High-mass MALDI-MS unravels ligand-mediated G protein–coupling selectivity to GPCRs

G protein–coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR–G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand–GPCR–partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [β1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using β1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαiβγ and β-arrestin-1 and showed that carvedilol induces an increase in coupling of β-arrestin-1 and Gαiβγ to β1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.

Download Full-text

Pathological AT1R-B2R Protein Aggregation and Preeclampsia

Cells ◽

10.3390/cells10102609 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2609

Author(s):

Ursula Quitterer ◽

Said AbdAlla

Keyword(s):

Risk Factors ◽

Complex Formation ◽

Protein Aggregation ◽

G Protein ◽

Protein Complex ◽

G Protein Coupled Receptors ◽

Protein Complex Formation ◽

The Impact ◽

G Protein Coupled ◽

Gpcr Protein

Preeclampsia is one of the most frequent and severe complications of pregnancy. Symptoms of preeclampsia usually occur after 20 weeks of pregnancy and include hypertension and kidney dysfunction with proteinuria. Up to now, delivery of the infant has been the most effective and life-saving treatment to alleviate symptoms of preeclampsia because a causative treatment does not exist, which could prolong a pregnancy complicated with preeclampsia. Preeclampsia is a complex medical condition, which is attributed to a variety of different risk factors and causes. Risk factors account for insufficient placentation and impaired vasculogenesis and finally culminate in this life-threatening condition of pregnancy. Despite progress, many pathomechanisms and causes of preeclampsia are still incompletely understood. In recent years, it was found that excessive protein complex formation between G-protein-coupled receptors is a common sign of preeclampsia. Specifically, the aberrant heteromerization of two vasoactive G-protein-coupled receptors (GPCRs), the angiotensin II AT1 receptor and the bradykinin B2 receptor, is a causative factor of preeclampsia symptoms. Based on this knowledge, inhibition of abnormal GPCR protein complex formation is an experimental treatment approach of preeclampsia. This review summarizes the impact of pathological GPCR protein aggregation on symptoms of preeclampsia and delineates potential new therapeutic targets.

Download Full-text

Pharmacological Characterization of Receptor Redistribution and β-Arrestin Recruitment Assays for the Cannabinoid Receptor 1

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109337937 ◽

2009 ◽

Vol 14 (7) ◽

pp. 811-823 ◽

Cited By ~ 30

Author(s):

Miranda M.C. van der Lee ◽

Marion Blomenröhr ◽

Antoon A. van der Doelen ◽

Jesse W.Y. Wat ◽

Niels Smits ◽

...

Keyword(s):

G Protein ◽

Cannabinoid Receptor ◽

Receptor Activation ◽

Binding Affinities ◽

Cannabinoid Receptor 1 ◽

Pharmacological Characterization ◽

Cellular Effects ◽

Partial Agonists ◽

C Terminus ◽

G Protein Coupled

Receptor redistribution and β-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular β-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution® assay and 2 nonimaging-based β-arrestin recruitment assays, Tango™ and PathHunter ™, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of Gαi coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the 3 assays, and are comparable with the measured ligand binding affinities. The inverse agonist SR141716 decreased basal signal in all 3 assays, but only in the Tango bla assay a reliable EC50 could be determined for this compound, suggesting that Tango is the most suitable assay for the identification of new inverse agonists. Both the Redistribution and the PathHunter assay could discriminate partial agonists from full agonists, whereas in the Tango assay partial agonists behaved as full agonists. Only the PathHunter cells allowed detection of cannabinoid receptor activation via β-arrestin recruitment and Gαi-protein-mediated inhibition of cAMP, thus enabling the identification of biased ligands that differ in these cellular effects. The characteristics and limitations of the different assays are discussed. ( Journal of Biomolecular Screening 2009:811-823)

Download Full-text

S (+)-Ketamine Suppresses Desensitization of γ-Aminobutyric Acid Type B Receptor-mediated Signaling by Inhibition of the Interaction of γ-Aminobutyric Acid Type B Receptors with G Protein–coupled Receptor Kinase 4 or 5

Anesthesiology ◽

10.1097/aln.0b013e318204e003 ◽

2011 ◽

Vol 114 (2) ◽

pp. 401-411 ◽

Cited By ~ 9

Author(s):

Yuko Ando ◽

Minoru Hojo ◽

Masato Kanaide ◽

Masafumi Takada ◽

Yuka Sudo ◽

...

Keyword(s):

Complex Formation ◽

G Protein ◽

Protein Complex ◽

Protein Complexes ◽

Aminobutyric Acid ◽

K Channel ◽

Type B ◽

Protein Complex Formation ◽

Acid Type ◽

G Protein Coupled

Background Intrathecal baclofen therapy is an established treatment for severe spasticity. However, long-term management occasionally results in the development of tolerance. One of the mechanisms of tolerance is desensitization of γ-aminobutyric acid type B receptor (GABABR) because of the complex formation of the GABAB2 subunit (GB2R) and G protein-coupled receptor kinase (GRK) 4 or 5. The current study focused on S(+)-ketamine, which reduces the development of morphine tolerance. This study was designed to investigate whether S(+)-ketamine affects the GABABR desensitization processes by baclofen. Methods The G protein-activated inwardly rectifying K channel currents induced by baclofen were recorded using Xenopus oocytes coexpressing G protein-activated inwardly rectifying K channel 1/2, GABAB1a receptor subunit, GB2R, and GRK. Translocation of GRKs 4 and 5 and protein complex formation of GB2R with GRKs were analyzed by confocal microscopy and fluorescence resonance energy transfer analysis in baby hamster kidney cells coexpressing GABAB1a receptor subunit, fluorescent protein-tagged GB2R, and GRKs. The formation of protein complexes of GB2R with GRKs was also determined by coimmunoprecipitation and Western blot analysis. Results Desensitization of GABABR-mediated signaling was suppressed by S(+)-ketamine in a concentration-dependent manner in the electrophysiologic assay. Confocal microscopy revealed that S(+)-ketamine inhibited translocation of GRKs 4 and 5 to the plasma membranes and protein complex formation of GB2R with the GRKs. Western blot analysis also showed that S(+)-ketamine inhibited the protein complex formation of GB2R with the GRKs. Conclusion S(+)-Ketamine suppressed the desensitization of GABABR-mediated signaling at least in part through inhibition of formation of protein complexes of GB2R with GRK 4 or 5.

