Significance G protein-coupled receptors (GPCRs), one of the largest cell surface receptor families, transmit their signals through the coupling of intracellular partners, such as the G proteins. ...Knowing how this coupling occurs is essential, because it governs the entire signaling process. To address this open question, we used a purified GPCR as a model to which we applied various state-of-the-art biochemical and biophysical approaches. By doing so, we provide direct experimental evidence of a signaling mechanism in which receptor conformational changes are directly linked to a rearrangement of a preassembled complex between the receptor and its cognate Gq protein. This sheds light on the way in which a GPCR interacts with G proteins to trigger signaling.
How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.
In the aim of testing tools for tracing cell trafficking of exogenous cholesterol, two fluorescent derivatives of cholesterol, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and ...21-methylpyrenyl-cholesterol (Pyr-met-Chol), with distinctive chemico-physical characteristics, have been compared for their cell incorporation properties, using two cell models differently handling cholesterol, with two incorporation routes. In the Caco-2 cell model, the cholesterol probes were delivered in bile salt micelles, as a model of intestinal absorption. The two probes displayed contrasting behaviors for cell uptake characteristics, cell staining, and efflux kinetics. In particular, Pyr-met-Chol cell incorporation involved SR-BI, while that of NBD-Chol appeared purely passive. In the PC-3 cell model, which overexpresses lipoprotein receptors, the cholesterol probes were delivered via the serum components, as a model of systemic delivery. We showed that Pyr-met-Chol-labelled purified LDL or HDL were able to specifically deliver Pyr-met-Chol to the PC-3 cells, while NBD-Chol incorporation was independent of lipoproteins. Observations by fluorescence microscopy evidenced that, while NBD-Chol readily stained the cytosolic lipid droplets, Pyr-met-Chol labelling led to the intense staining of intracellular structures of membranous nature, in agreement with the absence of detectable esterification of Pyr-met-Chol. A 48 h incubation of PC-3 cells with either Pyr-met-Chol-labelled LDL or HDL gave same staining patterns, mainly colocalizing with Lamp1, caveolin-1 and CD63. These data indicated convergent trafficking downwards their respective receptors, LDL-R and SR-BI, toward the cholesterol-rich internal membrane compartments, late endosomes and multivesicular bodies. Interestingly, Pyr-met-Chol staining of these structures exhibited a high excimer fluorescence emission, revealing their ordered membrane environment, and indicating that Pyr-met-Chol behaves as a fair cholesterol tracer regarding its preferential incorporation into cholesterol-rich domains. We conclude that, while NBD-Chol is a valuable marker of cholesterol esterification, Pyr-met-Chol is a reliable new lipoprotein fluorescent marker which allows to probe specific intracellular trafficking of cholesterol-rich membranes.
The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the G
protein, promoting kidney water reabsorption. Molecular mechanisms underlying ...activation of this critical G protein-coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo-electron microscopy structure of the AVP-V2R-G
complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and G
binding modes. They reveal an original receptor-G
interface in which the Gα
subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.
T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs ...during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.
This review addresses the question of fluorescent detection of ordered membrane (micro) domains in living (cultured) cells, with a "practical" point of view since the situation is much more ...complicated than for studying model membranes. We first briefly recall the bases of model membrane structural organization involving liquid-ordered and -disordered phases, and the main features of their counterparts in cell membranes that are the various microdomains. We then emphasize the utility of the fluorescent probes derived from cholesterol, and delineate the respective advantages, limitations and drawbacks of the existing ones. In particular, besides their intramembrane behavior, their relevant characteristics should integrate their different cellular fates for membrane turn-over, trafficking and metabolism, in order to evaluate and improve their efficiency for in-situ probing membrane microdomains in the cell physiology context. Finally, at the present stage, it appears that Bdp-Chol and Pyr-met-Chol display well complementary properties, allowing to use them in combination to improve the reliability of the current experimental approaches. But the field is still open, and there remains much work to perform in this research area.
