Ligand-regulated oligomerization of β2-adrenoceptors in a model lipid bilayer

The β2-adrenoceptor (β2AR) was one of the first Family A G protein-coupled receptors (GPCRs) shown to form oligomers in cellular membranes, yet we still know little about the number and arrangement of protomers in oligomers, the influence of ligands on the organization or stability of oligomers, or the requirement for other proteins to promote oligomerization. We used fluorescence resonance energy transfer (FRET) to characterize the oligomerization of purified β2AR site-specifically labelled at three different positions with fluorophores and reconstituted into a model lipid bilayer. Our results suggest that the β2AR is predominantly tetrameric following reconstitution into phospholipid vesicles. Agonists and antagonists have little effect on the relative orientation of protomers in oligomeric complexes. In contrast, binding of inverse agonists leads to significant increases in FRET efficiencies for most labelling pairs, suggesting that this class of ligand promotes tighter packing of protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers. The results provide new structural insights into β2AR oligomerization and suggest a possible mechanism for the functional effects of inverse agonists

Current and future advances in fluorescence-based visualization of plant cell wall components and cell wall biosynthetic machineries

Plant cell wall-derived biomass serves as a renewable source of energy and materials with increasing importance. The cell walls are biomacromolecular assemblies defined by a fine arrangement of different classes of polysaccharides, proteoglycans, and aromatic polymers and are one of the most complex structures in Nature. One of the most challenging tasks of cell biology and biomass biotechnology research is to image the structure and organization of this complex matrix, as well as to visualize the compartmentalized, multiplayer biosynthetic machineries that build the elaborate cell wall architecture. Better knowledge of the plant cells, cell walls, and whole tissue is essential for bioengineering efforts and for designing efficient strategies of industrial deconstruction of the cell wall-derived biomass and its saccharification. Cell wall-directed molecular probes and analysis by light microscopy, which is capable of imaging with a high level of specificity, little sample processing, and often in real time, are important tools to understand cell wall assemblies. This review provides a comprehensive overview about the possibilities for fluorescence label-based imaging techniques and a variety of probing methods, discussing both well-established and emerging tools. Examples of applications of these tools are provided. We also list and discuss the advantages and limitations of the methods. Specifically, we elaborate on what are the most important considerations when applying a particular technique for plants, the potential for future development, and how the plant cell wall field might be inspired by advances in the biomedical and general cell biology fields

The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the β2 adrenergic receptor (β2AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The β2AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the β2AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the β2AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the β2AR and the structural basis for ligand efficacy

Conformational changes in the G protein Gs induced by the β2 adrenergic receptor

G protein-coupled receptors (GPCRs) represent the largest family of membrane receptors(1) that instigate signaling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely GDP dissociation from the G protein α-subunit, is the key step toward G protein activation and initiation of downstream signaling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism we applied peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to probe changes in the structure of the heterotrimeric G protein Gs (the stimulatory G protein for adenylyl cyclase) upon formation of a complex with agonist-bound β(2) adrenergic receptor (β(2)AR). Our studies reveal structural links between the receptor binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange (HX) than would be predicted from the crystal structure of the β(2)AR-Gs complex. Together with x-ray crystallographic and electron microscopic data of the β(2)AR-Gs complex (ref 2 and Westfield et al, manuscript submitted), we provide a rationale for a mechanism of nucleotide exchange whereby the receptor perturbs the structure of the amino-terminal region of α-subunit of Gs and consequently alters the ‘P-loop’ that binds the β-phosphate in GDP. As with the ras-family of small molecular weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor

<div><p>Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S <i>GnTl</i>^- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate G_s protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.</p></div

G -protein activity assay.

<p>(A) The fluorescence intensity monitored at excitation/emission 500 nm/512 nm with slit widths 2.5 nm/5 nm for the reaction mixture of BODIPY-FL-GTPγS and G_s: addition of ligand GLP-1 alone shows no G_s activation (black), addition of GLP1R-ND alone shows an increase in fluorescence intensity, indicating the basal activity (brown), and addition of GLP1R-ND together with the GLP-1-(7–37) ligand (red) and addition of GLP1R-ND together with the Ex-4 ligand (blue) show an increase in intensity above basal level; (B) The activation of G_s by GLP1R-ND upon binding to GLP-1 and Ex-4 after subtraction of the basal activity.</p

Characterization of GLP1R.

<p>(A) SDS-PAGE gel of purified GLP1R-ND, with two major bands at ~28 kDa and ~50 kDa representing MSP and GLP1R respectively; (B) Transmission electron microscopy (TEM) images of GLP1R-ND. The average size is ~18 nm; (C) Mass-based size distribution of isolated GLP1R-ND and wash-off unbound nanodiscs collected during chromatographic purification measured by dynamic light scattering (DLS).</p

Scheme of a receptor (blue) incorporated in a nanodisc.

<p>A nanodisc is a lipid bilayer (grey) surrounded by two membrane scaffold proteins (green).</p

Fluorescently labeled peptides for ligand binding assays.

<p>(A) Sequences of FAM-labeled GLP-1-(7–37) and Ex-4; (B) Chemical structure of the fluorescent dye 5(6)-carboxyfluorescein (FAM); (C) Chemical structure of BODIPY-FL-GTPγS.</p