Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system

AbstractSchistosomiasis is an acute and chronic disease caused by parasitic worms of the genus Schistosoma. Treatment is solely dependent on praziquantel. In the face of the worldwide dimension, projects have been initiated to develop new chemotherapies. Due to their proven druggability, G protein-coupled receptors (GPCRs) are promising targets for anthelmintics. However, to identify candidate receptors, a deeper understanding of GPCR signalling in schistosome biology is essential. Comparative transcriptomics of paired and unpaired worms and their gonads revealed 59 differentially regulated GPCR-coding genes putatively involved in neuronal processes. In general, the diversity among GPCRs and their integral membrane topology make it difficult to characterize and deorphanize these receptors. To overcome existing limitations, we performed a pilot approach and utilized the innovative Membrane-Anchored Ligand And Receptor yeast two-hybrid system (MALAR-Y2H) to associate potential neuropeptide ligands with their cognate receptors. Here, we demonstrated the ability to express full-length GPCRs of Schistosoma mansoni in a heterologous yeast-based system. Additionally, we localized GPCRs and chimeras of neuropeptides fused to the WBP1 transmembrane domain of yeast to the plasma membrane of yeast cells. Reporter gene assays indicated ligand-receptor binding, which allowed us to identify certain neuropeptides as potential ligands for two GPCRs, which had been found before to be differentially expressed in schistosomes in a pairing-dependent manner. Thus, the MALAR-Y2H system appears suitable to unravel schistosome GPCR–ligand interactions. Besides its relevance for understanding schistosome biology, identifying and characterizing GPCR–ligand interaction will also contribute to applied research aspects.

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Rapid, Facile Detection of Heterodimer Partners for Target Human G-Protein-Coupled Receptors Using a Modified Split-Ubiquitin Membrane Yeast Two-Hybrid System

PLoS ONE ◽

10.1371/journal.pone.0066793 ◽

2013 ◽

Vol 8 (6) ◽

pp. e66793 ◽

Cited By ~ 12

Author(s):

Yasuyuki Nakamura ◽

Jun Ishii ◽

Akihiko Kondo

Keyword(s):

G Protein ◽

Hybrid System ◽

G Protein Coupled Receptors ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Split Ubiquitin ◽

G Protein Coupled ◽

Two Hybrid

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Novel Protein Interactions of G Protein-Coupled Receptor Kinase 5 (GRK5) Searched with Yeast Two-Hybrid System

Korean Circulation Journal ◽

10.4070/kcj.2002.32.7.613 ◽

2002 ◽

Vol 32 (7) ◽

pp. 613 ◽

Cited By ~ 1

Author(s):

Byung Cheol Jin ◽

Tae Joon Park ◽

Eun Ji Kim ◽

Ji Eun Lee ◽

Jung Hoon Lee ◽

...

Keyword(s):

G Protein ◽

Hybrid System ◽

Protein Interactions ◽

Receptor Kinase ◽

Yeast Two Hybrid ◽

G Protein Coupled Receptor ◽

Yeast Two Hybrid System ◽

G Protein Coupled ◽

Two Hybrid ◽

Novel Protein

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Functional Analyses of the Pto Resistance Gene Family in Tomato and the Identification of a Minor Resistance Determinant in a Susceptible Haplotype

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.3.281 ◽

2002 ◽

Vol 15 (3) ◽

pp. 281-291 ◽

Cited By ~ 54

Author(s):

Jeff H. Chang ◽

Yin-Shan Tai ◽

Adriana J. Bernal ◽

Daniel T. Lavelle ◽

Brian J. Staskawicz ◽

...

Keyword(s):

