Improved ligand-binding- and signaling-competent human NK2R yields in yeast using a chimera with the rat NK2R C-terminus enable NK2R-G protein signaling platform

Abstract The tachykinin 2 receptor (NK2R) plays critical roles in gastrointestinal, respiratory and mental disorders and is a well-recognized target for therapeutic intervention. To date, therapeutics targeting NK2R have failed to meet regulatory agency approval due in large part to the limited characterization of the receptor-ligand interaction and downstream signaling. Herein, we report a protein engineering strategy to improve ligand-binding- and signaling-competent human NK2R that enables a yeast-based NK2R signaling platform by creating chimeras utilizing sequences from rat NK2R. We demonstrate that NK2R chimeras incorporating the rat NK2R C-terminus exhibited improved ligand-binding yields and downstream signaling in engineered yeast strains and mammalian cells, where observed yields were better than 4-fold over wild type. This work builds on our previous studies that suggest exchanging the C-termini of related and well-expressed family members may be a general protein engineering strategy to overcome limitations to ligand-binding and signaling-competent G protein-coupled receptor yields in yeast. We expect these efforts to result in NK2R drug candidates with better characterized signaling properties.

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The Specificity of Downstream Signaling for A1 and A2AR Does Not Depend on the C-Terminus, Despite the Importance of This Domain in Downstream Signaling Strength

Biomedicines ◽

10.3390/biomedicines8120603 ◽

2020 ◽

Vol 8 (12) ◽

pp. 603

Author(s):

Abhinav R. Jain ◽

Claire McGraw ◽

Anne S. Robinson

Keyword(s):

Crystal Structures ◽

G Protein ◽

Mammalian Cells ◽

Full Length ◽

Surprising Result ◽

G Protein Coupling ◽

Hek 293 ◽

Protein Coupling ◽

Downstream Signaling ◽

C Terminus

Recent efforts to determine the high-resolution crystal structures for the adenosine receptors (A1R and A2AR) have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been utilized for all the adenosine receptor crystal structures obtained to date. Ligand binding for this truncated receptor has been shown to be similar to full-length receptor for A2AR. However, the C-terminus has been identified as a location for protein-protein interactions that may be critical for the physiological function of these important drug targets. We show that variants with A2AR C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptor constructs transfected into mammalian cells (HEK-293). In addition, we show that in a humanized yeast system, the absence of the full-length C-terminus affected downstream signaling using a yeast MAPK response-based fluorescence assay, though full-length receptors showed native-like G-protein coupling. To further study the G protein coupling, we used this humanized yeast platform to explore coupling to human-yeast G-protein chimeras in a cellular context. Although the C-terminus was essential for Gα protein-associated signaling, chimeras of A1R with a C-terminus of A2AR coupled to the A1R-specific Gα (i.e., Gαi1 versus Gαs). This surprising result suggests that the C-terminus is important in the signaling strength, but not specificity, of the Gα protein interaction. This result has further implications in drug discovery, both in enabling the experimental use of chimeras for ligand design, and in the cautious interpretation of structure-based drug design using truncated receptors.

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A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity

Science Signaling ◽

10.1126/scisignal.aaw5885 ◽

2020 ◽

Vol 13 (617) ◽

pp. eaaw5885 ◽

Cited By ~ 5

Author(s):

Marta Sanchez-Soto ◽

Ravi Kumar Verma ◽

Blair K. A. Willette ◽

Elizabeth C. Gonye ◽

Annah M. Moore ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

G Protein ◽

Structural Basis ◽

Protein Signaling ◽

Ligand Binding Site ◽

Intracellular Loop ◽

Gpcr Signaling ◽

Biased Signaling ◽

Dynamics Simulations

Signaling bias is the propensity for some agonists to preferentially stimulate G protein–coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein–biased agonist of the D2 dopamine receptor (D2R) that results in impaired β-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with β-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired β-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the β2-adrenergic receptor (β2R) to build β2R-WT and β2R-Y1995.38A models in complex with the full β2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in β2R-Y1995.38A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in β2R-Y1995.38A, which is predicted to affect its interactions with β-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.

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Probing the mutational landscape of regulators of G protein signaling proteins in cancer

Science Signaling ◽

10.1126/scisignal.aax8620 ◽

2020 ◽

Vol 13 (617) ◽

pp. eaax8620 ◽

Cited By ~ 7

Author(s):

Vincent DiGiacomo ◽

Marcin Maziarz ◽

Alex Luebbers ◽

Jillian M. Norris ◽

Pandu Laksono ◽

...

