scholarly journals Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

2021 ◽  
Author(s):  
Yusman Manchanda ◽  
Zenouska Ramchunder ◽  
Maria M Shchepinova ◽  
Guy A Rutter ◽  
Asuka Inoue ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pancreatic Beta Cell ◽  
Ease Of Use ◽  
Intracellular Signalling ◽  
Receptor Coupling ◽  
Cell Class ◽  
Class B ◽  
Cell Assays ◽  
Glucagon Like Peptide 1

Mini-G proteins are engineered thermostable variants of Gα subunits designed to specifically stabilise G protein-coupled receptors (GPCRs) in their active conformation for structural analyses. Due to their smaller size and ease of use, they have become popular tools in recent years to assess specific GPCR behaviours in cells, both as reporters of receptor coupling to each G protein subtype and for in-cell assays designed to quantify compartmentalised receptor signalling from a range of subcellular locations. Here, we describe a previously unappreciated consequence of the co-expression of mini-G proteins with their cognate GPCRs, namely a profound disruption in GPCR trafficking and intracellular signalling caused by the co-expression of the specific mini-G subtype coupled to the affected receptor. We studied the Gαs-coupled pancreatic beta cell class B GPCR glucagon-like peptide-1 receptor (GLP-1R) as a model to describe in detail the molecular consequences derived from this effect, including a complete halt in β-arrestin-2 recruitment and receptor internalisation, despite near-normal levels of receptor GRK2 recruitment and lipid nanodomain segregation, as well as the disruption of endosomal GLP-1R signalling by mini-Gs co-expression. We also extend our analysis to a range of other prototypical GPCRs covering the spectrum of Gα subtype coupling preferences, to unveil a widely conserved phenomenon of GPCR internalisation blockage by specific mini-G proteins coupled to a particular receptor. Our results have important implications for the design of methods to assess intracellular GPCR signalling. We also present an alternative adapted bystander intracellular signalling assay for the GLP-1R in which we substitute the mini-Gs by a nanobody, Nb37, with specificity for active Gαs:GPCR complexes and no deleterious effect on the capacity for GLP-1R internalisation.

scholarly journals Glucagon-Like Peptide-1 and Its Class B G Protein–Coupled Receptors: A Long March to Therapeutic Successes

2016 ◽  
Vol 68 (4) ◽  
pp. 954-1013 ◽  
Author(s):  
Chris de Graaf ◽  
Dan Donnelly ◽  
Denise Wootten ◽  
Jesper Lau ◽  
Patrick M. Sexton ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Class B ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
G Protein Coupled

scholarly journals Conserved 2nd Residue of Helix 8 of GPCR May Confer the Subclass-Characteristic and Distinct Roles through a Rapid Initial Interaction with Specific G Proteins

2019 ◽  
Vol 20 (7) ◽  
pp. 1752 ◽  
Author(s):  
Sato
Keyword(s):  
Amino Acids ◽  
G Protein ◽  
G Proteins ◽  
Specific Interactions ◽  
G Protein Coupled Receptor ◽  
Conserved Residues ◽  
Trace Amine ◽  
Class B ◽  
Initial Interaction ◽  
G Protein Coupled

To obtain a systematic view of the helix-8-second residue responsible for G protein-coupled receptor (GPCR)–G protein initial specific interactions, 786 human GPCRs were subclassified based on the pairs of agonist groups and target G proteins and compared with their conserved second residue of helix 8. Of 314 non-olfactory and deorphanized GPCRs, 273 (87%) conserved single amino acids in the subclasses, while 93 (58%) of the 160 subclasses possessed only a single GPCR member. Class B, C, Frizzled, and trace amine-associated GPCRs demonstrated 100% conservation, whereas class Ⅰ and Ⅱ olfactory and vomeronasal 1 receptors demonstrated much lower rates of conservation (20–47%). These conserved residues are characteristic of GPCR classes and G protein subtypes and confer their functionally-distinct roles.

scholarly journals Structural basis for GLP-1 receptor activation by LY3502970, an orally active nonpeptide agonist

2020 ◽  
Vol 117 (47) ◽  
pp. 29959-29967 ◽  
Author(s):  
Takahiro Kawai ◽  
Bingfa Sun ◽  
Hitoshi Yoshino ◽  
Dan Feng ◽  
Yoshiyuki Suzuki ◽  
...  
Keyword(s):  
Oral Administration ◽  
G Protein ◽  
Partial Agonist ◽  
Pharmacokinetic Profile ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Structural Basis ◽  
Protein Activation ◽  
Class B ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved GLP-1R agonists are peptide based, and it has proven difficult to obtain small-molecule activators possessing optimal pharmaceutical properties. We report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over β-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein–coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule. In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.

A study of M1 receptor coupling to inhibitory G proteins in CHO cells via GTPγS binding determined with anti-G protein antibodies

Life Sciences ◽  
1999 ◽  
Vol 64 (6-7) ◽  
pp. 569 ◽  
Author(s):  
N.W. DeLapp ◽  
D. McClure ◽  
J. McKinzie ◽  
A. Silbernagel ◽  
B.D. Sawyer ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Cho Cells ◽  
Receptor Coupling ◽  
M1 Receptor ◽  
Gtpγs Binding

Coupling of ATP-sensitive K+ channels to A1 receptors by G proteins in rat ventricular myocytes

1990 ◽  
Vol 259 (3) ◽  
pp. H820-H826 ◽  
Author(s):  
G. E. Kirsch ◽  
J. Codina ◽  
L. Birnbaumer ◽  
A. M. Brown
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Extracellular Adenosine ◽  
Rat Ventricular Myocytes ◽  
Receptor Coupling ◽  
Neonatal Rat Ventricular Myocytes ◽  
Gamma S ◽  
Ischemic Muscle