Lipid monolayers are often considered as model membranes, but they are also the physiologic lipid part of the peripheral envelope of lipoproteins and cytosolic lipid bodies. However, their structural ...organization is still rather elusive, in particular when both cholesterol and sphingomyelin are present. To investigate such structural organization of hemimembranes, we measured, using alternative current voltammetry, the differential capacitance of condensed phosphatidylcholine-based monolayers as a function of applied potential, which is sensitive to their lipid composition and molecular arrangement. Especially, monolayers containing both sphingomyelin and cholesterol, at 15% w/w, presented specific characteristics of the differential capacitance versus potential curves recorded, which was indicative of specific interactions between these two lipid components. We then compared the behavior of two cholesterol derivatives (at 15% w/w), 21-methylpyrenyl-cholesterol (Pyr-met-Chol) and 22-nitrobenzoxadiazole-cholesterol (NBD-Chol), with that of cholesterol when present in model monolayers. Indeed, these two probes were chosen because of previous findings reporting opposite behaviors within bilayer membranes regarding their interaction with ordered lipids, with only Pyr-met-Chol mimicking cholesterol well. Remarkably, in monolayers containing sphingomyelin or not, Pyr-met-Chol and NBD-Chol presented contrasting behaviors, and Pyr-met-Chol mimicked cholesterol only in the presence of sphingomyelin. These two observations (i.e., optimal amounts of sphingomyelin and cholesterol, and the ability to discriminate between Pyr-met-Chol and NBD-Chol) can be interpreted by the existence of heterogeneities including ordered patches in sphingomyelin- and cholesterol-containing monolayers. Since such monolayer lipid arrangement shares some properties with the raft-type lipid microdomains well-described in sphingomyelin- and cholesterol-containing bilayer membranes, our data thus strongly suggest the existence of compact and ordered microdomains in model lipid monolayers.
Human immunodeficiency virus entry into target cells requires sequential interactions of the viral glycoprotein envelope gp120 with CD4 and chemokine receptors CCR5 or CXCR4. CD4 interaction with the ...chemokine receptor is suggested to play a critical role in this process but to what extent such a mechanism takes place at the surface of target cells remains elusive. To address this issue, we used a confocal microspectrofluorimetric approach to monitor fluorescence resonance energy transfer at the cell plasma membrane between enhanced blue and green fluorescent proteins fused to CD4 and CCR5 receptors. We developed an efficient fluorescence resonance energy transfer analysis from experiments carried out on individual cells, revealing that receptors constitutively interact at the plasma membrane. Binding of R5-tropic HIV gp120 stabilizes these associations thus highlighting that ternary complexes between CD4, gp120, and CCR5 occur before the fusion process starts. Furthermore, the ability of CD4 truncated mutants and CCR5 ligands to prevent association of CD4 with CCR5 reveals that this interaction notably engages extracellular parts of receptors. Finally, we provide evidence that this interaction takes place outside raft domains of the plasma membrane.
Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of ...cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168–9178. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR⁎) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR⁎ is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR⁎ was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor–G-protein interaction.
Heterodimerization of G protein-coupled receptors has an impact on their signaling properties, but the molecular mechanisms underlying heteromer-directed selectivity remain elusive. Using purified ...monomers and dimers reconstituted into lipid discs, we explored how dimerization impacts the functional and structural behavior of the ghrelin receptor. In particular, we investigated how a naturally occurring truncated splice variant of the ghrelin receptor exerts a dominant negative effect on ghrelin signaling upon dimerization with the full-length receptor. We provide direct evidence that this dominant negative effect is due to the ability of the non-signaling truncated receptor to restrict the conformational landscape of the full-length protein. Indeed, associating both proteins within the same disc blocks all agonist- and signaling protein-induced changes in ghrelin receptor conformation, thus preventing it from activating its cognate G protein and triggering arrestin 2 recruitment. This is an unambiguous demonstration that allosteric conformational events within dimeric assemblies can be directly responsible for modulation of signaling mediated by G protein-coupled receptors.
Previous single-molecule studies have shown a long-term diffusion superimposed to a short-term confinement of the human mu opioid (hMOP) receptors at the surface of heterologous cells. However, ...additional ensemble average measurements are required to reach a complete understanding of the undergoing process. Here, we analyse, by fluorescence recovery after photobleaching measurements, the lateral diffusion of fully functional T7-EGFP-hMOP receptors in neuroblastoma SH-SY5Y cells naturally expressing a low level of the wild-type receptor. Experiments carried out at variable observation radii demonstrate the restriction of the receptors diffusion to sub-micrometer sized domains. Furthermore, consistently with the long-term single-molecule data, the domains are found permeable.
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