Determinants of thetrans-Dominant Negative Effect of Truncated Forms of the CCR5 Chemokine Receptor

The human immunodeficiency virus, type 1 (HIV-1) entry process is triggered by interaction between the viral envelope and a seven membrane-spanning domain receptor at the cell surface, usually the CCR5 chemokine receptor. Different naturally occurring mutations in theCCR5gene abolish receptor function, the most frequent being a 32-nucleotide deletion resulting in a truncated protein (Δ32) lacking the last three transmembrane domains (TM5–7). This mutant is retained in the endoplasmic reticulum and exerts atrans-dominant negative (TDN) effect on the wild type, preventing its exit from this compartment. This TDN effect is often considered as evidence for the oligomerization of CCR5 during transport to the cell surface. Here we use a genetic approach to define the structural determinants of the TDN effect of the Δ32 mutant. It was abolished by certain deletions and by mutations of cysteine residues preventing formation of a disulfide link between the first and second extracellular loops, suggesting that conformation of Δ32 is important for its interaction with CCR5. To circumvent this problem, we used chimeric forms of the Δ32 and wild type CCR5, consisting in substitutions with homologous domains from the mouse CCR5. All chimeric full-length receptors were expressed at the cell surface and were functional for interaction with HIV-1 or with a chemokine ligand, when assayed. The TDN effect was only observed if both the TM3 domain in CCR5 and the TM4 domain in Δ32 were from human origin, whereas the rest of the proteins could be from either origin. This suggests that the TDN effect involves some form of interaction between these transmembrane domains. Alternatively, but less likely to us, substitutions in TM4 could affect the conformation of CCR5 in the endoplasmic reticulum but not at the cell surface. However that may be, it seems that the TDN effect of the Δ32 mutant has no bearing to the issue of CCR5 dimerization and to its possible role in the processing of the receptor to the cell surface.

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Human Loss-of-Function Gonadotropin-Releasing Hormone Receptor Mutants Retain Wild-Type Receptors in the Endoplasmic Reticulum: Molecular Basis of the Dominant-Negative Effect

Molecular Endocrinology ◽

10.1210/me.2004-0091 ◽

2004 ◽

Vol 18 (7) ◽

pp. 1787-1797 ◽

Cited By ~ 78

Author(s):

Shaun P. Brothers ◽

Anda Cornea ◽

Jo Ann Janovick ◽

P. Michael Conn

Keyword(s):

Endoplasmic Reticulum ◽

Hormone Receptor ◽

Molecular Basis ◽

Gonadotropin Releasing Hormone ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Loss Of Function ◽

Wild Type ◽

Negative Effect ◽

Gonadotropin Releasing Hormone Receptor

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Mutant voltage-gated Na+ channels can exert a dominant negative effect through coupled gating

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00721.2017 ◽

2018 ◽

Vol 315 (5) ◽

pp. H1250-H1257 ◽

Cited By ~ 12

Author(s):

Jérôme Clatot ◽

Yang Zheng ◽

Aurore Girardeau ◽

Haiyan Liu ◽

Kenneth R. Laurita ◽

...

Keyword(s):

