Functional characterization of ecdysone receptor gene switches in mammalian cells

Dynamin proteins containing a GTPase domain, a pleckstrin homology motif and a proline-rich tail participate in receptor-mediated endocytosis in organisms ranging from insects to vertebrates. In addition, dynamin-related GTPases, such as the yeast Golgi protein Vps1p, which lack both the pleckstrin homology motif and the proline-rich region, participate in vesicular transport within the secretory pathway in lower eukaryotes. However, no data is available on the existence of Vps1p-like proteins in mammalian cells. In this study, we report the identification and characterization of a novel gene encoding a human dynamin-related protein, DRP1, displaying high similarity to the Golgi dynamin-like protein Vps1p from yeast and to a Caenorhabditis elegans protein deposited in the databank. These proteins are highly conserved in their N-terminal tripartite GTPase domain but lack the pleckstrin homology motif and proline-rich region. Northern blot analysis reveals that the DRP1 mRNA is detected at high levels in human muscle, heart, kidney and brain. Immunolocalization studies in Chinese hamster ovary (CHO) cells using an epitope-tagged form of DRP1 and confocal microscopy show that this protein is concentrated in a perinuclear region that labels with the endoplasmic reticulum marker DiOC6(3) and the Golgi marker C5-DMB-Cer. In addition, the localization of DRP1 is highly similar to the localization of the endoplasmic reticulum and cis-Golgi GTPase Rab1A, but not to the staining for the trans-Golgi GTPase Rab6. Furthermore, overexpression of a cDNA encoding a GTP binding site mutant of DRP1 (DRP1(K38E)) in CHO cells decreases the amount of a secreted luciferase reporter protein, whereas the overexpression of wild-type DRP1 increases the secretion of this marker. Together, these results constitute the first structural and functional characterization of a mammalian protein similar to the yeast dynamin-related GTPase Vps1p and indicate that the participation of these proteins in secretion has been conserved throughout evolution.

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Functional characterization of naturally occurring NR3C2 gene mutations in Italian patients suffering from pseudohypoaldosteronism type 1

Acta Endocrinologica ◽

10.1530/eje.1.02330 ◽

2007 ◽

Vol 156 (2) ◽

pp. 249-256 ◽

Cited By ~ 17

Author(s):

Antonio Balsamo ◽

Alessandro Cicognani ◽

Monia Gennari ◽

Wolfgang G Sippell ◽

Soara Menabò ◽

...

Keyword(s):

Receptor Gene ◽

Functional Characterization ◽

Clinical Signs ◽

Gene Mutations ◽

Ethnic Origin ◽

Snp Markers ◽

Italian Population ◽

Pseudohypoaldosteronism Type

Objective: The renal form of pseudohypoaldosteronism type 1 (PHA1) is a rare disease caused by mutations in the human mineralocorticoid receptor gene (NR3C2). Design: Aim of the study was to analyze the NR3C2 gene in three Italian patients with clinical signs of renal PHA1 and to evaluate the distribution of the -2G > C, c.538A > G, and c.722C > T single nucleotide polymorphism (SNP) pattern in the PHA1 patients and in 90 controls of the same ethnic origin. Methods: Analysis of the NR3C2 gene sequence and of the polymorphic SNP markers. Functional characterization of the detected novel NR3C2 mutations utilizing aldosterone-binding assays and reporter gene transactivation assays. Results: One novel nonsense (Y134X) and one novel frameshift (2125delA) mutation were detected. They exhibited no aldosterone binding and no transactivation abilities. No mutation was detected in the third patient. Haploinsufficiency of NR3C2 was ruled out by microsatellite analysis in this patient. The c.722T SNP was detected in 97% of alleles in the Italian population which is significantly different from the general German or US population. Conclusions: Molecular analysis of the NR3C2 gene in PHA1 patients is warranted to detect novel mutations in order to clarify the underlying genetic cause, which may extend the insight into relevant functional regions of the hMR protein. The effect the different distribution of the c.722T SNP is not clear to date. Further studies are necessary to provide evidence as to a possible advantage of a less sensitive hMR in southern countries.

