Endogenously Expressed Muscarinic Receptors in HEK293 Cells Augment Up-regulation of Stably Expressed α4β2 Nicotinic Receptors

Hussmann, G. Patrick; Yasuda, Robert P.; Xiao, Yingxian; Wolfe, Barry B.; Kellar, Kenneth J.

doi:10.1074/jbc.m111.289546

Cited by 13 publications

(13 citation statements)

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“…Rats were sacrificed 10 min after the last injection of the chronic study. both up-regulated and untreated nAChRs (Hussmann et al, 2011b). Moreover, the key finding in this experiment was that nAChRs from saz-A-treated rats, which were not upregulated, were indeed occupied by the drug.…”

Section: Resultsmentioning

confidence: 53%

“…The predominant nAChR increased by nicotine in rodents is the ␣4␤2 subtype (Flores et al, 1992;Mao et al, 2008;Moretti et al, 2010;Marks et al, 2011), and based on the ligand used to measure the receptors, that is probably also the case in humans. Moreover, in cultured cells that express nAChRs either endogenously or heterologously these receptors are increased after incubation with those agonists, as well as with a wide variety of other nicotinic ligands (Gopalakrishnan et al, 1996(Gopalakrishnan et al, , 1997Whiteaker et al, 1998;Dá vila-García et al, 1999;Xiao and Kellar, 2004;Gahring et al, 2010;Hussmann et al, 2011b).…”

Section: Discussionmentioning

confidence: 99%

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Chronic Sazetidine-A at Behaviorally Active Doses Does Not Increase Nicotinic Cholinergic Receptors in Rodent Brain

Hussmann

Turner

Lomazzo

et al. 2012

J Pharmacol Exp Ther

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Chronic nicotine administration increases ␣4␤2 neuronal nicotinic acetylcholine receptor (nAChR) density in brain. This upregulation probably contributes to the development and/or maintenance of nicotine dependence. nAChR up-regulation is believed to be triggered at the ligand binding site, so it is not surprising that other nicotinic ligands also up-regulate nAChRs in the brain. These other ligands include varenicline, which is currently used for smoking cessation therapy. Sazetidine-A (saz-A) is a newer nicotinic ligand that binds with high affinity and selectivity at ␣4␤2* nAChRs. In behavioral studies, saz-A decreases nicotine self-administration and increases performance on tasks of attention. We report here that, unlike nicotine and varenicline, chronic administration of saz-A at behaviorally active and even higher doses does not up-regulate nAChRs in rodent brains. We used a newly developed method involving radioligand binding to measure the concentrations and nAChR occupancy of saz-A, nicotine, and varenicline in brains from chronically treated rats. Our results indicate that saz-A reached concentrations in the brain that were ϳ150 times its affinity for ␣4␤2* nAChRs and occupied at least 75% of nAChRs. Thus, chronic administration of saz-A did not upregulate nAChRs despite it reaching brain concentrations that are known to bind and desensitize virtually all ␣4␤2* nAChRs in brain. These findings reinforce a model of nicotine addiction based on desensitization of up-regulated nAChRs and introduce a potential new strategy for smoking cessation therapy in which drugs such as saz-A can promote smoking cessation without maintaining nAChR up-regulation, thereby potentially increasing the rate of long-term abstinence from nicotine.

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Section: Resultsmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 99%

Chronic Sazetidine-A at Behaviorally Active Doses Does Not Increase Nicotinic Cholinergic Receptors in Rodent Brain

Hussmann

Turner

Lomazzo

et al. 2012

J Pharmacol Exp Ther

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“…The mutual requirement of mGlu1/5 and GluD1 to generate currents was further tested by stimulating other Gq-coupled receptors in GluD1-expressing HEK cells. No response was observed upon stimulation of the endogenous muscarinic M3 receptor 44 , 45 , 46 by carbachol (100 μ M ) in DHPG-responsive HEK cells co-expressing mGlu1 and GluD1 ( n =5, I DHPG : 80±19 pA; Supplementary Figure S1 ). Similarly, application of the 5-HT2C serotonin receptor agonist 2,5-dimethoxy-4-iodoamphetamine (50 μ M ) failed to induce appreciable response in HEK cells co-expressing 5-HT2C and GluD1 (1.7±2.6 pA, n =10; Supplementary Figure S1 ).…”