Cell Surface ◽

Single Channel ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Wild Type ◽

Voltage Gated ◽

Cell Surface Biotinylation ◽

Negative Effect ◽

Coupled Gating ◽

Α Subunits

Mutations in voltage-gated Na+ channels have been linked to several channelopathies leading to a wide variety of diseases including cardiac arrhythmias, epilepsy, and myotonia. We have previously demonstrated that voltage-gated Na+ channel (Nav)1.5 trafficking-deficient mutant channels could lead to a dominant negative effect by impairing trafficking of the wild-type (WT) channel. We also reported that voltage-gated Na+ channels associate as dimers with coupled gating properties. Here, we hypothesized that the dominant negative effect of mutant Na+ channels could also occur through coupled gating. This was tested using cell surface biotinylation and single channel recordings to measure the gating probability and coupled gating of the dimers. As previously reported, coexpression of Nav1.5-L325R with WT channels led to a dominant negative effect, as reflected by a 75% reduction in current density. Surprisingly, cell surface biotinylation showed that Nav1.5-L325R mutant is capable of trafficking, with 40% of Nav1.5-L325R reaching the cell surface when expressed alone. Importantly, even though a dominant negative effect on the Na+ current is observed when WT and Nav1.5-L325R are expressed together, the total Nav channel cell surface expression was not significantly altered compared with WT channels alone. Thus, the trafficking deficiency could not explain the 75% decrease in inward Na+ current. Interestingly, single channel recordings showed that Nav1.5-L325R exerted a dominant negative effect on the WT channel at the gating level. Both coupled gating and gating probability of WT:L325R dimers were drastically impaired. We conclude that dominant negative suppression exerted by Nav1.5 mutants can also be caused by impairing the WT gating probability, a mechanism resulting from the dimerization and coupled gating of voltage-gated Na+ channel α-subunits. NEW & NOTEWORTHY The presence of dominant negative mutations in the Na+ channel gene leading to Brugada syndrome was supported by our recent findings that Na+ channel α-subunits form dimers. Up until now, the dominant negative effect was thought to be caused by the interaction of the wild-type Na+ channel with trafficking-deficient mutant channels. However, the present study demonstrates that coupled gating of voltage-gated Na+ channels can also be responsible for the dominant negative effect leading to arrhythmias.

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The B-Oligomer of Pertussis Toxin Deactivates Cc Chemokine Receptor 5 and Blocks Entry of M-Tropic HIV-1 Strains

Journal of Experimental Medicine ◽

10.1084/jem.190.5.597 ◽

1999 ◽

Vol 190 (5) ◽

pp. 597-606 ◽

Cited By ~ 73

Author(s):

Massimo Alfano ◽

Helena Schmidtmayerova ◽

Carol-Ann Amella ◽

Tatiana Pushkarsky ◽

Michael Bukrinsky

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

Pertussis Toxin ◽

Chemokine Receptor ◽

Viral Envelope ◽

Cell Surface Expression ◽

Target Cells ◽

Peripheral Blood Mononuclear ◽

Hiv 1

Infection of target cells by HIV-1 requires initial binding interactions between the viral envelope glycoprotein gp120, the cell surface protein CD4, and one of the members of the seven-transmembrane G protein–coupled chemokine receptor family. Most primary isolates (R5 strains) use chemokine receptor CCR5, but some primary syncytium-inducing, as well as T cell line–adapted, strains (X4 strains) use the CXCR4 receptor. Signaling from both CCR5 and CXCR4 is mediated by pertussis toxin (PTX)-sensitive Gi proteins and is not required for HIV-1 entry. Here, we show that the PTX holotoxin as well as its binding subunit, B-oligomer, which lacks Gi-inhibitory activity, blocked entry of R5 but not X4 strains into primary T lymphocytes. Interestingly, B-oligomer inhibited virus production by peripheral blood mononuclear cell cultures infected with either R5 or X4 strains, indicating that it can affect HIV-1 replication at both entry and post-entry levels. T cells treated with B-oligomer did not initiate signal transduction in response to macrophage inflammatory protein (MIP)-1β or RANTES (regulated upon activation, normal T cell expressed and secreted); however, cell surface expression of CCR5 and binding of MIP-1β or HIV-1 to such cells were not impaired. The inhibitory effect of B-oligomer on signaling from CCR5 and on entry of R5 HIV-1 strains was reversed by protein kinase C (PKC) inhibitors, indicating that B-oligomer activity is mediated by signaling events that involve PKC. B-oligomer also blocked cocapping of CCR5 and CD4 induced by R5 HIV-1 in primary T cells, but did not affect cocapping of CXCR4 and CD4 after inoculation of the cultures with X4 HIV-1. These results suggest that the B-oligomer of PTX cross-deactivates CCR5 to impair its function as a coreceptor for HIV-1.

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A Novel Mutation 774del4 in IFNGR1, Which Produces Truncated Form of Interferon γ Receptor 1, Causes a Dominant-Negative Effect on Interferon γ Signal Transduction.

Blood ◽

10.1182/blood.v108.11.1246.1246 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1246-1246

Author(s):

Satoshi Okada ◽

Nobutsune Ishikawa ◽

Kenichirou Shirao ◽

Hiroshi Kawaguchi ◽

Miyuki Tsumura ◽

...

