Identification of an Ascaris G protein-coupled acetylcholine receptor with atypical muscarinic pharmacology

Abstract

Acetylcholine (ACh) is a neurotransmitter/neuromodulator in the nematode nervous system and induces its effects through interaction with both ligand-gated ion channels (LGICs) and G proteincoupled receptors (GPCRs). The structure, pharmacology and physiological importance of LGICs have been appreciably elucidated in model nematodes, including parasitic species where they are targets for anthelmintic drugs. Significantly less, however, is understood about nematode ACh GPCRs, termed GARs (G protein-linked ACh receptors). What is known comes from the free-living Caenorhabditis elegans as no GARs have been characterized from parasitic species. Here we clone a putative GAR from the pig gastrointestinal nematode Ascaris suum with high structural homology to the C. elegans receptor GAR-1. Our GPCR, dubbed AsGAR-1, isalternatively spliced and expressed in the head and tail of adult worms but not in dorsal or ventralbody wall muscle, or the ovijector. ACh activated AsGAR-1 in a concentration-dependent manner but the receptor was not activated by other small neurotransmitters. The classical muscarinic agonists carbachol, arecoline, oxotremorine M and bethanechol were also AsGAR-1 agonists but pilocarpine was ineffective. AsGAR-1 activation by ACh was partially antagonized by the muscarinic blocker atropine but pirenzepine and scopolamine were largely ineffective. Certain biogenic amine GPCR antagonists were also found to block AsGAR-1. Our conclusion is that Ascaris possesses G protein-coupled ACh receptors that are homologous in structure to thosepresent in C. elegans, and that although they have some sequence homology to vertebrate muscarinic receptors, their pharmacology is atypically muscarinic.

Quantitative Structure-Activity Relationships (QSARs) of Monoterpenoids at an Expressed American Cockroach Octopamine Receptor

“…Therefore, when the receptor is in the active state, histidine will be produced, and yeast cells will grow when they are present in histidine-deficient medium. The histidine-auxotrophic assay was performed similarly to previous reports from our laboratories (23,25).Briefly, 2 mL of selective medium (-Leu) was inoculated with transformed yeast cells and allowed to grow overnight on an orbital shaker (30°C and 250 RPM (OD 600 1.0-2.0)). Cells were pelleted at 5,000 x g at room temperature and washed three times with medium which was deficient in leucine 99 and histidine.…”

“…Saccharomyces cerevisiae, referred to as yeast hereafter, has emerged as an important organism for the study of heterologously expressed GPCRs (21,22). Functional expression of heterotrimeric GPCRs can be achieved by linking the expressed receptor to the endogenous pheromone response pathway, which has been performed for analysis of multiple mammalian GPCRs (22) and some invertebrate GPCRs (23)(24)(25). Previously, we have reported the expression of Pa oa1 in yeast, which resulted in a ligand-independent (constitutive) expression system (25).…”

Evidence for a Bacterial Lipopolysaccharide-Recognizing G-Protein-Coupled Receptor in the Bacterial Engulfment by Entamoeba histolytica

“…The gene was cloned into the pUC57 vector, and the cDNA was amplified with forward and reverse primers, adding restriction sites for NcoI and BamHI (5=-GC CATACCATGGATCAATCATTCGGTAATCAA-3= and 5=-GCCATAGG ATCCTTAAGTCAAGTTAATTTCTCTTGAA-3=) to the 5= and 3= ends of the amplicon, respectively. Purified amplicons and the linearized yeast expression vector Cp4258, which contains a leucine auxotrophic marker and encodes ampicillin resistance (18,19), were codigested with NcoI and BamHI restriction endonucleases. The EhGPCR-1 gene was then ligated into Cp4258 by using T4 DNA ligase (New England BioLabs).…”

“…Saccharomyces cerevisiae strain CY 18043 (J. Broach, Princeton University) was used as the yeast recipient, since this strain is a histidine auxotroph that exhibits histidine prototrophism upon GPCR activation, even for exogenous receptors (18,19). Nontransfected CY 18043 yeast was grown in yeast extract-peptone-dextrose medium supplemented with all essential amino acids.…”

Basic and modern concepts on cholinergic receptor: A review

“…LGICs in model nematodes, including parasitic species where they are targets for anthelmintic drugs [20] , and we have also investigated the ability of the M 2 /M 4 muscarinic ACh receptor antagonist N, N′-bis [6- (methoctramine) to induce and modulate synaptic plasticity in the CA1 area of the hippocampus in urethane-anesthetized rats [21] .…”