Successful expression of a functional yeast G-protein-coupled receptor (Ste2) in mammalian cells

Yin, Dameng; Gavi, Shai; Shumay, Elena; Duell, Ken; Konopka, James B.; Malbon, Craig C.; Wang, Hsien‐yu

doi:10.1016/j.bbrc.2005.01.130

Cited by 18 publications

(18 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Possibly, the number of receptors is tightly controlled by a self-regulating mechanism that interferes with induced overproduction. In contrast, Ste2p could be functionally overexpressed in Sf9 as well as in mammalian cells (Table 4), in agreement with results obtained in other laboratories (Lundstrom et al, 2006;Yin et al, 2005).…”

Section: Discussionsupporting

confidence: 92%

Functional expression of mammalian receptors and membrane channels in different cells

Eifler

Duckely

Sumanovski

et al. 2007

Journal of Structural Biology

Self Cite

View full text Add to dashboard Cite

Section: Discussionsupporting

confidence: 92%

Functional expression of mammalian receptors and membrane channels in different cells

Eifler

Duckely

Sumanovski

et al. 2007

Journal of Structural Biology

Self Cite

View full text Add to dashboard Cite

“…This result is consistent with rapid degradation of PRE-2 in sexually differentiated tissues, the site where pheromone receptors are assumed to be most active. Such a scenario is observed in S. cerevisiae, with ubiquitination leading to rapid turnover of pheromone receptors (Yin et al 2005). …”

Section: Resultsmentioning

confidence: 96%

Roles for Receptors, Pheromones, G Proteins, and Mating Type Genes During Sexual Reproduction in Neurospora crassa

Kim

Wright²,

Park³

et al. 2012

Genetics

View full text Add to dashboard Cite

Here we characterize the relationship between the PRE-2 pheromone receptor and its ligand, CCG-4, and the general requirements for receptors, pheromones, G proteins, and mating type genes during fusion of opposite mating-type cells and sexual sporulation in the multicellular fungus Neurospora crassa. PRE-2 is highly expressed in mat a cells and is localized in male and female reproductive structures. Dpre-2 mat a females do not respond chemotropically to mat A males (conidia) or form mature fruiting bodies (perithecia) or meiotic progeny (ascospores). Strains with swapped identity due to heterologous expression of pre-2 or ccg-4 behave normally in crosses with opposite mating-type strains. Coexpression of pre-2 and ccg-4 in the mat A background leads to self-attraction and development of barren perithecia without ascospores. Further perithecial development is achieved by inactivation of Sad-1, a gene required for meiotic gene silencing. Findings from studies involving forced heterokaryons of opposite mating-type strains show that presence of one receptor and its compatible pheromone is necessary and sufficient for perithecial development and ascospore production. Taken together, the results demonstrate that although receptors and pheromones control sexual identity, the mating-type genes (mat A and mat a) must be in two different nuclei to allow meiosis and sexual sporulation to occur.

show abstract

“…Recent advances in the expression and purification of membrane proteins have been described for various expression hosts, for example: Escherichia coli (Drew et al 2003(Drew et al , 2005Grisshammer et al 2005), yeast (Wedekind et al 2006;Lee et al 2007), insect cells (Massotte 2003), mammalian cells (Yin et al 2005;Werner et al 2008) and cell-free systems (Klammt et al 2007). However, from approximately 1000 known GPCRs, only five high-resolution 3-D structures of two distinct receptor types have been reported: bovine rhodopsin (Palczewski et al 2000) and opsin (Park et al 2008), squid rhodopsin (Murakami and Kouyama 2008), the human b2-adrenergic receptor Rosenbaum et al 2007) and the turkey b1-adrenergic receptor (Warne et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

2008

View full text Add to dashboard Cite

The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His) 6 tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.

show abstract

Successful expression of a functional yeast G-protein-coupled receptor (Ste2) in mammalian cells

Cited by 18 publications

References 35 publications

Functional expression of mammalian receptors and membrane channels in different cells

Functional expression of mammalian receptors and membrane channels in different cells

Roles for Receptors, Pheromones, G Proteins, and Mating Type Genes During Sexual Reproduction in Neurospora crassa

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

Contact Info

Product

Resources

About