Partial purification of a putative proctolin receptor from the African migratory locust

Puiroux, Jacques; Pedelaborde, Anne; Loughton, B.G.

doi:10.1016/s0965-1748(98)00064-2

Cited by 4 publications

(4 citation statements)

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“…Although absolute levels of CG6986 mRNA appeared low, they were comparable to those of the neuropeptide F GPCR, CG1147 (30). Proctolin binding sites from locust hindgut and oviducal muscles display an estimated size Ϸ50 kDa after partial purification (40). We estimated the size of the CG6986 protein at Ϸ60 kDa by Western Blot analysis, a size consistent with the computational prediction.…”

Section: Discussionsupporting

confidence: 71%

Identification and characterization of a G protein-coupled receptor for the neuropeptide proctolin in Drosophila melanogaster

Johnson

Garczynski

Park

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Proctolin is a bioactive neuropeptide that modulates interneuronal and neuromuscular synaptic transmission in a wide variety of arthropods. We present several lines of evidence to propose that the orphan G protein-coupled receptor CG6986 of Drosophila is a proctolin receptor. When expressed in mammalian cells, CG6986 confers second messenger activation after proctolin application, with an EC 50 of 0.3 nM. In competition-based studies, the CG6986 receptor binds proctolin with high affinity (IC50 ‫؍‬ 4 nM). By microarray analysis, CG6986 transcript is consistently detected in head mRNA of different genotypes, and under different environmental conditions. By blot analysis, anti-CG6986 antibodies detect a band in tissue homogenates similar to the predicted size of the protein. Proctolin receptor immunosignals are found in the hindgut, heart, and in distinct neuronal populations of the CNS; such patterns correlate with previous demonstrations of proctolin biological activity, and in several instances, with areas of proctolin peptide immunosignals. The identification of a bona fide proctolin receptor provides the basis for a mechanistic analysis of this critical synaptic modulator.T he pentapeptide proctolin (RYLPT) was the first neuropeptide to be isolated and sequenced from insects (1). It was purified based on its myotropic actions on the insect hindgut, and was subsequently found to have wide distribution throughout the arthropods (2-4). In the crab Cancer, proctolin has potent effects on defined neural circuits within the stomatogastric system (5) and at the neuromuscular junction (6). In insects, proctolin is a major peptide cotransmitter and it modulates contractions of both somatic and visceral muscles (7,8). Proctolin-like immunosignals are found within a small number of neurons in the larval CNS of Drosophila and these include efferents to bodywall muscles, heart, and viscera (9).The mechanisms of actions of proctolin have also been investigated. Proctolin elevates levels of inositol trisphosphate (IP 3 ) and cAMP, and increases calcium entry through voltagegated ion channels (10-13). Several studies have suggested the existence of multiple proctolin receptor subtypes based on binding studies and activities of proctolin (14, 15) or its structural analogues (16-19). However, more recent studies (15,20,21) of proctolin binding support a model featuring a single proctolin receptor. The identification of a specific proctolin receptor will help address issues regarding possibilities of multiple receptors, the precise nature of proctolin-dependent signaling pathways, and phylogenetic differences in proctolin signaling. The significance of such studies is substantial because proctolin exerts such a widespread role as a synaptic modulator in arthropod physiology.Analysis of the completed Drosophila genomic sequences (22, 23) identified Ͼ100 genes encoding G protein-coupled receptors (GPCRs). Further analysis indicated that 44 such receptors are likely to have peptide ligands and that the majority of these are derive...

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Section: Discussionsupporting

confidence: 71%

Identification and characterization of a G protein-coupled receptor for the neuropeptide proctolin in Drosophila melanogaster

Johnson

Garczynski

Park

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…In the locust, similar experiments were conducted after digitonin solubilization and iso‐electric focusing of proctolin‐binding sites. They revealed very close K d values for oviduct (19.1 ± 3.8 n m ) and hindgut (22.9 ± 3.4 n m ) proctolin‐binding proteins [18]. In both cases, the affinity found on partially purified proctolin‐binding sites is stronger than that found on membranes and is closer to the concentrations required to induce contractions.…”

Section: Discussionmentioning

confidence: 99%

“…Previous work was undertaken to purify proctolin‐binding proteins from locust and the most efficient rate of solubilization from hindgut or oviduct membranes was achieved with membrane preparations at a low protein concentration (1 mg·mL −1 ) using 1% digitonin [18]. We checked the efficacy of Chaps and digitonin, two detergents used frequently for membrane protein solubilization, on cockroach foregut membrane preparations at 1 mg protein·mL −1 .…”

Section: Discussionmentioning

confidence: 99%

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Purification of proctolin‐binding proteins from the foregut of the insect Blaberus craniifer

Mazzocco

Puiroux

2000

European Journal of Biochemistry

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A membrane protein that specifically binds the insect neuropeptide proctolin was purified using standard chromatography from cockroach foregut membranes. Proctolin-binding sites were efficiently solubilized with either the nonionic detergent digitonin or the zwitterionic detergent Chaps, as indicated by the specific binding of 3 H-proctolin to solubilized samples. A solubilized sample obtained from 1600 foregut membranes was subjected to a five-step chromatographic purification including chromatofocusing, anion-exchange and size-exclusion chromatographies. The final size-exclusion separation resulted in the isolation of < 100 pmol of purified proctolin-binding proteins, eluting as a single peak at < 74 kDa. Analysis of the purified sample using SDS/PAGE and silver staining showed two bands at 80 kDa and 76 kDa. Densitometric analysis of the gel indicated that each band contained < 7±8 mg of protein, suggesting that one band corresponds to the proctolinbinding activity. Proctolin-binding proteins were thus purified 1800-fold using standard chromatography.

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Proctolin in the post-genomic era: new insights and challenges

et al. 2004

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Complete understanding of how neuropeptides operate as neuromodulators and neurohormones requires integration of knowledge obtained at different levels of biology, including molecular, biochemical, physiological and whole organism studies. Major advances have recently been made in the understanding of the molecular basis of neuropeptide action in invertebrates by analysis of data generated from sequencing the genomes of several insect species, especially that of Drosophila melanogaster. This approach has quickly led to the identification of genes encoding: (1) novel neuropeptide sequences, (2) neuropeptide receptors and (3) peptidases that might be responsible for the processing and inactivation of neuropeptides. In this article, we review our current knowledge of the biosynthesis, receptor interaction and metabolic inactivation of the arthropod neuropeptide, proctolin, and how the analysis and exploitation of genome sequencing projects has provided new insights.

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Partial purification of a putative proctolin receptor from the African migratory locust

Cited by 4 publications

References 14 publications

Identification and characterization of a G protein-coupled receptor for the neuropeptide proctolin in Drosophila melanogaster

Identification and characterization of a G protein-coupled receptor for the neuropeptide proctolin in Drosophila melanogaster

Purification of proctolin‐binding proteins from the foregut of the insect Blaberus craniifer

Proctolin in the post-genomic era: new insights and challenges

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