Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in<i>Dictyostelium discoideum</i>

Linskens, Maarten H.K.; Grootenhuis, Peter D. J.; Blaauw, Mieke; Winkel, Bianca Huisman‐de; Ravestein, Arno van; Haastert, Peter J.M. van; Heikoop, Judith C.

doi:10.1096/fasebj.13.6.639

Cited by 20 publications

(20 citation statements)

References 29 publications

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“…To express the wild-type amtA gene, a plasmid containing amtA was constructed by inserting a PCR fragment containing the complete amtA open reading frame into the Dictyostelium extrachromosomal expression vector HK12neo, a derivative of MB12neo (43).…”

Section: Strainsmentioning

confidence: 99%

Regulation of Ammonia Homeostasis by the Ammonium Transporter AmtA in Dictyostelium discoideum

et al. 2007

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Ammonia has been shown to function as a morphogen at multiple steps during the development of the cellular slime mold Dictyostelium discoideum; however, it is largely unknown how intracellular ammonia levels are controlled. In the Dictyostelium genome, there are five genes that encode putative ammonium transporters: amtA, amtB, amtC, rhgA, and rhgB. Here, we show that AmtA regulates ammonia homeostasis during growth and development. We found that cells lacking amtA had increased levels of ammonia/ammonium, whereas their extracellular ammonia/ ammonium levels were highly decreased. These results suggest that AmtA mediates the excretion of ammonium. In support of a role for AmtA in ammonia homeostasis, AmtA mRNA is expressed throughout the life cycle, and its expression level increases during development. Importantly, AmtA-mediated ammonia homeostasis is critical for many developmental processes. amtA ؊ cells are more sensitive to NH 4 Cl than wild-type cells in inhibition of chemotaxis toward cyclic AMP and of formation of multicellular aggregates. Furthermore, even in the absence of exogenously added ammonia, we found that amtA ؊ cells produced many small fruiting bodies and that the viability and germination of amtA ؊ spores were dramatically compromised. Taken together, our data clearly demonstrate that AmtA regulates ammonia homeostasis and plays important roles in multiple developmental processes in Dictyostelium.

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Section: Strainsmentioning

confidence: 99%

Regulation of Ammonia Homeostasis by the Ammonium Transporter AmtA in Dictyostelium discoideum

et al. 2007

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“…Finally, the complete gbpD gene was released with BamHI and XbaI, and cloned between the actin 15 promoter and the actin 8 terminator of MB74 using its BglII and SpeI sites. MB74 is an extrachromosomal shuttle plasmid for Dictyostelium derived from MB12NEO (Linskens et al, 1999). Approximately 1-4 µg plasmid was electroporated to the cells as described above.…”

Section: Construction Of Overexpression Plasmidsmentioning

confidence: 99%

RasGEF-containing proteins GbpC and GbpD have differential effects on cell polarity and chemotaxis in Dictyostelium

Bosgraaf

Waijer

Engel

et al. 2005

Journal of Cell Science

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The regulation of cell polarity plays an important role in chemotaxis. Previously, two proteins termed GbpC and GbpD were identified in Dictyostelium, which contain RasGEF and cyclic nucleotide binding domains. Here we show that gbpC-null cells display strongly reduced chemotaxis, because they are unable to polarise effectively in a chemotactic gradient. However, gbpD-null mutants exhibit the opposite phenotype: cells display improved chemotaxis and appear hyperpolar, because cells make very few lateral pseudopodia, whereas the leading edge is continuously remodelled. Overexpression of GbpD protein results in severely reduced chemotaxis. Cells extend many bifurcated and lateral pseudopodia, resulting in the absence of a leading edge. Furthermore, cells are flat and adhesive owing to an increased number of substrate-attached pseudopodia. This GbpD phenotype is not dependent on intracellular cGMP or cAMP, like its mammalian homolog PDZ-GEF. Previously we showed that GbpC is a high-affinity cGMP-binding protein that acts via myosin II. We conclude that cGMP activates GbpC, mediating the chemoattractant-induced establishment of cell polarity through myosin. GbpD induces the formation of substrate-attached pseudopodia, resulting in increased attachment and suppression of polarity.

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“…All PCR products generated were TOPO-cloned into pcDNA3.1/V5-His plasmid (Invitrogen) for sequence analysis. The mutant seat-belt ␤-subunit cDNA inserts were then transferred into the Dictyostelium extra-chromosomal expression vector MB12neo, using their BglII and SpeI endonuclease restriction sites (Linskens et al, 1999).…”

Section: Construction and Expression Of Cassette-substituted Gonadotrmentioning

confidence: 99%

Receptor-selective determinants in catfish gonadotropin seat-belt loops

Vischer

Marques

Granneman

et al. 2004

Molecular and Cellular Endocrinology

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Mammalian gonadotropins are highly selective. Charge differences between the Cys 10-11 sequence of FSH␤ and LH␤/CG␤ seat-belt loops determine the ability of these hormones to interact with the LH-R. Selective FSH-R binding is mainly dependent on the presence of an FSH␤-specific sequence between Cys 11-12 of the seat-belt loop. Intriguingly, African catfish LH␤ (cfLH␤) lacks a positively charged Cys 10-11 region and stimulates both catfish LH-R and FSH-R with comparable potencies. Our studies on the promiscuous behaviour of cfLH using chimeric gonadotropins revealed that the Cys 10-11 region of cfLH␤ contains cfLH-R-selective determinants, whereas the Cys 11-12 region of cfLH␤ confers FSH-R-stimulating activity to cfLH. Hence, the location of receptor-selective determinants appeared to be fairly well conserved throughout evolution, despite the low sequence identity between mammalian and catfish seat-belt loops. Moreover, various structure-function differences between gonadotropins are discussed in the context of the different (female) reproductive strategies between mammalian and non-mammalian species that required the divergence to a more specific LH-R-stimulating activity of one of the gonadotropins in mammals.

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Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins inDictyostelium discoideum

Cited by 20 publications

References 29 publications

Regulation of Ammonia Homeostasis by the Ammonium Transporter AmtA in Dictyostelium discoideum

Regulation of Ammonia Homeostasis by the Ammonium Transporter AmtA in Dictyostelium discoideum

RasGEF-containing proteins GbpC and GbpD have differential effects on cell polarity and chemotaxis in Dictyostelium

Receptor-selective determinants in catfish gonadotropin seat-belt loops

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