Keyword(s):

G Protein ◽

G Proteins ◽

Mammalian Cells ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Loss Of Function ◽

Gpcr Signaling ◽

Mutational Landscape ◽

Binding Interface

The advent of deep-sequencing techniques has revealed that mutations in G protein–coupled receptor (GPCR) signaling pathways in cancer are more prominent than was previously appreciated. An emergent theme is that cancer-associated mutations tend to cause enhanced GPCR pathway activation to favor oncogenicity. Regulators of G protein signaling (RGS) proteins are critical modulators of GPCR signaling that dampen the activity of heterotrimeric G proteins through their GTPase-accelerating protein (GAP) activity, which is conferred by a conserved domain dubbed the “RGS-box.” Here, we developed an experimental pipeline to systematically assess the mutational landscape of RGS GAPs in cancer. A pan-cancer bioinformatics analysis of the 20 RGS domains with GAP activity revealed hundreds of low-frequency mutations spread throughout the conserved RGS domain structure with a slight enrichment at positions that interface with G proteins. We empirically tested multiple mutations representing all RGS GAP subfamilies and sampling both G protein interface and noninterface positions with a scalable, yeast-based assay. Last, a subset of mutants was validated using G protein activity biosensors in mammalian cells. Our findings reveal that a sizable fraction of RGS protein mutations leads to a loss of function through various mechanisms, including disruption of the G protein–binding interface, loss of protein stability, or allosteric effects on G protein coupling. Moreover, our results also validate a scalable pipeline for the rapid characterization of cancer-associated mutations in RGS proteins.

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High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors

CrossRef Listing of Deleted DOIs ◽

10.1177/108705719900400105 ◽

1999 ◽

Vol 4 (1) ◽

pp. 27-32 ◽

Cited By ~ 36

Author(s):

Ilona Kariv ◽

Michelle E. Stevens ◽

Davette L. Behrens ◽

Kevin R. Oldenburg

Keyword(s):

Ligand Binding ◽

G Protein ◽

High Throughput ◽

High Throughput Screening ◽

Transmembrane Domain ◽

Receptor Function ◽

Camp Production ◽

Ligand Interaction ◽

G Protein Coupled ◽

Native Ligand

Impairment of G protein—coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate™ (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within ≤20 μm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein—coupled receptor function as measured by cAMP production and is suitable for high throughput screening.

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New small molecule fluorescent probes for G protein-coupled receptors: valuable tools for drug discovery

Future Medicinal Chemistry ◽

10.4155/fmc-2019-0327 ◽

2021 ◽

Vol 13 (1) ◽

pp. 63-90

Author(s):

Joshua W Conner ◽

Daniel P Poole ◽

Manuela Jörg ◽

Nicholas A Veldhuis

Keyword(s):

Drug Discovery ◽

Ligand Binding ◽

G Protein ◽

Small Molecule ◽

G Protein Coupled Receptors ◽

Signaling Proteins ◽

Drug Candidates ◽

Compound Screening ◽

G Protein Coupled ◽

Single Approach

G protein-coupled receptors (GPCRs) are essential signaling proteins and tractable therapeutic targets. To develop new drug candidates, GPCR drug discovery programs require versatile, sensitive pharmacological tools for ligand binding and compound screening. With the availability of new imaging modalities and proximity-based ligand binding technologies, fluorescent ligands offer many advantages and are increasingly being used, yet labeling small molecules remains considerably more challenging relative to peptides. Focusing on recent fluorescent small molecule studies for family A GPCRs, this review addresses some of the key challenges, synthesis approaches and structure–activity relationship considerations, and discusses advantages of using high-resolution GPCR structures to inform conjugation strategies. While no single approach guarantees successful labeling without loss of affinity or selectivity, the choice of fluorophore, linker type and site of attachment have proved to be critical factors that can significantly affect their utility in drug discovery programs, and as discussed, can sometimes lead to very unexpected results.

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Identification of Critical Residues Involved in Ligand Binding and G Protein Signaling in Human Somatostatin Receptor Subtype 2

Endocrinology ◽

10.1210/en.2011-1662 ◽

2012 ◽

Vol 153 (6) ◽

pp. 2747-2755 ◽

Cited By ~ 5

Author(s):

Jesse J. Parry ◽

Ronald Chen ◽

Rebecca Andrews ◽

Kimberly A. Lears ◽

Buck E. Rogers

Keyword(s):

