Monoclonal antibodies specific for low-affinity interleukin-3 (IL-3) binding protein AIC2A: evidence that AIC2A is a component of a high- affinity IL-3 receptor

Abstract Mouse interleukin-3 (IL-3) binds to its receptor with high and low affinities. Using anti-Aic2 antibody, two distinct cDNAs (AIC2A and AIC2B) were isolated. The AIC2A gene encodes a protein of 120 Kd that binds IL-3 with low affinity, whereas the AIC2B gene encodes a protein that is 91% identical to AIC2A at the amino acid level, but which does not bind IL-3. To study the structure of the functional high-affinity IL-3 receptor (IL-3R), we generated specific monoclonal antibodies against the AIC2A protein. We produced a soluble AIC2A protein by inserting a termination codon at the beginning of the transmembrane domain of the AIC2A cDNA. Soluble AIC2A protein expressed in COS7 cells was purified to homogeneity and three anti-AIC2A monoclonal antibody- producing hybridomas (3D1, 3D4, and 9D3) were obtained from a rat immunized with the purified soluble AIC2A protein. The antibodies were specific for the AIC2A protein and did not bind to the AIC2B protein. Using chimeric receptors between AIC2A and AIC2B, recognition sites of the antibodies were mapped. The antibodies immunoprecipitated a 120-Kd protein from IL-3-dependent PT18 cells. The N-terminal sequence of the 120-Kd protein was consistent with the predicted processing site of the signal sequence of the AIC2A protein. Staining of IL-3-dependent and IL- 3-independent cell lines with the 9D3 antibody were consistent with the IL-3 binding. The 9D3 antibody inhibited both the high-affinity IL-3 binding and the low-affinity binding, as well as IL-3-dependent proliferation. These results indicate that the AIC2A protein is a binding component of a high-affinity IL-3R.

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Monoclonal antibodies specific for low-affinity interleukin-3 (IL-3) binding protein AIC2A: evidence that AIC2A is a component of a high- affinity IL-3 receptor

Blood ◽

10.1182/blood.v79.4.895.bloodjournal794895 ◽

1992 ◽

Vol 79 (4) ◽

pp. 895-903 ◽

Cited By ~ 12

Author(s):

T Ogorochi ◽

T Hara ◽

HM Wang ◽

K Maruyama ◽

A Miyajima

Keyword(s):

Monoclonal Antibodies ◽

Transmembrane Domain ◽

Signal Sequence ◽

Amino Acid Level ◽

Terminal Sequence ◽

Chimeric Receptors ◽

Interleukin 3 ◽

High Affinity ◽

Processing Site ◽

Binding Component

Mouse interleukin-3 (IL-3) binds to its receptor with high and low affinities. Using anti-Aic2 antibody, two distinct cDNAs (AIC2A and AIC2B) were isolated. The AIC2A gene encodes a protein of 120 Kd that binds IL-3 with low affinity, whereas the AIC2B gene encodes a protein that is 91% identical to AIC2A at the amino acid level, but which does not bind IL-3. To study the structure of the functional high-affinity IL-3 receptor (IL-3R), we generated specific monoclonal antibodies against the AIC2A protein. We produced a soluble AIC2A protein by inserting a termination codon at the beginning of the transmembrane domain of the AIC2A cDNA. Soluble AIC2A protein expressed in COS7 cells was purified to homogeneity and three anti-AIC2A monoclonal antibody- producing hybridomas (3D1, 3D4, and 9D3) were obtained from a rat immunized with the purified soluble AIC2A protein. The antibodies were specific for the AIC2A protein and did not bind to the AIC2B protein. Using chimeric receptors between AIC2A and AIC2B, recognition sites of the antibodies were mapped. The antibodies immunoprecipitated a 120-Kd protein from IL-3-dependent PT18 cells. The N-terminal sequence of the 120-Kd protein was consistent with the predicted processing site of the signal sequence of the AIC2A protein. Staining of IL-3-dependent and IL- 3-independent cell lines with the 9D3 antibody were consistent with the IL-3 binding. The 9D3 antibody inhibited both the high-affinity IL-3 binding and the low-affinity binding, as well as IL-3-dependent proliferation. These results indicate that the AIC2A protein is a binding component of a high-affinity IL-3R.

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Conformation as the Therapeutic Target for Neurodegenerative Diseases

Current Alzheimer Research ◽

10.2174/1567205014666170116152622 ◽

2017 ◽

Vol 14 (4) ◽

pp. 393-402 ◽

Cited By ~ 6

Author(s):

Rajaraman Krishnan ◽

Franz Hefti ◽

Haim Tsubery ◽

Michal Lulu ◽

Ming Proschitsky ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neurodegenerative Diseases ◽

Protein Misfolding ◽

Specific Binding ◽

Drug Candidate ◽

High Affinity ◽

Novel Approach ◽

Multiple Protein ◽

Clinical Benefits ◽

Effective Drugs

Therapeutic strategies that target pathways of protein misfolding and the toxicity of intermediates along these pathways are mainly at discovery and early development stages, with the exception of monoclonal antibodies that have mainly failed to produce convincing clinical benefits in late stage trials. The clinical failures represent potentially critical lessons for future neurodegenerative disease drug development. More effective drugs may be achieved by pursuing the following two strategies. First, conformational targeting of aggregates of misfolded proteins, rather than less specific binding that includes monomer subunits, which vastly outnumber the toxic targets. Second, since neurodegenerative diseases frequently include more than one potential protein pathology, generic targeting of aggregates by shape might also be a crucial feature of a drug candidate. Incorporating both of these critical features into a viable drug candidate along with high affinity binding has not been achieved with small molecule approaches or with antibody fragments. Monoclonal antibodies developed so far are not broadly acting through conformational recognition. Using GAIM (General Amyloid Interaction Motif) represents a novel approach that incorporates high affinity conformational recognition for multiple protein assemblies, as well as recognition of an array of assemblies along the misfolding pathway between oligomers and fibers. A GAIM-Ig fusion, NPT088, is nearing clinical testing.

