically and have distinct outcomes in immune clearance and modulation of cell function. However, it is unclear how the same receptors (FcγR) binding to identical ligands (IgG) can elicit such distinct responses. We and others have shown that Syk kinase, Src‐related tyrosine kinases (SRTKs) and phosphatidyl inositol 3‐kinases (PI3K) play important roles in FcγR phagocytosis. Herein, we demonstrate that these kinases are not required for FcγR endocytosis. Endocytosis of heat‐aggregated IgG (HA‐IgG) by COS‐1 cells stably transfected with FcγRIIA or chimeric FcγRI‐γ‐γ (EC‐TM‐CYT) was not significantly altered by PP2, piceatannol, or wortmannin. In contrast, phagocytosis of large opsonized particles (IgG‐sensitized sheep erythrocytes, EA) was markedly reduced by these inhibitors. These results were confirmed in primary mouse bone marrow‐derived macrophages and freshly isolated human monocytes. Levels of receptor phosphorylation were similar when FcγRIIA was cross‐linked using HA‐IgG or EA. However, inhibition of FcγR phosphorylation prevented only FcγR phagocytosis. Finally, biochemical analyses of PI3K(p85)‐Syk binding indicated that direct interactions between native Syk and PI3K proteins are differentially regulated during FcγR phagocytosis and endocytosis. Overall, our results indicate that FcγR endocytosis and phagocytosis differ dramatically in their requirement for Syk, SRTKs, and PI3K, pointing to striking differences in their signal transduction mechanisms. We propose a competitive inhibition‐based model in which PI3K and c‐Cbl play contrasting roles in the induction of phagocytosis or endocytosis signaling cascades.

The effect of phosphatases SHP-1 and SHIP-1 on signaling by the ITIM- and ITAM-containing Fcγ receptors FcγRIIB and FcγRIIA

Inositol and tyrosine phosphatases have been implicated in inhibitory signaling by an Fc receptor for immunoglobulin G, FcγRIIB, in B cells, mast cells, and monocytes. Here, we propose a role for the Src homology 2 (SH2)‐containing tyrosine phosphatase‐1 (SHP‐1) in FcγRIIB‐mediated inhibition of FcγR signaling. Coexpression of SHP‐1 enhances FcγRIIB‐mediated inhibition of FcγRIIA phagocytosis in COS‐1 cells. SHP‐1 also enhances the reduction in FcγRIIA tyrosine phosphorylation that accompanies this inhibition. Significantly, tyrosine phosphorylation of Syk kinase is substantially inhibited by SHP‐1. Furthermore, the activation of SHP‐1 tyrosine phosphorylation is observed following stimulation of FcγRII in COS‐1 cells and in human monocytes. The SH2 domain containing inositol phosphatase (SHIP), SHIP‐1 also enhances FcγRIIB‐mediated inhibition of FcγRIIA, indicating that FcγRIIB can use more than one pathway for its inhibitory action. In addition, SHP‐1 and SHIP‐1 can inhibit FcγRIIA phagocytosis and signal transduction in the absence of FcγRIIB. The data support emerging evidence that SH2‐containing phosphatases, such as SHP‐1 and SHIP‐1, can modulate signaling by “activating” receptors.

Structure/function relationships of Fcγ receptors in phagocytosis

Abstract An important function of Fc γ receptors is the ingestion or phagocytosis of IgG sensitized cells. It has been difficult to clearly define the individual function of each receptor in phagocytosis because hematopoietic cells express multiple Fc γ receptor isoforms. To examine this issue, an in-vitro model system in COS-1 cells has been developed. When transfected with an appropriate Fc γ receptor, COS-1 cells which lack endogeneous Fc receptors, ingest IgG-sensitized cells. Using this model, a single class of human Fc γ receptor in the absence of other Fc receptors was observed to mediate phagocytosis. Furthermore, isoforms from each Fc γ receptor class can mediate phagocytosis although the requirements for phagocytosis differ. Investigation of the relationship between structure and function for Fc receptor-mediated phagocytosis established the importance of the cytoplasmic tyrosines of the receptor or its associated γ chain. For example, two cytoplasmic YXXL sequences, in a configuration similar to the conserved tyrosine containing motif found in immunoglobulin gene family receptors, are important for phagocytosis by the human Fc γ receptor, Fc γ RIIA. Fc γ RI and Fc γ RIIIA do not possess cytoplasmic tyrosines, but transmit a phagocytic signal through interaction with an associated γ -subunit which contains two YXXL sequences in a conserved motif required for phagocytosis. The human Fc γ RII isoforms, Fc γ RIIB1 and Fc γ RIIB2, do not induce phagocytosis and have only a single YXXL sequence. Crosslinking of the phagocytic Fc γ receptors induces tyrosine phosphorylation of either Fc γ RIIA or the γ chain and treatment with tyrosine kinase inhibitors reduces both phagocytosis and phosphorylation of the receptor tyrosine residues. The protein tyrosine kinase Syk, which is associated with the γ chain in monocytes/macrophages, dramatically enhances phagocytosis mediated by Fc γ RI and Fc γ RIIIA and also induces non-phagocytic Fc γ RI or Fc γ RIIIA expressing cells to acquire phagocytic capability.

