scholarly journals Quantitative internalization kinetics of the green fluorescence protein‐tagged guanylyl (guanylate) cyclase/natriuretic peptide receptor‐A in Human Embryonic Kidney‐293 cells

2013 ◽  
Vol 27 (S1) ◽  
Author(s):  
INDRA MANI ◽  
Renu Garg ◽  
Vickey A Nguyen ◽  
Kailash N Pandey
Keyword(s):  
Guanylate Cyclase ◽  
Green Fluorescence Protein ◽  
Natriuretic Peptide Receptor ◽  
Human Embryonic Kidney ◽  
Green Fluorescence ◽  
Peptide Receptor ◽  
293 Cells ◽  
Natriuretic Peptide Receptor A ◽  
Fluorescence Protein ◽  
Kinetics Of

scholarly journals Internalization and trafficking of guanylyl (guanylate) cyclase/natriuretic peptide receptor A is regulated by an acidic tyrosine-based cytoplasmic motif GDAY

2005 ◽  
Vol 388 (1) ◽  
pp. 103-113 ◽  
Author(s):  
Kailash N. PANDEY ◽  
Huong T. NGUYEN ◽  
Renu GARG ◽  
Madan L. KHURANA ◽  
Jude FINK
Keyword(s):  
Guanylate Cyclase ◽  
Receptor Internalization ◽  
Natriuretic Peptide Receptor ◽  
Wild Type ◽  
Hek 293 ◽  
Peptide Receptor ◽  
293 Cells ◽  
Natriuretic Peptide Receptor A ◽  
Mutant Receptors

We have identified a GDAY motif in the C-terminal domain of guanylyl cyclase (guanylate cyclase)/NPRA (natriuretic peptide receptor A) sequence, which serves a dual role as an internalization signal and a recycling signal. To delineate the role of the GDAY motif in receptor internalization and sequestration, we mutated Gly920, Asp921 and Tyr923 to alanine residues (GDAY/AAAA) in the NPRA cDNA sequence. The cDNAs encoding wild-type and mutant receptors were transfected in HEK-293 cells (human embryonic kidney 293 cells). The internalization studies of ligand–receptor complexes revealed that endocytosis of 125I-ANP by HEK-293 cells expressing G920A, Y923A or GDAY/AAAA mutant receptor was decreased by almost 50% (P<0.001) when compared with cells expressing the wild-type receptor. However, the effect of D921A mutation on receptor internalization was minimal. Ligand-mediated down-regulation of G920A, Y923A and GDAY/AAAA mutant receptors was decreased by 35–40% when compared with wild-type NPRA. Subsequently, the recycling of internalized D921A and GDAY/AAAA mutant receptors from the intracellular pool was decreased by more than 40±4% when compared with wild-type NPRA. Recycling of G920A and Y923A mutant receptors was also decreased, but to a significantly lesser extent compared with the D921A or GDAY/AAAA mutant receptors. We conclude that the Gly920 and Tyr923 residues within the GDAY consensus motif are necessary for internalization, and that residue Asp921 is important for recycling of NPRA. The current results provide new evidence for a dual role of the GDAY sequence motif in ligand-mediated internalization, recycling and down-regulation of a single-transmembrane receptor protein NPRA.

scholarly journals Kinesin-1 and dynein at the nuclear envelope mediate the bidirectional migrations of nuclei

2010 ◽  
Vol 191 (1) ◽  
pp. 115-128 ◽  
Author(s):  
Heidi N. Fridolfsson ◽  
Daniel A. Starr
Keyword(s):  
Nuclear Envelope ◽  
Green Fluorescence Protein ◽  
Time Lapse ◽  
Nuclear Migration ◽  
Differential Interference Contrast ◽  
Wild Type ◽  
Green Fluorescence ◽  
Fluorescence Protein ◽  
Bidirectional Movement ◽  
Kinetics Of

Kinesin-1 and dynein are recruited to the nuclear envelope by the Caenorhabditis elegans klarsicht/ANC-1/Syne homology (KASH) protein UNC-83 to move nuclei. The mechanisms of how these motors are coordinated to mediate nuclear migration are unknown. Time-lapse differential interference contrast and fluorescence imaging of embryonic hypodermal nuclear migration events were used to characterize the kinetics of nuclear migration and determine microtubule dynamics and polarity. Wild-type nuclei display bidirectional movements during migration and are also able to roll past cytoplasmic granules. unc-83, unc-84, and kinesin-1 mutants have severe nuclear migration defects. Without dynein, nuclear migration initiates normally but lacks bidirectional movement and shows defects in nuclear rolling, implicating dynein in resolution of cytoplasmic roadblocks. Microtubules are highly dynamic during nuclear migration. EB1::green fluorescence protein imaging demonstrates that microtubules are polarized in the direction of nuclear migration. This organization of microtubules fits with our model that kinesin-1 moves nuclei forward and dynein functions to move nuclei backward for short stretches to bypass cellular roadblocks.

scholarly journals Regulation of natriuretic peptide receptor-A gene expression and stimulation of its guanylate cyclase activity by transcription factor Ets-1

2009 ◽  
Vol 29 (1) ◽  
pp. 57-70 ◽  
Author(s):  
Prerna Kumar ◽  
Gevoni Bolden ◽  
Kiran K. Arise ◽  
Stephen T. Krazit ◽  
Kailash N. Pandey
Keyword(s):  
Natriuretic Peptide ◽  
Gene Transcription ◽  
Guanylate Cyclase ◽  
Promoter Activity ◽  
Target Cells ◽  
Mrna Levels ◽  
Natriuretic Peptide Receptor ◽  
Peptide Receptor ◽  
Natriuretic Peptide Receptor A ◽  
Npr1 Gene

ANP (atrial natriuretic peptide) exerts its biological effects by binding to GC (guanylate cyclase)-A/NPR (natriuretic peptide receptor)-A, which generates the second messenger cGMP. The molecular mechanism mediating Npr1 (coding for GC-A/NPRA) gene regulation and expression is not well understood. The objective of the present study was to elucidate the mechanism by which Ets-1 [Ets (E twenty-six) transformation-specific sequence] contributes to the regulation of Npr1 gene transcription and expression. Chromatin immunoprecipitation and gel-shift assays confirmed the in vivo and in vitro binding of Ets-1 to the Npr1 promoter. Overexpression of Ets-1 enhanced significantly Npr1 mRNA levels, protein expression, GC activity and ANP-stimulated intracellular accumulation of cGMP in transfected cells. Depletion of endogenous Ets-1 by siRNA (small interfering RNA) dramatically decreased promoter activity by 80%. Moreover, methylation of the Npr1 promoter region (−356 to +55) reduced significantly the promoter activity and hypermethylation around the Ets-1 binding sites directly reduced Ets-1 binding to the Npr1 promoter. Collectively, the present study demonstrates that Npr1 gene transcription and GC activity of the receptor are critically controlled by Ets-1 in target cells.

scholarly journals Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites

2021 ◽  
Author(s):  
Mahsa Babaei ◽  
Luisa Sartori ◽  
Alexey Karpukhin ◽  
Dmitrii Abashkin ◽  
Elena Matrosova ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Dna ◽  
Green Fluorescence Protein ◽  
Cellular Growth ◽  
Green Fluorescence ◽  
Biotechnological Production ◽  
Yeast Saccharomyces Cerevisiae ◽  
Integration Sites ◽  
Fluorescence Protein ◽  
Integration Efficiency

Abstract Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

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