The Arginine of the DRY Motif in Transmembrane Segment III Functions as a Balancing Micro-switch in the Activation of the β2-Adrenergic Receptor

Valentin-Hansen, Louise; Groenen, Marleen; Nygaard, Rie; Frimurer, Thomas M.; Holliday, Nicholas D.; Schwartz, Thue W.

doi:10.1074/jbc.m112.348565

Cited by 39 publications

(42 citation statements)

References 35 publications

(27 reference statements)

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“…3). Following stimulation with NPY, receptor-GFP or SNAP-Y1 receptor fluorescence intensities were then measured within internal compartments identified by a transferrin marker (Valentin-Hansen et al, 2012)-an analysis that offered improved sensitivity compared with granularity for both SNAP-Y1 and Y1-GFP responses in these cells (illustrative granularity data are shown in Supplemental Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

“…Finally, we examined the ability of structurally unrelated agonists (NPY and isoprenaline) to stimulate endocytosis of the Y1/b2AR BiFC dimer, and in this case both were full agonists (Supplemental Fig. 5), with respective potencies that did not differ significantly from the SNAP-Y1-Yn population responses (NPY) or our previous assessment of isoprenaline-induced internalization of SNAP-tagged b2ARs (Valentin-Hansen et al, 2012).…”

Section: Resultsmentioning

confidence: 99%

“…A similar protocol was adopted (without SNAP labeling) to image Y receptor-GFP internalization in 293TR cell lines seeded 48 hours prior to experiments, with a tetracycline (1 mg/ml) induction period of 18-21 hours. For assessment of NPY-induced internalization of the noncomplemented SNAP-Y1/Y1-GFP cell lines, the SNAP-Y1 labeling step used SNAP-Surface AF546 (0.2 mM), and transferrin-AF633 (5 mg/ml) was also included during NPY stimulation in the assay medium as a marker for internal compartments to aid analysis (Valentin-Hansen et al, 2012). During IX Ultra acquisition, the TexasRed channel (561 nm; 593/20-nm band pass) was used to acquire SNAP-Y1 images, and the Cy5 channel visualized transferrin labeling.…”

Section: Methodsmentioning

confidence: 99%

“…While several granule parameters (count, area, intensity) yielded equivalent response data, the figures presented are based on average granule count per cell. To measure receptor internalization in the noncomplemented SNAP-Y1/ Y1-GFP cell lines, an alternative method quantified either labeled SNAP-Y1 or Y1-GFP fluorescence intensities within the same 3-mm internal compartments identified by transferrin (using the MetaXpress multi-wavelength cell scoring algorithm; Valentin-Hansen et al, 2012). This improved sensitivity of measurement compared with granularity analysis in these experiments ( Statistical analysis was by two-way analysis of variance (ANOVA) with Bonferroni post-test, to test significance differences between pooled concentration-response and time course relationships; or by Student's unpaired t test, to assess differences between R max or pEC 50 values.…”

Section: Methodsmentioning

confidence: 99%

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A G Protein–Coupled Receptor Dimer Imaging Assay Reveals Selectively Modified Pharmacology of Neuropeptide Y Y1/Y5 Receptor Heterodimers

2015

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The ability of G protein-coupled receptors (GPCRs) to form dimers, and particularly heterodimers, offers potential for targeted therapeutics with improved selectivity. However, studying dimer pharmacology is challenging, because of signaling cross-talk or because dimerization may often be transient in nature. Here we develop a system to isolate the pharmacology of precisely defined GPCR dimers, trapped by bimolecular fluorescence complementation (BiFC). Specific effects of agonist activation on such dimers are quantified using automated imaging and analysis of their internalization, controlled for by simultaneous assessment of endocytosis of one coexpressed protomer population. We applied this BiFC system to study example neuropeptide Y (NPY) Y1 receptor dimers. Incorporation of binding-site or phosphorylation-site mutations into just one protomer of a Y1/Y1 BiFC homodimer had no impact on efficient NPY-stimulated endocytosis, demonstrating that single-site agonist occupancy, and one phosphorylated monomer within this dimer, was sufficient. For two Y1 receptor heterodimer combinations (with the Y4 receptor or b2-adrenoceptor), agonist and antagonist pharmacology was explained by independent actions on the respective orthosteric binding sites. However, Y1/Y5 receptor BiFC dimers, compared with the constituent subtypes, were characterized by reduced potency and efficacy of Y5-selective peptide agonists, the inactivity of Y1-selective antagonists, and a change from surmountable to nonsurmountable antagonism for three unrelated Y5 antagonists. Thus, allosteric interactions between Y1 and Y5 receptors modify the pharmacology of the heterodimer, with implications for potential antiobesity agents that target centrally coexpressed Y1 and Y5 receptors to suppress appetite.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A G Protein–Coupled Receptor Dimer Imaging Assay Reveals Selectively Modified Pharmacology of Neuropeptide Y Y1/Y5 Receptor Heterodimers