ATP-sensitive K+ (K+[ATP]) current is thought to be regulated by GTP-binding proteins (G proteins), but the pathways that couple receptor, G protein, and channel have not been defined. We studied regulation of tolbutamide-sensitive K+[ATP] current in neonatal rat ventricular myocytes. Application of 0.1 mM ATP to the intracellular side of membrane patches reduced K+ [ATP] channel activity, and addition of the nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at 0.1 mM restored activity. Application of 0.1 mM intracellular GTP plus 10 microM extracellular adenosine or 100 nM N6-cyclohexyladenosine had the same effect as GTP gamma S; hence K+[ATP] channels may be coupled to adenosine receptors via G proteins. To determine which G protein, we applied G alpha subunits, preactivated with GTP gamma S to the cytoplasmic side of membrane patches, and found that alpha i1, alpha i2, and alpha i3 mimicked the effect of GTP gamma S, but not alpha o or Gs, suggesting that Gi alpha acts via a membrane-delimited pathway. Adenosine receptor coupling may be important for activating K+[ATP] channels in ischemic muscle.

Structural basis of Gs and Gi recognition by the human glucagon receptor

Science ◽  
2020 ◽  
Vol 367 (6484) ◽  
pp. 1346-1352 ◽  
Author(s):  
Anna Qiao ◽  
Shuo Han ◽  
Xinmei Li ◽  
Zhixin Li ◽  
Peishen Zhao ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Structural Basis ◽  
Heterotrimeric G Proteins ◽  
Glucagon Receptor ◽  
Functional Studies ◽  
Class B ◽  
Interaction Interface ◽  
Binding Cavity ◽  
Intracellular Loops

Class B G protein–coupled receptors, an important class of therapeutic targets, signal mainly through the Gs class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo–electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, Gs or Gi1. These two structures adopt a similar open binding cavity to accommodate Gs and Gi1. The Gs binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect Gi more than Gs binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.

scholarly journals Local membrane charge regulates ல2 adrenergic receptor coupling to Gi

2018 ◽  
Author(s):  
M.J. Strohman ◽  
S. Maeda ◽  
D. Hilger ◽  
M. Masureel ◽  
Y. Du ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Adrenergic Receptor ◽  
Mutational Analysis ◽  
Neutral Lipids ◽  
Phospholipid Composition ◽  
Transmembrane Receptors ◽  
Receptor Coupling ◽  
Membrane Charge ◽  
Biochemical Assays

AbstractG protein coupled receptors (GPCRs) are transmembrane receptors that signal through heterotrimeric G proteins. Lipid modifications anchor G proteins to the plasma membrane; however, little is known about the effect of phospholipid composition on GPCR-G protein coupling. The β2 adrenergic receptor (β2AR) signals through both Gs and Gi in cardiac myocytes where studies suggest that Gi signaling may be cardioprotective. However, Gi coupling is much less efficient than Gs coupling in most cell-based and biochemical assays, making it difficult to study β2AR-Gi interactions. To investigate the role of phospholipid composition on Gs and Gi coupling, we reconstituted β2AR in detergent/lipid mixed micelles and found that negatively charged phospholipids (PS and PG) inhibit β2AR-Gi3 coupling. Replacing negatively charged lipids with neutral lipids (PC or PE) facilitated the formation of a functional β2AR-Gi3 interaction that activated Gi3. Ca2+, known to interact with negatively charged PS, facilitated β2AR-Gi3 interaction in PS. Mutational analysis suggested that Ca2+ interacts with the negatively charged EDGE motif on the carboxyl-terminal end of the αN helix of Gi3 and coordinates an EDGE-PS interaction. These results were confirmed in β2AR reconstituted into nanodisc phospholipid bilayers. β2AR-Gi3 interaction was favored in neutral lipids (PE and PC) over negatively charged lipids (PG and PS). In contrast, basal β2AR-Gs interaction was favored in negatively charged lipids over neutral lipids. In negatively-charged lipids, Ca2+ and Mg2+ facilitated β2AR-Gi3 interaction. Taken together, our observations suggest that local membrane charge modulates the interaction between β2AR and competing G protein subtypes.

scholarly journals Cryo-EM structure of the human PAC1 receptor coupled to an engineered heterotrimeric G protein

2019 ◽  
Author(s):  
Kazuhiro Kobayashi ◽  
Wataru Shihoya ◽  
Tomohiro Nishizawa ◽  
Francois Marie Ngako Kadji ◽  
Junken Aoki ◽  
...  
Keyword(s):  
G Protein ◽  
Structural Information ◽  
Receptor Activation ◽  
Peptide Ligand ◽  
Pac1 Receptor ◽  
Peripheral Tissues ◽  
Class B ◽  
Neuropeptide Hormone ◽  
The Central Nervous System ◽  
Glucagon Like Peptide 1

AbstractPituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide hormone functioning in the central nervous system and peripheral tissues. The PACAP receptor PAC1R, which belongs to the class B G-protein-coupled receptors (GPCRs), is a drug target for mental disorders and dry eye syndrome. Here we present a cryo-electron microscopy structure of human PAC1R bound to PACAP and an engineered Gs heterotrimer. The structure revealed that TM1 plays an essential role in PACAP recognition. The ECD (extracellular domain) of PAC1R tilts by ~40° as compared to that of the glucagon-like peptide-1 receptor (GLP1R), and thus does not cover the peptide ligand. A functional analysis demonstrated that the PAC1R-ECD functions as an affinity trap and is not required for receptor activation, whereas the GLP1R-ECD plays an indispensable role in receptor activation, illuminating the functional diversity of the ECDs in the class B GPCRs. Our structural information will facilitate the design and improvement of better PAC1R agonists for clinical applications.This article is a preprint version and has not been certified by peer review.

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