A Mutant (Arg327→His) GPIIb Associated to Thrombasthenia Exerts a Dominant Negative Effect in Stably Transfected CHO Cells

1996 ◽  
Vol 76 (03) ◽  
pp. 292-301 ◽  
Author(s):  
Milagros Ferrer ◽  
Marta Fernandez-Pinel ◽  
Consuelo Gonzalez-Manchon ◽  
Jose Gonzalez ◽  
Matilde S Ayuso ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cho Cells ◽  
Functional Characterization ◽  
Surface Expression ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Glycoprotein Complex ◽  
Negative Effect

SummaryThis work reports the structural and functional characterization of the platelet glycoprotein complex GPIIb-IIIa (integrin αIIbβ3) in a patient of type II Glanzmann thrombasthenia, bearing a homozygous G→A base transition at position 1074 of GPIIb that results in an Arg327→His substitution.CHO cells stably transfected with cDNA encoding His327GPIIb showed a drastic reduction in the surface expression of αIIbβ3 complex relative to control cells transfected with wild type GPIIb. Immunopre-cipitation analysis demonstrated that GPIIb synthesis, heterodimeriza-tion, and short term maturation were not impeded, suggesting that conformational changes dependent on Arg327 of GPIIb may play an essential role in either the rate of maturation and/or transport of heterodimers to the cell surface.Cotransfection of CHO cells with equimolar amounts of cDNAs encoding wild type and mutant His327-GPIIb led to a marked reduction in the surface expression of αIIbβ3. This novel observation of a dominant-negative effect of the mutant His327αIIb subunit provides a molecular basis for the reduced platelet αIIbβ3 content observed in the heterozygous offspring.

Novel REST Truncation Mutations Causing Hereditary Gingival Fibromatosis

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.

A Missense Mutation (Tyr88 to Cys) in the Platelet Membrane Glycoprotein Ibβ Gene Affects GPIb/IX Complex Expression

2001 ◽  
Vol 86 (11) ◽  
pp. 1249-1256 ◽  
Author(s):  
Yumi Kurokawa ◽  
Takehiko Kamijo ◽  
Shinji Kunishima ◽  
Dermot Kenny ◽  
Kiyoshi Kitano ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Platelet Glycoprotein ◽  
Homozygous Mutation ◽  
Sequencing Analysis ◽  
Giant Platelets ◽  
Giant Platelet ◽  
Negative Effect ◽  
Dna Sequencing Analysis

SummaryThis study examined the molecular basis of a missense mutation of the platelet glycoprotein (GP) Ibβ gene in two families. In the propositus with a novel form of Bernard-Soulier syndrome (BSS) from Family I, only GPIbα was detectable in reduced amounts on platelet surfaces by flow cytometry. There were no GPIX or GPIbβ found by immunoblotting. DNA sequencing analysis showed a homozygous mutation in the GPIbβ gene which changed Tyr (TAC) to Cys (TGC) at residue 88. Her parents were heterozygous for Tyr88Cys in the GPIbβ gene. In transient transfection studies on 293T cells, both Tyr88Cys and Tyr88Ala mutations suppressed the expression of GPIb/IX complexes. In addition, Tyr88Cys GPIbβ mutation was found to exert a dominant negative effect on the GPIb expression.Five individuals from Family II, four of whom reported elsewhere as having giant platelet disorders with normal aggregation (BLOOD, 1997; 89: 2404) and one newly analyzed in this study, were heterozygous for Tyr88Cys in the GPIb gene. Microsatellite analysis of chromosome 22 showed a common haplotype in 8 of the individuals with Tyr88Cys mutations in Families I and II. Tyr88 in the GPIbβ gene plays a significant role in the GPIb/IX expression; the defect causes BSS in a homozygous form and possibly giant platelets in a heterozygous form.

scholarly journals A Mutation Linked with Bartter's Syndrome Locks Kir 1.1a (Romk1) Channels in a Closed State

1999 ◽  
Vol 114 (5) ◽  
pp. 685-700 ◽  
Author(s):  
Thomas P. Flagg ◽  
Margaret Tate ◽  
Jean Merot ◽  
Paul A. Welling
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
K Channel ◽  
Bartter’S Syndrome ◽  
Wild Type ◽  
Bartter's Syndrome ◽  
Closed State ◽  
Negative Effect

Mutations in the inward rectifying renal K+ channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332–391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP–wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332–351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.

scholarly journals The Role of Subunit Assembly in Peripherin-2 Targeting to Rod Photoreceptor Disk Membranes and Retinitis Pigmentosa