The monocyte Fcγ receptors FcγRI/γ and FcγRIIA differ in their interaction with Syk and with Src‐related tyrosine kinases

There are important differences in signaling between the Fc receptor for immunoglobulin G (IgG) FcγRIIA, which uses the Ig tyrosine‐activating motif (ITAM) within its own cytoplasmic domain, and FcγRI, which transmits signals by means of an ITAM located within the cytoplasmic domain of its associated γ‐chain. For example, in transfected epithelial cells and COS‐1 cells, FcγRIIA mediates phagocytosis of IgG‐coated red blood cells more efficiently than does FcγRI/γ, and enhancement of phagocytosis by Syk kinase is more pronounced for FcγRI/γ than for FcγRIIA. In addition, structure/function studies indicate that the γ‐chain ITAM and the FcγRIIA ITAM have different requirements for mediating the phagocytic signal. To study the differences between FcγRIIA and FcγRI/γ, we examined the interaction of FcγRIIA and the FcγRI/γ chimera FcγRI‐γ‐γ (extracellular domain–transmembrane domain–cytoplasmic domain) with Syk kinase and with the Src‐related tyrosine kinases (SRTKs) Hck and Lyn in transfected COS‐1 cells. Our data indicate that FcγRIIA interacts more readily with Syk than does FcγRI‐γ‐γ and suggest that one consequence may be the greater phagocytic efficiency of FcγRIIA compared with FcγRI/γ. Furthermore, individual SRTKs affect the efficiency of phagocytosis differently for FcγRI‐γ‐γ and FcγRIIA and also influence the ability of these receptors to interact with Syk kinase. Taken together, the data suggest that differences in signaling by FcγRIIA and FcγRI‐γ‐γ are related in part to interaction with Syk and Src kinases and that individual SRTKs play different roles in FcγR‐mediated phagocytosis.

The Cytoplasmic Domain of Human FcγRIa Alters the Functional Properties of the FcγRI·γ-Chain Receptor Complex

The γ/ζ-chain family of proteins mediate cell activation for multiple immunoglobulin receptors. However, the recognition that these receptors may have distinct biologic functions suggests that additional signaling elements may contribute to functional diversity. We hypothesized that the cytoplasmic domain (CY) of the ligand binding α-chain alters the biological properties of the receptor complex. Using macrophage FcγRIa as a model system, we created stable transfectants expressing a full-length or a CY deletion mutant of human FcγRIa. Both receptors functionally associate with the endogenous murine γ-chain. However, we have established that the CY of FcγRIa directly contributes to the functional properties of the receptor complex. Deletion of the FcγRIa CY leads to slower kinetics of receptor-specific phagocytosis and endocytosis as well as lower total phagocytosis despite identical levels of receptor expression. Deletion of the CY also converts the phenotype of calcium independent FcγRIa-specific phagocytosis to a calcium-dependent phenotype. Finally, deletion of the CY abrogates FcγRIa-specific secretion of interleukin-6 but does not affect production of interleukin-1β. These results demonstrate a functional role for the CY of FcγRIa and provide a general model for understanding how multiple receptors that utilize the γ-chain can generate diversity in function.

Interaction of Two Phagocytic Host Defense Systems Fcγ RECEPTORS AND COMPLEMENT RECEPTOR 3

Phagocytosis of foreign pathogens by cells of the immune system is a vitally important function of innate immunity. The phagocytic response is initiated when ligands on the surface of invading microorganisms come in contact with receptors on the surface of phagocytic cells such as neutrophils, monocytes/macrophages, and dendritic cells. The complement receptor CR3 (CD11b/CD18, Mac-1) mediates the phagocytosis of complement protein (C3bi)-coated particles. Fcγ receptors (FcγRs) bind IgG-opsonized particles and provide a mechanism for immune clearance and phagocytosis of IgG-coated particles. We have observed that stimulation of FcγRs modulates CR3-mediated phagocytosis and that FcγRIIA and FcγRI exert opposite (stimulatory and inhibitory) effects. We have also determined that an intact FcγR immunoreceptor tyrosine-based activation motif is required for these effects, and we have investigated the involvement of downstream effectors. The ability to up-regulate or down-regulate CR3 signaling has important implications for therapeutics in disorders involving the host defense system.

The CY Domain of the FcγRIa α-Chain (CD64) Alters γ-Chain Tyrosine-based Signaling and Phagocytosis

Although the cytoplasmic domain of the human FcγRIa α-chain lacks tyrosine-based phosphorylation motifs, it modulates receptor cycling and receptor-specific cytokine production. The cytoplasmic domain of FcγRIa is constitutively phosphorylated, and the inhibition of dephosphorylation with okadaic acid, an inhibitor of type 1 and type 2A protein serine/threonine phosphatase, inhibits both receptor-induced activation of the early tyrosine phosphorylation cascade and receptor-specific phagocytosis. To explore the basis for these effects of the cytoplasmic domain of FcγRIa, we developed a series of human FcγRIa molecular variants, expressed in the murine macrophage cell line P388D1, and demonstrate that serine phosphorylation of the cytoplasmic domain is an important regulatory mechanism. Truncation of the cytoplasmic domain and mutation of the cytoplasmic domain serine residues to alanine abolish the okadaic acid inhibition of phagocytic function. In contrast, the serine mutants did not recapitulate the selective effects of cytoplasmic domain truncation on cytokine production. These results demonstrate for the first time a direct functional role for serine phosphorylation in the α-chain of FcγRIa and suggest that the cytoplasmic domain of FcγRI regulates the different functional capacities of the FcγRIa-receptor complex.