scholarly journals Mutagenesis facilitated crystallization of GLP-1R

IUCrJ ◽  
2019 ◽  
Vol 6 (6) ◽  
pp. 996-1006 ◽  
Author(s):  
Yueming Xu ◽  
Yuxia Wang ◽  
Yang Wang ◽  
Kaiwen Liu ◽  
Yao Peng ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Crystal Packing ◽  
Peptide Hormone ◽  
Current Data ◽  
Transmembrane Helices ◽  
Dynamic Simulations ◽  
Class B ◽  
Glucagon Like Peptide 1 ◽  
Diabetes Drug ◽  
G Protein Coupled

The class B family of G-protein-coupled receptors (GPCRs) has long been a paradigm for peptide hormone recognition and signal transduction. One class B GPCR, the glucagon-like peptide-1 receptor (GLP-1R), has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease. The previously determined structures of inactive GLP-1R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of 98 designed mutations. Here we systematically summarize all 98 mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical hydrophobic interactions shows the highest success rate. We further investigate four back mutations by thermal-shift assay, crystallization and molecular dynamic simulations, and conclude that mutation I1962.66bF increases thermal stability intrinsically and that mutation S2714.47bA decreases crystal packing entropy extrinsically, while mutations S1932.63bC and M2333.36bC may be dispensable since these two cysteines are not disulfide-linked. Our results indicate intrinsic connections between different regions of GPCR transmembrane helices and the current data suggest a general mutagenesis principle for structural determination of GPCRs and other membrane proteins.

scholarly journals A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor

Cell Research ◽  
2020 ◽  
Vol 30 (12) ◽  
pp. 1098-1108 ◽  
Author(s):  
Wen Sun ◽  
Li-Nan Chen ◽  
Qingtong Zhou ◽  
Li-Hua Zhao ◽  
Dehua Yang ◽  
...  
Keyword(s):  
Drug Targets ◽  
Receptor Activation ◽  
Ligand Specificity ◽  
Middle Region ◽  
Transmembrane Helices ◽  
Intestinal Hormones ◽  
Functional Studies ◽  
C Terminus ◽  
Class B ◽  
G Protein Coupled

AbstractGlucagon-like peptides (GLP-1 and GLP-2) are two proglucagon-derived intestinal hormones that mediate distinct physiological functions through two related receptors (GLP-1R and GLP-2R) which are important drug targets for metabolic disorders and Crohn’s disease, respectively. Despite great progress in GLP-1R structure determination, our understanding on the differences of peptide binding and signal transduction between these two receptors remains elusive. Here we report the electron microscopy structure of the human GLP-2R in complex with GLP-2 and a Gs heterotrimer. To accommodate GLP-2 rather than GLP-1, GLP-2R fine-tunes the conformations of the extracellular parts of transmembrane helices (TMs) 1, 5, 7 and extracellular loop 1 (ECL1). In contrast to GLP-1, the N-terminal histidine of GLP-2 penetrates into the receptor core with a unique orientation. The middle region of GLP-2 engages with TM1 and TM7 more extensively than with ECL2, and the GLP-2 C-terminus closely attaches to ECL1, which is the most protruded among 9 class B G protein-coupled receptors (GPCRs). Functional studies revealed that the above three segments of GLP-2 are essential for GLP-2 recognition and receptor activation, especially the middle region. These results provide new insights into the molecular basis of ligand specificity in class B GPCRs and may facilitate the development of more specific therapeutics.

scholarly journals The putative signal peptide of glucagon-like peptide-1 receptor is not required for receptor synthesis but promotes receptor expression

2014 ◽  
Vol 34 (6) ◽  
Author(s):  
Yunjun Ge ◽  
Dehua Yang ◽  
Antao Dai ◽  
Caihong Zhou ◽  
Yue Zhu ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Receptor Expression ◽  
Hek293 Cells ◽  
Putative Signal Peptide ◽  
293 Cells ◽  
Class B ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
G Protein Coupled

