Detergent-free extraction of a functional low-expressing GPCR from a human cell line

Juarez, Juan F. Bada; Muñoz–García, Juan C.; Reis, Rosana I.; Henry, Alistair J.; McMillan, David; Kriek, Marco; Wood, Martyn; Vandenplas, Catherine; Sands, Zara A.; Castro, Luis C. Misal; Taylor, Richard J. K.; Watts, Anthony

doi:10.1016/j.bbamem.2019.183152

Cited by 37 publications

(29 citation statements)

References 71 publications

(92 reference statements)

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“…The regenerated samples were stable and soluble for days at room temperature. These results highlight the ability of SMALPs to efficiently extract this model GPCR in a stable form from its native, mammalian tissue, as has been done in cell lines and lower species with other GPCRs (Jamshad et al, 2015;Gakhar et al, 2020;Ganapathy et al, 2020;Bada Juarez et al, 2020;Routledge et al, 2020;Ueta et al, 2020).…”

Section: Smalp-encapsulated Abca4 and Rhodopsin Retain Ligand Bindingmentioning

confidence: 61%

Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

Sander

Sears

Pinto

et al. 2021

Preprint

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Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to co-immunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample’s copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which were enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition provides a model for future studies of other complex cellular structures, and gives new insights into how intricate membrane structure and protein activity are balanced within the ROS.SUMMARYSander et al. have parsed the lipid composition of native-source photoreceptor disks and find large differences in fatty acid unsaturation and chain length between the center and rim regions. They selectively copurify membrane proteins and lipids from each region in SMALPs using nanobodies and antibodies.

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Section: Smalp-encapsulated Abca4 and Rhodopsin Retain Ligand Bindingmentioning

confidence: 61%

Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

Sander

Sears

Pinto

et al. 2021

Preprint

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“…In all cases of GPCR solubilisation by SMA, some degree of receptor functionality in SMA lipid-nanoparticles has been demonstrated. For the A 2A R and D 1 R, radioligand binding assays produced similar ligand binding profiles in both the original membranes and in the SMA-solubilised material (Jamshad et al 2015a;Bada Juarez et al 2020). Subsequent publications using A 2A R-SMALPs have also demonstrated ligand binding using fluorescence correlation spectroscopy (Grime et al 2020) and conformational changes in the receptor caused by inverse agonist binding (Routledge et al 2020), although in the latter case limited changes were observed upon agonist binding.…”

Section: Polymer Lipid Nanoparticlesmentioning

confidence: 81%

Lipid nanoparticle technologies for the study of G protein-coupled receptors in lipid environments

Lavington

Watts

2020

Biophys Rev

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G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins which conduct a wide range of biological roles and represent significant drug targets. Most biophysical and structural studies of GPCRs have been conducted on detergent-solubilised receptors, and it is clear that detergents can have detrimental effects on GPCR function. Simultaneously, there is increasing appreciation of roles for specific lipids in modulation of GPCR function. Lipid nanoparticles such as nanodiscs and styrene maleic acid lipid particles (SMALPs) offer opportunities to study integral membrane proteins in lipid environments, in a form that is soluble and amenable to structural and biophysical experiments. Here, we review the application of lipid nanoparticle technologies to the study of GPCRs, assessing the relative merits and limitations of each system. We highlight how these technologies can provide superior platforms to detergents for structural and biophysical studies of GPCRs and inform on roles for protein-lipid interactions in GPCR function.

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“…The dopamine D1 receptor is expressed at low levels in HEK cells and has been purified using SMA(3:1) copolymer. Once stably integrated in native nanodiscs, this GPCR is helical based on circular dichroism (CD) spectra, and binds the SCH23390 antagonist and neurotensin peptide based on radioligand and microscale thermophoresis (MST) assays [ 40 ]. The dopamine and ghrelin receptors form heteromultimeric complexes in hypothalamic neurons.…”

Section: Native Gpcr–lipid Complexesmentioning

confidence: 99%

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids

et al. 2021

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Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins).

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Detergent-free extraction of a functional low-expressing GPCR from a human cell line

Cited by 37 publications

References 71 publications

Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

Lipid nanoparticle technologies for the study of G protein-coupled receptors in lipid environments

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids

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