Ligand Binding ◽

G Protein ◽

Calcium Release ◽

Expression Profiles ◽

Somatostatin Receptor ◽

Receptor Subtype ◽

G Protein Signaling ◽

Protein Signaling ◽

Wild Type ◽

Somatostatin Receptor Subtype 2

G protein signaling through human somatostatin receptor subtype 2 (SSTR2) is well known, but the amino acids involved in stimulation of intracellular responses upon ligand binding have not been characterized. We constructed a series of point mutants in SSTR2 at amino acid positions 89, 139, and 140 in attempts to disrupt G protein signaling upon ligand binding. The aspartic acid changes at position 89 to either Ala, Leu, or Arg generated mutant receptors with varying expression profiles and a complete inability to bind somatostatin-14 (SST). Mutations to Asp 139 and Arg 140 also led to varying expression profiles with some mutants maintaining their affinity for SST. Mutation of Arg 140 to Ala resulted in a mutated receptor that had a Bmax and dissociation constant (Kd) similar to wild-type receptor but was still coupled to the G protein as determined in both a cAMP assay and a calcium-release assay. In contrast, mutation of Asp 139 to Asn resulted in a mutated receptor with Bmax and Kd values that were similar to wild type but was uncoupled from G protein-mediated cAMP signaling, but not calcium release. Thus, we identified mutations in SSTR2 that result in either receptor expression levels that are similar to wild type but is completely ablated for ligand binding or a receptor that maintains affinity for SST and is uncoupled from G protein-mediated cAMP signaling.

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Display of Heterologous Proteins on gp64null Baculovirus Virions and Enhanced Budding Mediated by a Vesicular Stomatitis Virus G-Stem Construct

Journal of Virology ◽

10.1128/jvi.02007-07 ◽

2007 ◽

Vol 82 (3) ◽

pp. 1368-1377 ◽

Cited By ~ 24

Author(s):

Jian Zhou ◽

Gary W. Blissard

Keyword(s):

Amino Acids ◽

G Protein ◽

Vesicular Stomatitis Virus ◽

Mammalian Cells ◽

Heterologous Proteins ◽

C Terminus ◽

Influenza Virus Hemagglutinin ◽

Severe Reduction ◽

Vesicular Stomatitis ◽

Ha Protein

ABSTRACT The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the gp64 gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a gp64null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV gp64null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the gp64null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on gp64null AcMNPV budded virions. The combined use of gp64null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production.

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Calcium-Dependent Ligand Binding and G-protein Signaling of Family B GPCR Parathyroid Hormone 1 Receptor Purified in Nanodiscs

ACS Chemical Biology ◽

10.1021/cb300466n ◽

2013 ◽

Vol 8 (3) ◽

pp. 617-625 ◽

Cited By ~ 25

Author(s):

Nivedita Mitra ◽

Yuting Liu ◽

Jian Liu ◽

Eugene Serebryany ◽

Victoria Mooney ◽

...

Keyword(s):

Parathyroid Hormone ◽

Ligand Binding ◽

G Protein ◽

G Protein Signaling ◽

Protein Signaling ◽

Calcium Dependent ◽

Parathyroid Hormone 1 Receptor

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C5a-Stimulated Recruitment of β-Arrestin2 to the Nonsignaling 7-Transmembrane Decoy Receptor C5L2

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109341407 ◽

2009 ◽

Vol 14 (9) ◽

pp. 1067-1075 ◽

Cited By ~ 31

Author(s):

Lambertus H.C. Van Lith ◽

Julia Oosterom ◽

Andrea Van Elsas ◽

Guido J.R. Zaman

Keyword(s):

G Protein ◽

Fluorescent Protein ◽

G Protein Signaling ◽

Dependent Manner ◽

Protein Signaling ◽

Ligand Interaction ◽

C5a Receptor ◽

Decoy Receptor ◽

Complementation Assay ◽

Fragment Complementation

C5L2 (or GPR77) is a high-affinity receptor for the complement fragment C5a and its desarginated product, C5a-desArg. Unlike the classical C5a receptor CD88, C5L2 does not couple to intracellular G-protein-signaling pathways but is thought to function as a decoy receptor. The authors show that stimulation of C5L2 with C5a and C5a-desArg induces redistribution of green fluorescent protein—labeled β-arrestin2 to cytoplasmic vesicles. C3a and C3a-desArg were inactive in the β-arrestin translocation assay. Direct interaction of ligand-stimulated C5L2 with β-arrestin was confirmed using a novel β-galactosidase fragment complementation assay. In this assay, C5L2 was labeled with a mutationally altered peptide fragment of β-galactosidase, whereas β-arrestin2 was labeled with a corresponding deletion mutant of the enzyme. Stable transfection of the modified C5L2 and subsequent stimulation with C5a or C5a-desArg restored β-galactosidase activity in a dose-dependent manner. The subnanomolar potency of β-arrestin coupling in the β-galactosidase fragment complementation assay is in agreement with the affinity of the receptor-ligand interaction. C5L2 is the first example of a 7-transmembrane decoy receptor that couples to β-arrestin in a ligand-dependent manner. This observation supports the notion that G-protein-signaling and β-arrestin coupling can be 2 separate activities of 7-transmembrane receptors. Furthermore, the β-arrestin assays described in this article provide methods of screening for selective C5L2 modulators. ( Journal of Biomolecular Screening 2009:1067-1075)

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