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Functional Association of FcɛRIγ With Arginine632 of Paired Immunoglobulin-Like Receptor (PIR)-A3 in Murine Macrophages

Blood ◽

10.1182/blood.v94.5.1790 ◽

1999 ◽

Vol 94 (5) ◽

pp. 1790-1796 ◽

Cited By ~ 22

Author(s):

Lynn S. Taylor ◽

Daniel W. McVicar

Keyword(s):

Transmembrane Domain ◽

Dependent Manner ◽

Macrophage Cell Line ◽

Inhibitory Pathway ◽

Macrophage Cell ◽

Complete Understanding ◽

Chimeric Receptors ◽

Extracellular Region ◽

Activating Receptors ◽

Ifn Γ

Abstract Paired immunoglobulin-like receptors (PIR) are expressed on B cells and macrophages and include inhibitory and putative activating receptors referred to as PIR-B and PIR-A, respectively. Although PIR-B’s inhibitory pathway has been described, it is unknown whether PIR-A receptors can deliver activation signals to macrophages, and if so, through what mechanism. Here we use chimeric receptors to address the mechanisms of PIR-A signaling. Cotransfection of chimeric receptors comprised of the extracellular region of human CD4 and the transmembrane and cytoplasmic domains of murine PIR-A3 showed the ability of PIR-A3 to physically interact with the FcɛRIγ chain in 293T cells. This interaction is dependent on Arg632 within the PIR-A3 transmembrane domain. We also demonstrate PIR-A3 interaction with the endogenous FcɛRIγ of the ANA-1 macrophage cell line, again in an Arg632-dependent manner. Furthermore, we show that crosslinking of these chimeric receptors synergizes with IFN-γ in the production of nitric oxide. Our data are the first to show the potential of PIR-A3 to deliver activation signals to macrophages and establish its dependence on Arg632. These findings suggest that further study of the PIR-A receptors should be aggressively pursued toward a complete understanding of the intricate regulation of macrophage biology.

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Asp383 in the second transmembrane domain of the lutropin receptor is important for high affinity hormone binding and cAMP production

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)98570-4 ◽

1991 ◽

Vol 266 (23) ◽

pp. 14953-14957

Author(s):

I. Ji ◽

T.H. Ji

Keyword(s):

Transmembrane Domain ◽

Hormone Binding ◽

Camp Production ◽

High Affinity ◽

Lutropin Receptor

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The PBN1 Gene of Saccharomyces cerevisiae: An Essential Gene That Is Required for the Post-translational Processing of the Protease B Precursor

Genetics ◽

10.1093/genetics/149.3.1277 ◽

1998 ◽

Vol 149 (3) ◽

pp. 1277-1292 ◽

Cited By ~ 1

Author(s):

Rajesh R Naik ◽

Elizabeth W Jones

Keyword(s):

Saccharomyces Cerevisiae ◽

Secretory Pathway ◽

Transmembrane Domain ◽

Essential Gene ◽

Signal Sequence ◽

Signal Peptidase ◽

Amino Terminal ◽

Autocatalytic Cleavage ◽

Protease B ◽

Chromosome Iii

Abstract The vacuolar hydrolase protease B in Saccharomyces cerevisiae is synthesized as an inactive precursor (Prb1p). The precursor undergoes post-translational modifications while transiting the secretory pathway. In addition to N- and O -linked glycosylations, four proteolytic cleavages occur during the maturation of Prb1p. Removal of the signal peptide by signal peptidase and the autocatalytic cleavage of the large aminoterminal propeptide occur in the endoplasmic reticulum (ER). Two carboxy-terminal cleavages of the post regions occur in the vacuole: the first cleavage is catalyzed by protease A and the second results from autocatalysis. We have isolated a mutant, pbn1-1, that exhibits a defect in the ER processing of Prb1p. The autocatalytic cleavage of the propeptide from Prb1p does not occur and Prb1p is rapidly degraded in the cytosol. PBN1 was cloned and is identical to YCL052c on chromosome III. PBN1 is an essential gene that encodes a novel protein. Pbn1p is predicted to contain a sub-C-terminal transmembrane domain but no signal sequence. A functional HA epitope-tagged Pbn1p fusion localizes to the ER. Pbn1p is N-glycosylated in its amino-terminal domain, indicating a lumenal orientation despite the lack of a signal sequence. Based on these results, we propose that one of the functions of Pbn1p is to aid in the autocatalytic processing of Prb1p.

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Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies

Blood ◽

10.1182/blood.v75.4.874.874 ◽

1990 ◽

Vol 75 (4) ◽

pp. 874-880 ◽

Cited By ~ 13

Author(s):

AD D'Andrea ◽

PJ Szklut ◽

HF Lodish ◽

EM Alderman

Keyword(s):

Monoclonal Antibodies ◽

Receptor Binding ◽

Sodium Dodecyl ◽

Dependent Manner ◽

Epo Receptor ◽

High Affinity ◽

Group I ◽

Dependent Cell ◽

Group Ii ◽

Murine Erythroleukemia

Abstract We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO). All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L. Two MoAbs, designated Group I MoAbs, bind to an epitope within the N- terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO. Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO. We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding. MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L). To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO. Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner. We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO- dependent cell line. Only Group II MoAbs inhibit a newly developed EPO- dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.

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