Download Full-text

Yeast-Based Directed-Evolution For High-Throughput Structural Stabilization of G Protein-Coupled Receptors (GPCRs)

10.21203/rs.3.rs-1066896/v1 ◽

2021 ◽

Author(s):

May Meltzer ◽

Zvagelsky Tatiana ◽

Niv Papo ◽

Stanislav Engel

Keyword(s):

G Protein ◽

Resistant Cell ◽

Single Step ◽

G Protein Coupled Receptors ◽

Structural Basis ◽

Diffusion Method ◽

Structural Stabilization ◽

C Terminus ◽

Screening Platform ◽

G Protein Coupled

Abstract The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we presented a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method. The detergent-resistant cell wall of yeast provides a unique compartmentalization opportunity to physically link the receptor phenotype to its encoding DNA, and thus enable discovery of stable GPCR variants with unprecedent efficiency. The scope of mutations identified by the method offers important insights into the structural basis of GPCR stability, questioning the inherent instability of the GPCR scaffold, and revealing the potential role of the C-terminus in receptor stabilization.

Download Full-text

Chemical Synthesis of a Full-Length G-Protein-Coupled Receptor β2-Adrenergic Receptor with Defined Modification Patterns at the C-Terminus

Journal of the American Chemical Society ◽

10.1021/jacs.1c07369 ◽

2021 ◽

Author(s):

Yulei Li ◽

Jie Heng ◽

Demeng Sun ◽

Baochang Zhang ◽

Xin Zhang ◽

...

Keyword(s):

Chemical Synthesis ◽

G Protein ◽

Adrenergic Receptor ◽

Full Length ◽

G Protein Coupled Receptor ◽

C Terminus ◽

Β2 Adrenergic Receptor ◽

G Protein Coupled

Download Full-text

Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014631 ◽

2020 ◽

Vol 295 (51) ◽

pp. 17486-17496

Author(s):

Christopher T. Schafer ◽

Anthony Shumate ◽

David L. Farrens

Keyword(s):

G Protein ◽

Protein Activation ◽

Canonical Class ◽

Equilibrium Binding ◽

C Terminus ◽

G Protein Activation ◽

Ligand Discovery ◽

Pharmaceutical Agents ◽

G Protein Coupled ◽

Covalently Bound

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

Download Full-text

Structural Insights into the Process of GPCR-G Protein Complex Formation

Cell ◽

10.1016/j.cell.2019.04.021 ◽

2019 ◽

Vol 177 (5) ◽

pp. 1243-1251.e12 ◽

Cited By ~ 35

Author(s):

Xiangyu Liu ◽

Xinyu Xu ◽

Daniel Hilger ◽

Philipp Aschauer ◽

Johanna K.S. Tiemann ◽

...

Keyword(s):

Complex Formation ◽

G Protein ◽

Protein Complex ◽

Protein Complex Formation ◽

Structural Insights

Download Full-text

Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture

Scientific Reports ◽

10.1038/s41598-020-73813-7 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Alexei Yeliseev ◽

Arjen van den Berg ◽

Lioudmila Zoubak ◽

Kirk Hines ◽

Sam Stepnowski ◽

...

Keyword(s):

G Protein ◽

Mammalian Cell ◽

Rational Design ◽

Mammalian Cell Culture ◽

High Yield ◽

Pharmaceutical Drugs ◽

C Terminus ◽

Mammalian Cell Cultures ◽

High Level ◽

G Protein Coupled

Abstract Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles. The recombinant cannabinoid CB2 receptor was expressed at high yield in Expi293F mammalian cell cultures, solubilized and purified in Façade detergent. We report superior stability of the mammalian cell-expressed receptor compared to its E.coli-expressed counterpart, due to contributions from glycosylation of the N terminus and palmitoylation of the C terminus of CB2. Finally, we demonstrate that the mammalian Expi293F amino acid labelling kit is suitable for preparation of multi-milligram quantities of high quality, selectively stable isotope-labeled GPCR for studies by nuclear magnetic resonance.

Download Full-text

Plasma Membrane and Nuclear Localization of G Protein–coupled Receptor Kinase 6A

Molecular Biology of the Cell ◽

10.1091/mbc.e07-01-0013 ◽

2007 ◽

Vol 18 (8) ◽

pp. 2960-2969 ◽

Cited By ~ 32

Author(s):

Xiaoshan Jiang ◽

Jeffrey L. Benovic ◽

Philip B. Wedegaertner

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

G Protein ◽

Nuclear Localization ◽

Nuclear Export ◽

Amphipathic Helix ◽

G Protein Coupled Receptor ◽

C Terminus ◽

Acidic Residues ◽

G Protein Coupled

G protein–coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.

Download Full-text