GLP-1R (glucagon-like peptide-1 receptor) mediates the ‘incretin effect’ and many other anti-diabetic actions of its cognate ligand, GLP-1 (glucagon-like peptide-1). It belongs to the class B family of GPCRs (G protein-coupled receptors) and possesses an N-terminal putative SP (signal peptide). It has been reported that this sequence is required for the synthesis of GLP-1R and is cleaved after receptor synthesis. In the present study, we conducted an in-depth exploration towards the role of the putative SP in GLP-1R synthesis. A mutant GLP-1R without this sequence was expressed in HEK293 cells (human embryonic kidney 293 cells) and displayed normal functionality with respect to ligand binding and activation of adenylate cyclase. Thus the putative SP does not seem to be required for receptor synthesis. Immunoblotting analysis shows that the amount of GLP-1R synthesized in HEK293 cells is low when the putative SP is absent. This indicates that the role of the sequence is to promote the expression of GLP-1R. Furthermore, epitopes tagged at the N-terminal of GLP-1R are detectable by immunofluorescence and immunoblotting in our experiments. In conclusion, the present study points to different roles of SP in GLP-1R expression which broadens our understanding of the functionality of this putative SP of GLP-1R and possibly other Class B GPCRs.

Peptide binding at the GLP-1 receptor

2007 ◽  
Vol 35 (4) ◽  
pp. 713-716 ◽  
Author(s):  
R. Mann ◽  
N. Nasr ◽  
D. Hadden ◽  
J. Sinfield ◽  
F. Abidi ◽  
...  
Keyword(s):  
G Protein ◽  
Peptide Binding ◽  
Domain Model ◽  
Full Length ◽  
Transmembrane Helices ◽  
Core Domain ◽  
The Family ◽  
Loop Regions ◽  
Glucagon Like Peptide 1 ◽  
G Protein Coupled

The receptor for GLP-1 [glucagon-like peptide-1-(7–36)-amide] is a member of the ‘Family B’ of GPCRs (G-protein-coupled receptors) comprising an extracellular N-terminal domain containing six conserved cysteine residues (the N-domain) and a core domain (or J-domain) comprising the seven transmembrane helices and interconnecting loop regions. According to the two-domain model for peptide binding, the N-domain is primarily responsible for providing most of the peptide binding energy, whereas the core domain is responsible for binding the N-terminal region of the peptide agonists and transmitting the signal to the intracellular G-protein. Two interesting differences between the binding properties of two GLP-1 receptor agonists, GLP-1 and EX-4 (exendin-4), can be observed. First, while GLP-1 requires its full length to maintain high affinity, the eight N-terminal residues of EX-4 can be removed with little reduction in affinity. Secondly, EX-4 (but not GLP-1) can bind to the fully isolated N-domain of the receptor with an affinity matching that of the full-length receptor. In order to better understand these differences, we have studied the interaction between combinations of full-length or truncated ligands with full-length or truncated receptors.

scholarly journals The Interplay of Glucagon-Like Peptide-1 Receptor Trafficking and Signalling in Pancreatic Beta Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Amaara Marzook ◽  
Alejandra Tomas ◽  
Ben Jones
Keyword(s):  
Pancreatic Beta Cells ◽  
Metabolic Effects ◽  
Physiological Importance ◽  
Intracellular Signals ◽  
Class B ◽  
The Central Nervous System ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
G Protein Coupled

The glucagon-like peptide 1 receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) which mediates the effects of GLP-1, an incretin hormone secreted primarily from L-cells in the intestine and within the central nervous system. The GLP-1R, upon activation, exerts several metabolic effects including the release of insulin and suppression of appetite, and has, accordingly, become an important target for the treatment for type 2 diabetes (T2D). Recently, there has been heightened interest in how the activated GLP-1R is trafficked between different endomembrane compartments, controlling the spatial origin and duration of intracellular signals. The discovery of “biased” GLP-1R agonists that show altered trafficking profiles and selective engagement with different intracellular effectors has added to the tools available to study the mechanisms and physiological importance of these processes. In this review we survey early and recent work that has shed light on the interplay between GLP-1R signalling and trafficking, and how it might be therapeutically tractable for T2D and related diseases.