2003 ◽  
Vol 14 (8) ◽  
pp. 3400-3413 ◽  
Author(s):  
Christopher J.R. Loewen ◽  
Orson L. Moritz ◽  
Beatrice M. Tam ◽  
David S. Papermaster ◽  
Robert S. Molday
Keyword(s):  
Retinitis Pigmentosa ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Rod Photoreceptor ◽  
Wild Type ◽  
Photoreceptor Outer Segment ◽  
Negative Effect ◽  
Retinal Degenerative Diseases ◽  
Green Fluorescent

Peripherin-2 is a member of the tetraspanin family of membrane proteins that plays a critical role in photoreceptor outer segment disk morphogenesis. Mutations in peripherin-2 are responsible for various retinal degenerative diseases including autosomal dominant retinitis pigmentosa (ADRP). To identify determinants required for peripherin-2 targeting to disk membranes and elucidate mechanisms underlying ADRP, we have generated transgenic Xenopus tadpoles expressing wild-type and ADRP-linked peripherin-2 mutants as green fluorescent fusion proteins in rod photoreceptors. Wild-type peripherin-2 and P216L and C150S mutants, which assemble as tetramers, targeted to disk membranes as visualized by confocal and electron microscopy. In contrast the C214S and L185P mutants, which form homodimers, but not tetramers, were retained in the rod inner segment. Only the P216L disease mutant induced photoreceptor degeneration. These results indicate that tetramerization is required for peripherin-2 targeting and incorporation into disk membranes. Tetramerization-defective mutants cause ADRP through a deficiency in wild-type peripherin-2, whereas tetramerization-competent P216L peripherin-2 causes ADRP through a dominant negative effect, possibly arising from the introduction of a new oligosaccharide chain that destabilizes disks. Our results further indicate that a checkpoint between the photoreceptor inner and outer segments allows only correctly assembled peripherin-2 tetramers to be incorporated into nascent disk membranes.

A Mouse Model for Diamond-Blackfan Anemia Demonstrates a Dominant Negative Effect of a Point Mutation in the RPS19 Gene

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 38-38 ◽  
Author(s):  
Emily E. Devlin ◽  
Lydie DaCosta ◽  
Mohandas Narla ◽  
Gene Elliott ◽  
David M. Bodine
Keyword(s):  
Mouse Model ◽  
Macrocytic Anemia ◽  
Dominant Negative ◽  
Poly I:C ◽  
Dominant Negative Effect ◽  
Erythroid Cells ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Diamond Blackfan Anemia ◽  
Negative Effect

Abstract Diamond-Blackfan Anemia (DBA) is associated with mutations in several ribosomal protein genes, including Ribosomal Protein S19 (RPS19), which is mutated in approximately 25% of patients. Most RPS19 mutations are deletions of all or part of the RPS19 gene and are predicted to cause DBA by a haploinsufficiency mechanism. However, approximately 30% of RPS19 mutations are missense mutations in the RPS19 coding sequence, which we hypothesize act through a dominant negative mechanism. To test for a dominant negative effect, we generated a transgenic mouse model expressing a common and penetrant mutation at codon 62 that replaces an Arginine with a Tryptophan (R62W). The constructs contain the ubiquitous actin promoter linked to the wild-type or R62W human RPS19 cDNA followed by the 3′ region of the Gamma globin gene to provide RNA stability and intron splicing to facilitate RNA transport to the cytoplasm. The constructs are flanked by chicken HS4 barrier elements to ensure transgene expression regardless of the location in the genome. Eight lines of wild-type RPS19 transgenic mice were fertile, expressed RPS19 in all tissues, and had normal hematology. Twelve RPS19R62W founder animals were generated, six of which died before they reached 2 months of age. Two of these animals were analyzed and found to have a macrocytic anemia. None of the other 6 founder animals transmitted the RPS19R62W transgene to F1 pups or d13.5 embryos, suggesting either that the RPS19R62W transgene was not present in the germ line and/or that expression of the RPS19R62W protein may cause early lethality. Supporting this hypothesis, embryonic stem cells (ES) expressing wild-type RPS19 were viable, while ES cells expressing RPS19R62W were not viable. To circumvent potential embryonic lethality, we generated conditional RPS19R62W transgenic mice with stop sequences flanked by lox P sites inserted between the promoter and the RPS19 gene. In the presence of Cre recombinase, lox P sites are combined, excising the sequences between them. Adult mice carrying the conditional RPS19R62W transgene and the interferon inducible Mx1-Cre gene were treated with poly (I:C) to induce excision of the stop sequence. Following poly (I:C) administration, hematocrits dropped significantly in RPS19R62W/Mx1-Cre animals compared to controls, but rebounded to normal within two weeks, due to incomplete stop sequence excision and expansion of unexcised cells in the bone marrow. Colony-forming cell assays indicate that RPS19R62W-expressing bone marrow contains 2 to 3 fold fewer BFU-E and CFU-E (p<0.05) and similar numbers of CFU-GM compared to wild-type animals. The decrease in erythroid progenitors was variable, indicating different levels of excision as well as penetrance. When RPS19R62W mice were crossed to Prion-Cre mice, which express Cre at the early embryonic stage, small, anemic d13.5 embryos and occasional small, adult animals with macrocytic anemia were observed. Day 13.5 RPS19R62W/Prion-Cre fetal livers had reduced overall numbers of erythroid cells, and reduced numbers of BFU-E and CFU-E. The decrease in erythroid progenitors was variable, especially in the line carrying 1 copy of the transgene compared to the line carrying 4 copies of the transgene. FACS analysis of d13.5 fetal liver and adult RPS19R62W/Prion-Cre erythroid cells revealed a relative accumulation of erythroid progenitor cells and a relative decrease in the number of terminally differentiating erythroid cells, suggesting that terminal erythroid differentiation is delayed. These findings are consistent with the reticulocytopenia observed in adult RPS19R62W/Prion-Cre mice. In summary we have successfully generated a mouse model of DBA caused by ectopic expression of mutant human RPS19R62W. The development of a severe anemia following conditional expression of mutant RPS19 suggests that the R62W missense mutation has a dominant negative effect that delays erythropoiesis causing an overall reduction in erythroid cells.