Keyword(s):

Cell Surface ◽

Interferon Γ ◽

Dominant Negative ◽

Hek293 Cells ◽

Dominant Negative Effect ◽

Heterozygous Mutation ◽

Wild Type ◽

Truncated Form ◽

Mycobacterium Infections ◽

Ifn Γ

Abstract Patients with interferon γ receptor 1 (IFNγR1) deficiency are characterized by disseminated Bacilli Calmette-Guérin (BCG) infections and severe non-tuberculosis mycobacterium infections, and show remarkable genetic heterogeneity. It is known as one of inherited immunodeficiency disorders with recessive or dominant form. So far known dominant forms of IFNγR1 deficiency are associated with heterozygous mutation in the intracellular domain of IFNγR1, among of which 818del4 is known as a major mutation in IFNGR1, leading to frameshift and premature stop codon. We describe here a novel heterozygous IFNGR1 mutation in a patient with recurrent mycobacterium infections, resulting in impairment of receptor degradation. The proband, a 12-year old Japanese girl, developed multiple osteomyelitis due to M. avium infection. She had suffered from BCG lymphadenitis, which occurred 2 months after BCG vaccination, for 3 years. DNA sequence analysis of IFNGR1 demonstrated that the patient had a heterozygous mutation 774del4, producing a truncated protein lacking intracellular component of IFNγR1. IFNγR1 was overexpressed on the surface of CD14-positive cells in the peripheral blood of the patient, and STAT1 phosphorylation was partially defected in response to high dose IFN-γ stimulation. Impaired TNF-α production in response to IFN-γ stimulation was also observed. T cells from the patient presented decreased IFN-γ production in response to IL-12 stimulation. In order to define the characterization of this truncated form of IFNγR1, we cloned 774del4 and 818del4 mutations as well as the wild-type into mammalian expression vector. These constructs were transfected into HEK293 cells. Cycloheximide (CHX) treatment enabled detection of IFNγR1 expression by flow cytometry and Western blot analysis. In contrast to the wild-type IFNγR1 protein, which rapidly disappeared after CHX treatment, 774del4 mutant protein was stably retained on the cell surface as was observed in 818del4 mutant. These observations suggest that 774del4 mutant causes overexpression of dominant-negative form of IFNγR1 on the cell surface through impairment of receptor degradation.

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Revisiting PC1/3 Mutants: Dominant-Negative Effect of Endoplasmic Reticulum-Retained Mutants

Endocrinology ◽

10.1210/en.2015-1068 ◽

2015 ◽

Vol 156 (10) ◽

pp. 3625-3637 ◽

Cited By ~ 12

Author(s):

Elias H. Blanco ◽

Bruno Ramos-Molina ◽

Iris Lindberg

Keyword(s):

Endoplasmic Reticulum ◽

Peptide Hormone ◽

Biochemical Properties ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Wild Type ◽

Negative Effects ◽

Hormone Synthesis ◽

Type Protein ◽

Wild Type Protein

Prohormone convertase 1/3 (PC1/3), encoded by the gene PCSK1, is critical for peptide hormone synthesis. An increasing number of studies have shown that inactivating mutations in PCSK1 are correlated with endocrine pathologies ranging from intestinal dysfunction to morbid obesity, whereas the common nonsynonymous polymorphisms rs6232 (N221D) and rs6234–rs6235 (Q665E-S690T) are highly associated with obesity risk. In this report, we revisited the biochemical and cellular properties of PC1/3 variants in the context of a wild-type PC1/3 background instead of the S357G hypermorph background used for all previous studies. In the wild-type background the PC1/3 N221D variant exhibited 30% lower enzymatic activity in a fluorogenic assay than wild-type PC1/3; this inhibition was greater than that detected in an equivalent experiment using the PC1/3 S357G background. A PC1/3 variant with the linked carboxyl-terminal polymorphisms Q665E-S690T did not show this difference. We also analyzed the biochemical properties of 2 PC1/3 mutants, G209R and G593R, which are retained in the endoplasmic reticulum (ER), and studied their effects on wild-type PC1/3. The expression of ER-retained mutants induced ER stress markers and also resulted in dominant-negative blockade of wild-type PC1/3 prodomain cleavage and decreased expression of wild-type PC1/3, suggesting facilitation of the entry of wild-type protein to a degradative proteasomal pathway. Dominant-negative effects of PC1/3 mutations on the expression and maturation of wild-type protein, with consequential effects on PC1/3 availability, add a new element which must be considered in population and clinical studies of this gene.

