| DOI | Resolve DOI: https://doi.org/10.1016/S0166-6851(01)00400-5 |
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| Author | Search for: Hamdan, Fadi F.; Search for: Abramovitz, Mark; Search for: Mousa, Aisha; Search for: Xie, Jinling; Search for: Durocher, Yves1; Search for: Ribeiro, Paula |
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| Affiliation | - National Research Council of Canada. NRC Biotechnology Research Institute
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| Format | Text, Article |
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| Subject | amino acid sequence; base sequence; cloning, molecular; evolution, molecular; helminth proteins; Heterotrimeric GTP-binding proteins; histamine; luminescent measurements; microscopy, fluorescence; molecular sequence data; receptors, cell surface; receptors, G-protein-coupled; Schistosoma mansoni; sequence homology, amino acid |
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| Abstract | A new cDNA was cloned from the bloodfluke, Schistosoma mansoni and shown to encode a protein with structural characteristics of a biogenic amine G protein-coupled receptor (GPCR). At the amino acid level, the parasite receptor (SmGPCR) shared about the same level of sequence homology (approximately 30%) with all major types of amine GPCRs and could not be identified on the basis of sequence. SmGPCR exhibited several nonconservative substitutions at key GPCR positions, including an unusual asparagine substitution (Asn(111)) for the highly conserved aspartate of transmembrane (TM) 3. The full-length SmGPCR cDNA was double-tagged with N-terminal FLAG and C-terminal hexahistidine epitopes, and was codon-optimized for expression in cultured HEK293 and COS7 cells. In situ immunofluorescence analyses targeting the two N- and C-terminal epitopes demonstrated that the modified SmGPCR was expressed at high level in mammalian cells and assumed a typical GPCR topology, the N-terminus being extracellular and the C-terminus intracellular. Functional activity assays revealed that SmGPCR was responsive to histamine, which caused a dose-dependent elevation in intracellular Ca2+ (EC50=0.54+/-0.05 microM). An Asn(111)-->Asp mutation had no effect on the responsiveness to histamine, suggesting that SmGPCR does not require the TM3 aspartate for agonist activation, in contrast to most amine GPCRs. None of the other monoamines tested had any significant effect on receptor activity, using assays that measured both Ca2+- and cAMP-mediated signaling. The results suggest that SmGPCR is a novel structural class of histamine receptor that may be unique to flatworms. |
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| Publication date | 2002-01 |
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| In | |
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| Language | English |
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| NPARC number | 12333638 |
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| Export citation | Export as RIS |
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| Report a correction | Report a correction (opens in a new tab) |
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| Record identifier | b38afdbb-80f0-4997-ac9e-9791b9694db1 |
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| Record created | 2009-09-10 |
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| Record modified | 2020-03-30 |
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