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Cloning and functional characterization of the mouse C3a anaphylatoxin receptor gene

Immunogenetics ◽

10.1007/s002510050327 ◽

1997 ◽

Vol 47 (1) ◽

pp. 64-72 ◽

Cited By ~ 28

Author(s):

Matthew H. Hsu ◽

Julia A. Ember ◽

Meiying Wang ◽

Eric R. Prossnitz ◽

Tony E. Hugli ◽

...

Keyword(s):

Receptor Gene ◽

Functional Characterization

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Family-Based Clinical Associations and Functional Characterization of the Serotonin 2A Receptor Gene (HTR2A) in Autism Spectrum Disorder

Autism Research ◽

10.1002/aur.1383 ◽

2014 ◽

Vol 7 (4) ◽

pp. 459-467 ◽

Cited By ~ 15

Author(s):

Ryan M. Smith ◽

Wesley Banks ◽

Emily Hansen ◽

Wolfgang Sadee ◽

Gail E. Herman

Keyword(s):

Autism Spectrum Disorder ◽

Receptor Gene ◽

Functional Characterization ◽

Autism Spectrum ◽

Spectrum Disorder ◽

Serotonin 2A Receptor ◽

Clinical Associations ◽

Family Based ◽

Serotonin 2A

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Functional Characterization of Soybean Glyma04g39610 as a Brassinosteroid Receptor Gene and Evolutionary Analysis of Soybean Brassinosteroid Receptors

International Journal of Molecular Sciences ◽

10.3390/ijms17060897 ◽

2016 ◽

Vol 17 (6) ◽

pp. 897 ◽

Cited By ~ 8

Author(s):

Suna Peng ◽

Ping Tao ◽

Feng Xu ◽

Aiping Wu ◽

Weige Huo ◽

...

Keyword(s):

Receptor Gene ◽

Functional Characterization ◽

Evolutionary Analysis

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Genetic and functional characterization of putative Ras/Raf interaction inhibitors in C. elegans and mammalian cells

Journal of Molecular Signaling ◽

10.1186/1750-2187-5-2 ◽

2010 ◽

Vol 5 ◽

pp. 2 ◽

Cited By ~ 28

Author(s):

Vanessa González-Pérez ◽

David J Reiner ◽

Jamie K Alan ◽

Cicely Mitchell ◽

Lloyd J Edwards ◽

...

Keyword(s):

Mammalian Cells ◽

Functional Characterization ◽

C Elegans

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Characterization of small-molecule inhibitors of the sodium iodide symporter

Journal of Endocrinology ◽

10.1677/joe-08-0246 ◽

2008 ◽

Vol 200 (3) ◽

pp. 357-365 ◽

Cited By ~ 14

Author(s):

Sabine Lindenthal ◽

Nathalie Lecat-Guillet ◽

Alejandro Ondo-Mendez ◽

Yves Ambroise ◽

Bernard Rousseau ◽

...

Keyword(s):

Mammalian Cells ◽

Sodium Iodide ◽

Functional Characterization ◽

Screening Method ◽

Inhibitory Effect ◽

Independent Effect ◽

Induced Current ◽

Sodium Iodide Symporter ◽

Iodide Uptake

The sodium/iodide symporter (NIS) mediates the active transport of iodide from the bloodstream into thyrocytes. NIS function is strategic for the diagnosis and treatment of various thyroid diseases. In addition, a promising anti-cancer strategy based on targeted NIS gene transfer in non-thyroidal cells is currently developed. However, only little information is available concerning the molecular mechanism of NIS-mediated iodide translocation. Ten small molecules have recently been identified using a high-throughput screening method for their inhibitory effect on iodide uptake of NIS-expressing mammalian cells. In the present study, we analyzed these compounds for their rapid and reversible effects on the iodide-induced current in NIS-expressing Xenopus oocytes. Four molecules almost completely inhibited the iodide-induced current; for three of them the effect was irreversible, for one compound the initial current could be fully re-established after washout. Three molecules showed a rapid inhibitory effect of about 75%, half of which was reversible. Another three compounds inhibited the iodide-induced current from 10 to 50%. Some molecules altered the membrane conductance by themselves, i.e. in the absence of iodide. For one of these molecules the observed effect was also found in water-injected oocytes whereas for some others the iodide-independent effect was associated with NIS expression. The tested molecules show a surprisingly high variability in their possible mode of action, and thus are promising tools for further functional characterization of NIS on a molecular level, and they could be useful for medical applications.

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