Scintillation Proximity Assay (<scp>SPA</scp>) Technology to Study Biomolecular Interactions

Cook, Neil D.; Harris, Alison; Hopkins, A.; Hughes, Kelvin T.

doi:10.1002/0471140864.ps1908s27

Cited by 13 publications

(5 citation statements)

References 52 publications

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“…All the competition-binding experiments were performed using scintillation proximity assays (SPAs) ( 58 ) with Cu-YSi beads (1 mg ml −1 ) in SPA buffer containing 20 mM tris-HCl (pH 8), 100 mM NaCl, or 100 mM KCl. For the [ 3 H]ibogaine assays, SPA was performed with 50 nM SERT, 300 nM [ 3 H]ibogaine, and 1 μM to 10 mM cold 5-HT.…”

Section: Methodsmentioning

confidence: 99%

Illumination of serotonin transporter mechanism and role of the allosteric site

2021

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The serotonin transporter (SERT) terminates serotonin signaling by using sodium and chloride gradients to drive reuptake of serotonin into presynaptic neurons and is the target of widely used medications to treat neuropsychiatric disorders. Despite decades of study, the molecular mechanism of serotonin transport, the coupling to ion gradients, and the role of the allosteric site have remained elusive. Here, we present cryo-electron microscopy structures of SERT in serotonin-bound and serotonin-free states, in the presence of sodium or potassium, resolving all fundamental states of the transport cycle. From the SERT-serotonin complex, we localize the substrate-bound allosteric site, formed by an aromatic pocket positioned in the scaffold domain in the extracellular vestibule, connected to the central site via a short tunnel. Together with elucidation of multiple apo state conformations, we provide previously unseen structural understanding of allosteric modulation, demonstrating how SERT binds serotonin from synaptic volumes and promotes unbinding into the presynaptic neurons.

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Section: Methodsmentioning

confidence: 99%

Illumination of serotonin transporter mechanism and role of the allosteric site

2021

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“…The binding of the different insulins to the human IR-A and IR-B was analyzed in a competitive binding assay using the scintillation proximity assay as previously described [29] , [30] . Plasma membranes were enriched from CHO cells overexpressing either IR-A or IR-B (∼1.3×10 5 IR-A per cell and ∼2.2×10 5 IR-B per cell as determined by FACS analysis, unpublished data) by a series of differential centrifugations including a single flotation through a one-step sucrose gradient.…”

Section: Methodsmentioning

confidence: 99%

In Vitro Metabolic and Mitogenic Signaling of Insulin Glargine and Its Metabolites

et al. 2010

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BackgroundInsulin glargine (Lantus®) is a long-acting basal insulin analog that demonstrates effective day-long glycemic control and a lower incidence of hypoglycemia than NPH insulin. After subcutaneous injection insulin glargine is partly converted into the two main metabolites M1 ([GlyA21]insulin) and M2 ([GlyA21,des-ThrB30]insulin). The aim of this study was to characterize the glargine metabolites in vitro with regard to their insulin receptor (IR) and IGF-1 receptor (IGF1R) binding and signaling properties as well as their metabolic and mitogenic activities.MethodsThe affinity of human insulin, insulin glargine and its metabolites to the IR isoforms A and B or IGF1R was analyzed in a competitive binding assay using SPA technology. Receptor autophosphorylation activities were studied via In-Cell Western in CHO and MEF cells overexpressing human IR-A and IR-B or IGF1R, respectively. The metabolic response of the insulins was studied as stimulation of lipid synthesis using primary rat adipocytes. Thymidine incorporation in Saos-2 cells was used to characterize the mitogenic activity.ConclusionsThe binding of insulin glargine and its metabolites M1 and M2 to the IR were similar and correlated well with their corresponding autophosphorylation and metabolic activities in vitro. No differences were found towards the two IR isoforms A or B. Insulin glargine showed a higher affinity for IGF1R than insulin, resulting in a lower EC50 value for autophosphorylation of the receptor and a more potent stimulation of thymidine incorporation in Saos-2 cells. In contrast, the metabolites M1 and M2 were significantly less active in binding to and activation of the IGF1R and their mitogenicity in Saos-2 cells was equal to human insulin. These findings strongly support the idea that insulin glargine metabolites contribute with the same potency as insulin glargine to blood glucose control but lead to significantly reduced growth-promoting activity.

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“…A saturation binding experiment using [ 3 H]paroxetine was performed via the scintillation proximity assay (SPA) ( 77 ) using the lysate of porcine brain membranes in SPA buffer (20 mM Tris-HCl, pH 8, 100 mM NaCl, 1 mM DDM, 0.2 mM CHS). The membrane lysates were mixed with Cu-YSi beads (0.5 mg mL −1 ) in SPA buffer, and [ 3 H]paroxetine at a concentration of 0.3 to 40 nM.…”

Section: Methodsmentioning

confidence: 99%

Structures and membrane interactions of native serotonin transporter in complexes with psychostimulants

Yang

Zhao

Tajkhorshid

et al. 2023

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

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The serotonin transporter (SERT) is a member of the SLC6 neurotransmitter transporter family that mediates serotonin reuptake at presynaptic nerve terminals. SERT is the target of both therapeutic antidepressant drugs and psychostimulant substances such as cocaine and methamphetamines, which are small molecules that perturb normal serotonergic transmission by interfering with serotonin transport. Despite decades of studies, important functional aspects of SERT such as the oligomerization state of native SERT and its interactions with potential proteins remain unresolved. Here, we develop methods to isolate SERT from porcine brain (pSERT) using a mild, nonionic detergent, utilize fluorescence-detection size-exclusion chromatography to investigate its oligomerization state and interactions with other proteins, and employ single-particle cryo-electron microscopy to elucidate the structures of pSERT in complexes with methamphetamine or cocaine, providing structural insights into psychostimulant recognition and accompanying pSERT conformations. Methamphetamine and cocaine both bind to the central site, stabilizing the transporter in an outward open conformation. We also identify densities attributable to multiple cholesterol or cholesteryl hemisuccinate (CHS) molecules, as well as to a detergent molecule bound to the pSERT allosteric site. Under our conditions of isolation, we find that pSERT is best described as a monomeric entity, isolated without interacting proteins, and is ensconced by multiple cholesterol or CHS molecules.

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Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions

Cited by 13 publications

References 52 publications

Illumination of serotonin transporter mechanism and role of the allosteric site

Illumination of serotonin transporter mechanism and role of the allosteric site

In Vitro Metabolic and Mitogenic Signaling of Insulin Glargine and Its Metabolites

Structures and membrane interactions of native serotonin transporter in complexes with psychostimulants

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