Differential Pathogenesis of Bernard-Soulier Syndrome-Associated Mutations In the Platelet Glycoprotein Ibβ Ectodomain

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3202-3202
Author(s):  
Wenjun Yang ◽  
P.A McEwan ◽  
Xi Mo ◽  
Jonas Emsley ◽  
Renhao Li
Keyword(s):  
Cho Cells ◽  
Disulfide Bonds ◽  
Surface Expression ◽  
Full Length ◽  
Platelet Glycoprotein ◽  
Interfacial Region ◽  
Support Surface ◽  
Wild Type ◽  
Platelet Surface ◽  
Bernard Soulier Syndrome

Abstract Abstract 3202 Eight missense mutations in the ectodomain of glycoprotein (GP)Ibβ have been identified in patients with Bernard-Soulier syndrome (BSS) that is characterized by the deficiency of functional GPIb-IX complex on the platelet surface, clearly highlighting the importance of GPIbβ ectodomain in assembly of the GPIb-IX complex. To understand the molecular pathogenesis of these mutations, we have characterized their effects on the expression, secretion, folding of the isolated GPIbβ ectodomain as well as its interaction with GPIX ectodomain in the context of full-length complex. Each of the 8 mutations — C5Y, R17C, P29L, N64T, P74R, Y88C, P96S, and A108P — was constructed into genes encoding HA-tagged GPIbβ ectodomain or full-length GPIbβ subunit, and the mutant gene transfected transiently, along with GPIba and GPIX genes if desired, into Chinese hamster ovary (CHO) cells. Flow cytometry and Western blot analysis indicated that while all 8 mutations impeded formation of the disulfide bonds between GPIba and GPIbβ and significantly decreased the surface expression level of GPIb-IX complex comparing to the wild-type, the extent of disruption varies with each mutation. Further characterization in the context of isolated GPIbβ ectodomain revealed that the majority of 8 mutations — C5Y, R17C, P29L, N64T, Y88C, P96S — are detrimental to proper folding of the GPIbβ ectodomain, resulting in secretion defects and/or domain misfolding. In contrast, two mutations, P74R and A108P, preserved structural integrity of the GPIbβ ectodomain since the mutant ectodomains exhibited wild-type-like secretion levels and formed no inter-molecular disulfide bonds. However, neither of the two mutations, in the context of full-length GPIbβ, were able to support surface expression of GPIX in transfected CHO cells as the wild-type, indicating that P74R and A108P disrupt the interaction between GPIbβ and GPIX ectodomains. Thus, our results demonstrated although all 8 BSS mutations in GPIbβ share the same phenotype, they impair expression of the GPIb-IX complex by two different mechanisms — disrupting folding of the GPIbβ ectodomain or disrupting interactions between GPIb-IX subunits. Furthermore, our results suggest that Pro74 and Ala108 may be located in the interfacial region between GPIbβ and GPIX ectodomains, helping to shed light on the structure of GPIb-IX complex. Disclosures: No relevant conflicts of interest to declare.

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