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

Investigation of ECD conformational transition mechanism of GLP-1R by molecular dynamics simulations and Markov state model

2019 ◽  
Vol 21 (16) ◽  
pp. 8470-8481 ◽  
Author(s):  
Jintu Zhang ◽  
Qifeng Bai ◽  
Horacio Pérez-Sánchez ◽  
Shuxia Shang ◽  
Xiaoli An ◽  
...  
Keyword(s):  
Conformational Transition ◽  
G Protein Coupled Receptors ◽  
Markov State Model ◽  
Markov State ◽  
Transition Mechanism ◽  
Class B ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Dynamics Simulations ◽  
G Protein Coupled

As a member of the class B G protein-coupled receptors (GPCRs), the glucagon-like peptide-1 (GLP-1) can regulate the blood glucose level by binding to the glucagon-like peptide-1 receptor (GLP-1R).

scholarly journals Cryo-EM structure of human somatostatin receptor 2 complex with its agonist somatostatin delineates the ligand binding specificity

2022 ◽  
Author(s):  
Yunseok Heo ◽  
Eojin Yoon ◽  
Ye-Eun Jeon ◽  
Ji-Hye Yun ◽  
Naito Ishimoto ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
High Resolution ◽  
Ligand Binding ◽  
Neurological Disorders ◽  
Somatostatin Receptor ◽  
Somatostatin Receptors ◽  
Peptide Hormone ◽  
Transmembrane Helices ◽  
G Protein Coupled ◽  
Gi Proteins

Somatostatin is a peptide hormone regulating endocrine systems through binding to G-protein-coupled somatostatin receptors. somatostatin receptor 2 (SSTR2) is one of the human somatostatin receptors and highly implicated in cancers and neurological disorders. Here, we report the high resolution cryo-EM structure of full-length human SSTR2 bound to the agonist somatostatin (SST-14) complex with inhibitory G (Gi) proteins. Our structure shows that seven transmembrane helices form a deep pocket for ligand binding and that the highly conserved Trp-Lys motif of SST-14 positions at the bottom of the pocket. Furthermore, our sequence analysis combined with AlphaFold modeled structures of other SSTR isoforms provide how SSTR family proteins specifically interact with their cognate ligands. This work provides the first glimpse into the molecular recognition of somatostatin receptor and crucial resource to develop therapeutics targeting somatostatin receptors.

Bioinformatics Analysis and Validation of Differentially Expressed MicroRNAs with Their Target Genes Involved in GLP-1RA Facilitated Osteogenesis

2020 ◽  
Vol 15 ◽  
Author(s):  
Na Wang ◽  
Yukun Li ◽  
Sijing Liu ◽  
Liu Gao ◽  
Chang Liu ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Target Genes ◽  
Bioinformatics Analysis ◽  
Differentially Expressed ◽  
Functional Study ◽  
Regulation Network ◽  
Glucagon Like Peptide 1 ◽  
G Protein Coupled ◽  
Lower Expression

Background: Recent studies revealed that the hypoglycemic hormone, glucagon-like peptide-1 (GLP-1), acted as an important modulator in osteogenesis of bone marrow derived mesenchymal stem cells (BMSCs). Objectives: The aim of this study was to identify the specific microRNA (miRNA) using bioinformatics analysis and validate the presence of differentially expressed microRNAs with their target genes after GLP-1 receptor agonist (GLP-1RA) administration involved in ostogenesis of BMSCs. Methods: MiRNAs were extracted from BMSCs after 5 days’ treatment and sent for high-throughput sequencing for differentially expressed (DE) miRNAs analyses. Then the expression of the DE miRNAs verified by the real-time RT-PCR analyses. Target genes were predicted, and highly enriched GOs and KEGG pathway analysis were conducted using bioinformatics analysis. For the functional study, two of the target genes, SRY (sex determining region Y)-box 5 (SOX5) and G protein-coupled receptor 84 (GPR84), were identified. Results: A total of 5 miRNAs (miRNA-509-5p, miRNA-547-3p, miRNA-201-3p, miRNA-201-5p, and miRNA-novel-272-mature) were identified differentially expressed among groups. The expression of miRNA-novel-272-mature were decreased during the osteogenic differentiation of BMSCs, and GLP-1RA further decreased its expression. MiRNA-novel-272-mature might interact with its target mRNAs to enhance osteogenesis. The lower expression of miRNA-novel-272-mature led to an increase in SOX5 and a decrease in GPR84 mRNA expression, respectively. Conclusions: Taken together, these results provide further insights to the pharmacological properties of GLP-1RA and expand our knowledge on the role of miRNAs-mRNAs regulation network in BMSCs’ differentiation.

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