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Genotyping of the CCR5 Chemokine Receptor by Isothermal NASBA Amplification and Differential Probe Hybridization

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.6.6.959-965.1999 ◽

1999 ◽

Vol 6 (6) ◽

pp. 959-965 ◽

Cited By ~ 4

Author(s):

Joseph W. Romano ◽

Surya Tetali ◽

Eun Mi Lee ◽

Roxanne N. Shurtliff ◽

Xue Ping Wang ◽

...

Keyword(s):

Chemokine Receptor ◽

Mononuclear Cells ◽

Nucleic Acid Amplification ◽

Wild Type ◽

Dna Encoding ◽

Peripheral Blood Mononuclear ◽

Assay Sensitivity ◽

Ccr5 Chemokine Receptor ◽

Nucleic Acid Amplification Technology ◽

Hiv 1

ABSTRACT The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectibility of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

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A novel I551F variant of Na+/HCO3- cotransporter NBCe1-A shows reduced cell surface expression, resulting in diminished transport activity

AJP Renal Physiology ◽

10.1152/ajprenal.00584.2020 ◽

2021 ◽

Author(s):

Osamu Yamazaki ◽

Maho Yamashita ◽

Jinping Li ◽

Fumika Ochiai-Homma ◽

Tadashi Yoshida ◽

...

Keyword(s):

Cell Surface ◽

Mdck Cells ◽

Surface Expression ◽

Dominant Negative ◽

Hek293 Cells ◽

Cell Surface Expression ◽

Dominant Negative Effect ◽

Transport Activity ◽

Wild Type ◽

Single Nucleotide Variants

Homozygous mutations in SLC4A4, encoding the electrogenic Na+/HCO3- cotransporter NBCe1, cause proximal renal tubular acidosis (pRTA) associated with extrarenal symptoms. Although 17 mutated sites in SLC4A4 have thus far been identified among pRTA patients, physiological significance of other nonsynonymous single nucleotide variants (SNVs) remains largely undetermined. Here, we investigated the functional properties of SNVs in NBCe1. From NCBI dbSNP database, we identified 13 SNVs that have not previously been characterized in highly conserved, transmembrane domains of NBCe1-A. Immunocytochemical analysis revealed that I551F variant was present predominantly in the cytoplasm in HEK293 cells, whereas all other SNVs did not show as dramatic a change in subcellular distribution. Western blot analysis in HEK293 cells demonstrated that the I551F variant showed impaired glycosylation and a 69 % reduction in cell surface levels. To determine the role of I551 in more detail, we examined the significance of various artificial mutants both in non-polarized HEK293 cells and polarized MDCK cells, which indicated that only I551F substitution resulted in cytoplasmic retention. Moreover, functional analysis using Xenopus oocytes demonstrated that the I551F variant had a significantly reduced activity corresponding to 39 % of that of wild-type, whereas any other SNVs and artificial I551 mutants did not show significant changes in activity. Finally, immunofluorescence study in HEK293 cells indicated that the I551F variant retains wild-type NBCe1-A in the cytoplasm. These data demonstrate that I551F-NBCe1-A shows impaired transport activity predominantly through cytoplasmic retention, and suggest that the variant can have a dominant-negative effect by forming complexes with wild-type NBCe1-A.

