Opioid Receptors of Neuroblastoma Cells Are in Two Domains of the Plasma Membrane that Differ in Content of G Proteins

Ott, Susanne; Costa, Tommaso; Herz, A.

doi:10.1111/j.1471-4159.1989.tb09164.x

Cited by 9 publications

(2 citation statements)

References 25 publications

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“…In the present study, prolonged exposure of SH-SY5Y cells to DAMGO produced a typical 4.5-fold shift of its dose-response curve to the right, and Western blots revealed a specific reduction in the cellular content of G.. Considering the evidence for compartmentation of G proteins (44,45), the observed decrease of 26% could reflect a larger change in a discrete region of the plasma membrane. Previous studies have described altered levels of G protein following chronic opioid treatment: the effects were tissue dependent and resulted in different patterns of G-protein content in various regions of rat brain (46,47).…”

Section: Discussionsupporting

confidence: 58%

Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain.

Carter

Medzihradsky

1993

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

104

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In membranes from SH-SY5Y human neuroblastoma cells differentiated with retinoic acid, the ,u-selective agonist Tyr-D-Ala-Gly-N-Me,-Phe-Gly-ol (DAMGO) inhibited cAMP formation with an IC5o of 26 nM. Two separate antibodies raised against distinct regions of the Go. sequence attenuated the effect of DAMGO by 50-60%, whereas antibodies to Gial,2 or G1a3 reduced the ,u-opioid signal insignificantly or to a lesser extent. In contrast, inhibition of adenylyl cyclase by the 8-opioid agonist Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE; Pen = peniciliamine) was very sensitive to the Gial,2 antibody. In membranes from rat brain striatum, coupling of the 1t opioid receptor to adenylyl cyclase was also maximally blocked by antibodies to G... After long-term treatment of the cells with DAMGO, the content of Goa was reduced by 26%, whereas the levels of G1.1,2, G1.3, and Gsa were unaltered.

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Section: Discussionsupporting

confidence: 58%

Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain.

Carter

Medzihradsky

1993

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

104

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“…HEK-293 cells do not have caveoli, although ␤-adrenergic receptors and adenylyl cyclase V/VI localize to low buoyant density membrane domains in these cells (40). In addition, membrane fractionation studies have provided evidence for microdomains in the plasma membranes of neuroblastoma cells (41) and neutrophils (42) that have differences in their G protein content, and polarized epithelial cells differentially sort G protein ␣ subunits (43) and G protein-coupled receptors (44). It will be of interest to use the dominant negative ␣ s mutant described here to sort out the potential role of intracellular compartmentalization in regulating G protein signaling pathways in a wide range of cells and tissues.…”

Section: Discussionmentioning

confidence: 99%

A Highly Effective Dominant Negative αs Construct Containing Mutations That Affect Distinct Functions Inhibits Multiple Gs-coupled Receptor Signaling Pathways

Berlot

2002

Journal of Biological Chemistry

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To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative ␣ s mutant containing substitutions that alter distinct functions was produced and tested for its effects on G s -coupled receptor activity in HEK-293 cells. Mutations in the ␣3␤5 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for ␤␥, and A366S, which decreases affinity for GDP. This triple ␣ s mutant can inhibit signaling to G s from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this ␣ s mutant blocks all signaling from the calcitonin receptor to G q . These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein ␣ subunits can compete with each other for binding to the same receptor. This dominant negative ␣ s construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues.Stimulation of heterotrimeric G proteins by cell surface receptors activates signaling pathways that mediate specific responses to hormones and neurotransmitters. Cells express a wide variety of G protein-coupled receptors as well as numerous G protein ␣, ␤, and ␥ subunits. Many receptors can activate more than one type of G protein, and the G protein subunits can interact with many different types of receptors. The manner in which signaling specificity is maintained in the midst of this vast range of potential interactions is not well understood. This report investigates the interdependence of distinct signaling pathways activated by receptors with broad G protein specificities using a receptor-sequestering dominant negative G protein ␣ subunit.Many potential mechanisms could establish that distinct receptor-G protein interactions will be independent of each other. Among these, one possibility is that specific receptor-G protein complexes localize to separate membrane compartments (1, 2). Differential associations with particular proteins or lipids (3, 4) or covalent modifications such as phosphorylation (5) may result in subpopulations of receptors and G proteins that have restricted access to each other. Although G proteins are often expressed at much higher levels than their receptors are (6), there is evidence that different receptors utilize separate pools of G proteins (7,8). An alternative potential mechanism for isolating distinct receptor-G protein interactions is that receptors utilize separate regions for binding different G proteins. If this is the case, then multiple types of receptor-G protein interaction can occur simultaneously without affecting each other. Localization of G protein-binding sites have indicated that separate receptor regions may specify interactions with distinct G proteins (9, 10). Dominant negative G...

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Interactions Between Opioid Receptors and Guanine Nucleotide Regulatory Proteins in Intact Membranes: Studies in a Neurotumor Cell Line

Costa¹,

Ott²,

Vachon³

et al. 1991

Neurobiology of Opioids

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Opioid Receptors of Neuroblastoma Cells Are in Two Domains of the Plasma Membrane that Differ in Content of G Proteins

Cited by 9 publications

References 25 publications

Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain.

Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain.

A Highly Effective Dominant Negative αs Construct Containing Mutations That Affect Distinct Functions Inhibits Multiple Gs-coupled Receptor Signaling Pathways

Interactions Between Opioid Receptors and Guanine Nucleotide Regulatory Proteins in Intact Membranes: Studies in a Neurotumor Cell Line

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