Cell Death ◽

Amino Acid ◽

Hybrid System ◽

Dependent Manner ◽

Wild Type ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

A Minor ◽

Two Hybrid

Pto is a member of a multigene family and encodes a serine/threonine kinase that mediates gene-for-gene resistance to strains of Pseudomonas syringae pv. tomato expressing avrPto. The inferred amino acid sequence of the Pto homologs from both resistant (LpimPth2 to LpimPth4,) and susceptible (LescFen, LescPth2 to LescPth5) haplotypes suggested that most could encode functional serine/threonine kinases. In addition, the activation segments of the homologs are similar in sequence to that of Pto, and some have residues previously identified as required for binding of AvrPto by Pto in the yeast two-hybrid system. The Pto homologs were therefore characterized for transcription, for the ability of their products to interact with AvrPto in the yeast two-hybrid system, for their autophos-phorylation activity, and for their potential to elicit cell death in the presence of and absence of a ligand, as well as their dependence on Prf. LpimPth5, LpimPth4, and LescPth4 were not transcribed at levels detectable by reverse transcription-polymerase chain reaction. The interaction with AvrPto was unique to Pto in the yeast two-hybrid system. LescPth2 autophosphorylated in vitro as a fusion protein. LpimPth2, LpimPth3, LpimPth4, LescPth3, and LescPth4 did not autophosphorylate in vitro. Transient expression of wild-type Fen and wild-type LpimPth3, as well as LescFen, LescPth3, and LescPth5 with perturbations in their P+1 loop caused cell death in Nicotiana benthamiana. LpimPth3 and LescPth3 with amino acid substitutions in the P+1 loop also elicited cell death in tomato; this was dependent on the presence of wild-type Prf. Consequently, some homologs could potentially encode functional resistance proteins. LescPth5 induced cell death specifically in response to expression of AvrPto in tobacco in a Prf-dependent manner; this is consistent with a homolog from a ‘susceptible’ haplotype encoding a minor recognition determinant.

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Interactions between G-protein-coupled receptors and periplakin: a selective means to regulate G-protein activation

Biochemical Society Transactions ◽

10.1042/bst0320878 ◽

2004 ◽

Vol 32 (5) ◽

pp. 878-880 ◽

Cited By ~ 9

Author(s):

G. Milligan ◽

H. Murdoch ◽

E. Kellett ◽

J.H. White ◽

G.-J. Feng

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Interacting Proteins ◽

Yeast Two Hybrid ◽

Protein Activation ◽

G Protein Activation ◽

Melanin Concentrating Hormone ◽

Receptor Interacting Proteins ◽

G Protein Coupled ◽

Two Hybrid

A substantial number of G-protein-coupled receptor-interacting proteins have been identified initially by the use of yeast two-hybrid screens. Using the C-terminal tail of both opioid receptors and the melanin concentrating hormone receptor-1 as bait, the actin and intermediate filament-binding protein periplakin was isolated. In each case, the site of interaction is within helix VIII of the receptor and periplakin limits agonist-mediated G-protein activation potentially by competing with G-protein for this region of the receptor.

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GPR11, a Putative Seven-Transmembrane G Protein-Coupled Receptor, Controls Zoospore Development and Virulence of Phytophthora sojae

Eukaryotic Cell ◽

10.1128/ec.00265-09 ◽

2009 ◽

Vol 9 (2) ◽

pp. 242-250 ◽

Cited By ~ 19

Author(s):

Yonglin Wang ◽

Aining Li ◽

Xiaoli Wang ◽

Xin Zhang ◽

Wei Zhao ◽

...

Keyword(s):

G Protein ◽

Hyphal Growth ◽

Phytophthora Sojae ◽

G Protein Coupled Receptors ◽

Yeast Two Hybrid ◽

Content Type ◽

Signaling Mechanisms ◽

G Protein Coupled ◽

Two Hybrid ◽

Receptor Family

ABSTRACT G protein-coupled receptors (GPCRs) represent a large receptor family involved in a broad spectrum of cell signaling. To understand signaling mechanisms mediated by GPCRs in Phytophthora sojae, we identified and characterized the PsGPR11 gene, which encodes a putative seven-transmembrane GPCR. An expression analysis revealed that PsGPR11 was differentially expressed during asexual development. The highest expression level occurred in zoospores and was upregulated during early infection. PsGPR11-deficienct transformants were obtained by gene silencing strategies. Silenced transformants exhibited no differences in hyphal growth or morphology, sporangium production or size, or mating behavior. However, the release of zoospores from sporangia was severely impaired in the silenced transformants, and about 50% of the sporangia did not completely release their zoospores. Zoospore encystment and germination were also impaired, and zoospores of the transformants lost their pathogenicity to soybean. In addition, no interaction was observed between PsGPR11 and PsGPA1 with a conventional yeast two-hybrid assay, and the transcriptional levels of some genes which were identified as being negatively regulated by PsGPA1 were not clearly altered in PsGPR11-silenced mutants. These results suggest that PsGPR11-mediated signaling controls P. sojae zoospore development and virulence through the pathways independent of G protein.

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