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A novel PAX5-ELN fusion protein identified in B-cell acute lymphoblastic leukemia acts as a dominant negative on wild-type PAX5

Blood ◽

10.1182/blood-2006-05-025221 ◽

2006 ◽

Vol 109 (8) ◽

pp. 3417-3423 ◽

Cited By ~ 55

Author(s):

Marina Bousquet ◽

Cyril Broccardo ◽

Cathy Quelen ◽

Fabienne Meggetto ◽

Emilienne Kuhlein ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Quantitative Polymerase Chain Reaction ◽

Dominant Negative ◽

Leukemic Cells ◽

Dominant Negative Effect ◽

Wild Type ◽

Cell Acute Lymphoblastic Leukemia

Abstract We report a novel t(7;9)(q11;p13) translocation in 2 patients with B-cell acute lymphoblastic leukemia (B-ALL). By fluorescent in situ hybridization and 3′ rapid amplification of cDNA ends, we showed that the paired box domain of PAX5 was fused with the elastin (ELN) gene. After cloning the full-length cDNA of the chimeric gene, confocal microscopy of transfected NIH3T3 cells and Burkitt lymphoma cells (DG75) demonstrated that PAX5-ELN was localized in the nucleus. Chromatin immunoprecipitation clearly indicated that PAX5-ELN retained the capability to bind CD19 and BLK promoter sequences. To analyze the functions of the chimeric protein, HeLa cells were cotransfected with a luc-CD19 construct, pcDNA3-PAX5, and with increasing amounts of pcDNA3-PAX5-ELN. Thus, in vitro, PAX5-ELN was able to block CD19 transcription. Furthermore, real-time quantitative polymerase chain reaction (RQ-PCR) experiments showed that PAX5-ELN was able to affect the transcription of endogenous PAX5 target genes. Since PAX5 is essential for B-cell differentiation, this translocation may account for the blockage of leukemic cells at the pre–B-cell stage. The mechanism involved in this process appears to be, at least in part, through a dominant-negative effect of PAX5-ELN on the wild-type PAX5 in a setting ofPAX5 haploinsufficiency.

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Novel REST Truncation Mutations Causing Hereditary Gingival Fibromatosis

Journal of Dental Research ◽

10.1177/0022034521996620 ◽

2021 ◽

pp. 002203452199662

Author(s):

J.T. Chen ◽

C.H. Lin ◽

H.W. Huang ◽

Y.P. Wang ◽

P.C. Kao ◽

...

Keyword(s):

Nuclear Localization ◽

Genetic Disorder ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Wild Type ◽

Negative Effect ◽

Localization Signal ◽

Gingival Fibromatosis

Hereditary gingival fibromatosis (HGF) is a rare genetic disorder featured by nonsyndromic pathological overgrowth of gingiva. The excessive gingival tissues can cause dental, masticatory, and phonetic problems, which impose severe functional and esthetic burdens on affected individuals. Due to its high recurrent rate, patients with HGF have to undergo repeated surgical procedures of gingival resection, from childhood to adulthood, which significantly compromises their quality of life. Unraveling the genetic etiology and molecular pathogenesis of HGF not only gains insight into gingival physiology and homeostasis but also opens avenues for developing potential therapeutic strategies for this disorder. Recently, mutations in REST (OMIM *600571), encoding a transcription repressor, were reported to cause HGF (GINGF5; OMIM #617626) in 3 Turkish families. However, the functions of REST in gingival homeostasis and pathogenesis of REST-associated HGF remain largely unknown. In this study, we characterized 2 HGF families and identified 2 novel REST mutations, c.2449C>T (p.Arg817*) and c.2771_2793dup (p.Glu932Lysfs*3). All 5 mutations reported to date are nonsenses or frameshifts in the last exon of REST and would presumably truncate the protein. In vitro reporter gene assays demonstrated a partial or complete loss of repressor activity for these truncated RESTs. When coexpressed with the full-length protein, the truncated RESTs impaired the repressive ability of wild-type REST, suggesting a dominant negative effect. Immunofluorescent studies showed nuclear localization of overexpressed wild-type and truncated RESTs in vitro, indicating preservation of the nuclear localization signal in shortened proteins. Immunohistochemistry demonstrated a comparable pattern of ubiquitous REST expression in both epithelium and lamina propria of normal and HGF gingival tissues despite a reduced reactivity in HGF gingiva. Results of this study confirm the pathogenicity of REST truncation mutations occurring in the last exon causing HGF and suggest the pathosis is caused by an antimorphic (dominant negative) disease mechanism.

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