Section: Resultsmentioning

confidence: 99%

GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons

et al. 2017

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Human mutations of the GRID1 gene encoding the orphan delta1 glutamate receptor-channel (GluD1) are associated with schizophrenia but the explicit role of GluD1 in brain circuits is unknown. Based on the known function of its paralog GluD2 in cerebellum, we searched for a role of GluD1 in slow glutamatergic transmission mediated by metabotropic receptor mGlu1 in midbrain dopamine neurons, whose dysfunction is a hallmark of schizophrenia. We found that an mGlu1 agonist elicits a slow depolarizing current in HEK cells co-expressing mGlu1 and GluD1, but not in cells expressing mGlu1 or GluD1 alone. This current is abolished by additional co-expression of a dominant-negative GluD1 dead pore mutant. We then characterized mGlu1-dependent currents in dopamine neurons from midbrain slices. Both the agonist-evoked and the slow postsynaptic currents are abolished by expression of the dominant-negative GluD1 mutant, pointing to the involvement of native GluD1 channels in these currents. Likewise, both mGlu1-dependent currents are suppressed in GRID1 knockout mice, which reportedly display endophenotypes relevant for schizophrenia. It is known that mGlu1 activation triggers the transition from tonic to burst firing of dopamine neurons, which signals salient stimuli and encodes reward prediction. In vivo recordings of dopamine neurons showed that their spontaneous burst firing is abolished in GRID1 knockout mice or upon targeted expression of the dominant-negative GluD1 mutant in wild-type mice. Our results de-orphanize GluD1, unravel its key role in slow glutamatergic transmission and provide insights into how GRID1 gene alterations can lead to dopaminergic dysfunctions in schizophrenia.

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“…Given that it is unlikely that PfSR12 could be directly activated by both thrombin and carbamoyl choline; two unrelated ligands. The responses likely result from a potentiation of the endogenously expressed PAR and M3 muscarinc receptor in HEK-293 cells (Atwood et al 2011;Hussmann et al 2011). Two hypotheses emerged from our data: first PfSR12 could act as a partner protein, interacting with other receptors to increase the transduced signal, and second, PfSR12 could act as a chaperone and increase the expression of other GPCRs.…”

Section: Discussionmentioning

confidence: 83%

BRET sensors unravel thatPlasmodium falciparumserpentine receptor 12 (PfSR12) increases surface expression of mammalian GPCRs in HEK293 cells

Pereira

Brito

Moraes

et al. 2020

Preprint

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Malaria causes millions of deaths worldwide and is considered a huge public health problem for underdeveloped countries. The most severe cases of malaria present complications of the host circulatory system, which may cause clogging and rupture of blood vessels, leading to death or important sequelae. Because of the previously suggested role of thrombin and platelet aggregation in Plasmodium falciparum biology, we hypothesized that one of the GPCR-like proteins identified in the genome of the parasite, P. falciparum serpentine receptor 12 (PfSR12), could be a thrombin-activated GPCR. To test this hypothesis we used a series of Bioluminescence and Bioluminescence Resonance Energy Transfer (BRET)-based biosensors to investigate the signaling activity of PfSR12. Using an Obelin based biosensor, thrombin promoted a PfSR12-dependent cytosolic Ca 2+ rise in HEK293 cells. This Ca 2+ mobilization was accompanied by DAG formation and PKC activation as detected using DAG and PKC BRET-based biosensors indicating a Gq/PLC/IP3 signaling pathway. The role of Gq was confirm using Gq/11 knockout HEK293 cells as well as the Gq-selective inhibitor, YM254890. Further investigation revealed that PfSR12 is not itself a thrombin receptor but rather promotes the increase of cell surface expression of an endogenous thrombin receptor. This chaperone-like effect was not selective for thrombin receptors as PfSR12 expression also promoted an increased muscarinic type 3 receptor (M3R)-promoted DAG and PKC responses. This increase response was accompanied by an increase in surface expression of M3R. Our data indicate that PfSR12 acts as a chaperone and increases the expression of several GPCRs resulting in increased responsiveness to various hormones of mammalian cells that could contribute to the deleterious effects of Plasmodium falciparum infection.

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Endogenously Expressed Muscarinic Receptors in HEK293 Cells Augment Up-regulation of Stably Expressed α4β2 Nicotinic Receptors

Cited by 13 publications

References 62 publications

Chronic Sazetidine-A at Behaviorally Active Doses Does Not Increase Nicotinic Cholinergic Receptors in Rodent Brain

Chronic Sazetidine-A at Behaviorally Active Doses Does Not Increase Nicotinic Cholinergic Receptors in Rodent Brain

GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons

BRET sensors unravel thatPlasmodium falciparumserpentine receptor 12 (PfSR12) increases surface expression of mammalian GPCRs in HEK293 cells

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