2015

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“…In the case of ArgIII:26, loss of function is observed in most receptors. However, for example, in the B2AR, no effect is observed upon alanine substitution of ArgIII:26 with respect to G s stimulation, despite the fact that alanine substitutions of the interaction partners for ArgIII:26 in its active and inactive conformation had major loss of function or gain of function effects, respectively (33,34).…”

Section: Discussionmentioning

confidence: 99%

PheVI:09 (Phe6.44) as a Sliding Microswitch in Seven-transmembrane (7TM) G Protein-coupled Receptor Activation

Valentin-Hansen¹,

Holst

Frimurer

et al. 2012

Journal of Biological Chemistry

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Background: PheVI:09 (6.44) is highly conserved among 7TM receptors. Results: In the inactive state, PheVI:09 is locked against the backbone of TM-III but slides pass IleIII:16 (3.40) into a tight hydrophobic pocket between TM-III and TM-V during receptor activation. Conclusion:Mutational analysis and computational chemistry shows that PheVI:09 functions as a microswitch. Significance: This work clarifies the molecular mechanism of a crucial microswitch in 7TM receptor activation.

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Mechanistic basis of GPCR activation explored by ensemble refinement of crystallographic structures

Madsen

Frimurer

et al. 2022

Protein Science

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G protein‐coupled receptors (GPCRs) are important drug targets characterized by a canonical seven transmembrane (TM) helix architecture. Recent advances in X‐ray crystallography and cryo‐EM have resulted in a wealth of GPCR structures that have been used in drug design and formed the basis for mechanistic activation hypotheses. Here, ensemble refinement (ER) of crystallographic structures is applied to explore the impact of binding of agonists and antagonist/inverse agonists to selected structures of cannabinoid receptor 1 (CB1R), β2 adrenergic receptor (β2AR), and A2A adenosine receptor (A2AAR). To assess the conformational flexibility and its role in GPCR activation, hydrogen bond (H‐bond) networks are analyzed by calculating and comparing H‐bond propensities. Mapping pairwise propensity differences between agonist‐ and inverse agonist/antagonist‐bound structures for CB1R and β2AR shows that agonist binding destabilizes H‐bonds in the intracellular parts of TM 5–7, forming the G protein binding cavity, while H‐bonds of the extracellular segment of TMs surrounding the orthosteric site are conversely stabilized. Certain class A GPCRs, for example, A2AAR, bind an allosteric sodium ion that negatively modulates agonist binding. The impact of sodium‐excluding mutants (D522.50N, S913.39A) of A2AAR on agonist binding is examined by applying ER analysis to structures of wildtype and the two mutants in complex with a full agonist. While S913.39A exhibits normal activity, D522.50N quenches the downstream signaling. The mainchain H‐bond pattern of the latter is stabilized in the intracellular part of TM 7 containing the NPxxY motif, indicating that an induced rigidity of the mutation prevents conformational selection of G proteins resulting in receptor inactivation.

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The Arginine of the DRY Motif in Transmembrane Segment III Functions as a Balancing Micro-switch in the Activation of the β2-Adrenergic Receptor

Cited by 39 publications

References 35 publications

A G Protein–Coupled Receptor Dimer Imaging Assay Reveals Selectively Modified Pharmacology of Neuropeptide Y Y1/Y5 Receptor Heterodimers

A G Protein–Coupled Receptor Dimer Imaging Assay Reveals Selectively Modified Pharmacology of Neuropeptide Y Y1/Y5 Receptor Heterodimers

PheVI:09 (Phe6.44) as a Sliding Microswitch in Seven-transmembrane (7TM) G Protein-coupled Receptor Activation

Mechanistic basis of GPCR activation explored by ensemble refinement of crystallographic structures

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