scholarly journals Molecular Mechanism of Action of Pharmacoperone Rescue of Misrouted GPCR Mutants: The GnRH Receptor

2009 ◽  
Vol 23 (2) ◽  
pp. 157-168 ◽  
Author(s):  
Jo Ann Janovick ◽  
Akshay Patny ◽  
Ralph Mosley ◽  
Mark T. Goulet ◽  
Michael D. Altman ◽  
...  
Keyword(s):  
Quality Control ◽  
Salt Bridge ◽  
Gnrh Receptor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Pharmacological Chaperones ◽  
Naturally Occurring ◽  
Protein Mutants ◽  
Molecular Mechanism Of Action ◽  
G Protein Coupled

Abstract The human GnRH receptor (hGnRHR), a G protein-coupled receptor, is a useful model for studying pharmacological chaperones (pharmacoperones), drugs that rescue misfolded and misrouted protein mutants and restore them to function. This technique forms the basis of a therapeutic approach of rescuing mutants associated with human disease and restoring them to function. The present study relies on computational modeling, followed by site-directed mutagenesis, assessment of ligand binding, effector activation, and confocal microscopy. Our results show that two different chemical classes of pharmacoperones act to stabilize hGnRHR mutants by bridging residues D98 and K121. This ligand-mediated bridge serves as a surrogate for a naturally occurring and highly conserved salt bridge (E90–K121) that stabilizes the relation between transmembranes 2 and 3, which is required for passage of the receptor through the cellular quality control system and to the plasma membrane. Our model was used to reveal important pharmacophoric features, and then identify a novel chemical ligand, which was able to rescue a D98 mutant of the hGnRHR that could not be rescued as effectively by previously known pharmacoperones.

scholarly journals Isolation of Cry1Ab protein mutants ofBacillus thuringiensisby a highly efficient PCR site-directed mutagenesis system

1996 ◽  
Vol 145 (3) ◽  
pp. 333-339 ◽  
Author(s):  
Roberto Meza ◽  
Maria-Eugenia Nuñez-Valdez ◽  
Jorge Sanchez ◽  
Alejandra Bravo
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cry1ab Protein ◽  
Highly Efficient ◽  
Protein Mutants

scholarly journals Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action

1995 ◽  
Vol 310 (2) ◽  
pp. 553-558 ◽  
Author(s):  
Y Fukushima ◽  
Y Oka ◽  
T Saitoh ◽  
H Katagiri ◽  
T Asano ◽  
...  
Keyword(s):  
Molecular Mass ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Histamine H2 Receptor ◽  
H2 Receptor ◽  
H2 Receptors ◽  
G Protein Coupled

G-protein-coupled receptors generally share a similar structure containing seven membrane-spanning domains and extracellular site(s) for N-glycosylation. The histamine H2 receptor is a member of the family of G-protein-coupled receptors, and has three extracellular potential sites for N-glycosylation (Asn-4, Asn-162 and Asn-168). To date, however, no information has been presented regarding N-glycosylation of the H2 receptor. To investigate the presence, location and functional roles of N-glycosylation of the H2 receptor, site-directed mutagenesis was performed to eliminate the potential site(s) for N-glycosylation singly and collectively. The wild-type and mutated H2 receptors were expressed stably in Chinese hamster ovary (CHO) cells or transiently in COS7 cells. Immunoblotting of the wild-type and mutated H2 receptors with an antiserum directed against the C-terminus of the H2 receptor showed that mutation at Asn-162, but not at Asn-168, resulted in a substantial decrease in the molecular mass. A mutation at Asn-4 led to a further decrease in the molecular mass. Tunicamycin treatment of the transfected cells yielded a sharp band with a molecular mass identical to that of the mutant devoid of all three potential sites for N-glycosylation. These findings indicate that the H2 receptor is N-glycosylated, and that N-glycosylation takes place mainly at two sites, Asn-4 and Asn-162. Neither the affinity for tiotidine nor that for histamine was affected by the mutagenesis. Immunocytochemistry and tiotidine binding showed that the mutated receptors were exclusively distributed on the cell surface in a fashion similar to that of the wild-type. In addition, the glycosylation-defective receptor was capable of activating adenylate cyclase and elevating the intracellular Ca2+ concentration in response to histamine in stable CHO cell lines. Thus N-glycosylation of the H2 receptor is not required for cell surface localization, ligand binding or functional coupling to G-protein(s).

scholarly journals De novo Design of G Protein-Coupled Receptor 40 Peptide Agonists for Type 2 Diabetes Mellitus Based on Artificial Intelligence and Site-Directed Mutagenesis

2021 ◽  
Vol 9 ◽  
Author(s):  
Xu Chen ◽  
Zhidong Chen ◽  
Daiyun Xu ◽  
Yonghui Lyu ◽  
Yongxiao Li ◽  
...  
Keyword(s):  
Neural Network ◽  
Diabetes Mellitus ◽  
Machine Learning ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
G Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
G Protein Coupled

G protein-coupled receptor 40 (GPR40), one of the G protein-coupled receptors that are available to sense glucose metabolism, is an attractive target for the treatment of type 2 diabetes mellitus (T2DM). Despite many efforts having been made to discover small-molecule agonists, there is limited research focus on developing peptides acting as GPR40 agonists to treat T2DM. Here, we propose a novel strategy for peptide design to generate and determine potential peptide agonists against GPR40 efficiently. A molecular fingerprint similarity (MFS) model combined with a deep neural network (DNN) and convolutional neural network was applied to predict the activity of peptides constructed by unnatural amino acids (UAAs). Site-directed mutagenesis (SDM) further optimized the peptides to form specific favorable interactions, and subsequent flexible docking showed the details of the binding mechanism between peptides and GPR40. Molecular dynamics (MD) simulations further verified the stability of the peptide–protein complex. The R-square of the machine learning model on the training set and the test set reached 0.87 and 0.75, respectively; and the three candidate peptides showed excellent performance. The strategy based on machine learning and SDM successfully searched for an optimal design with desirable activity comparable with the model agonist in phase III clinical trials.

Glu2.53(90) of the GnRH receptor is part of the conserved G protein-coupled receptor structure and does not form a salt-bridge with Lys3.32(121)

2019 ◽  
Vol 481 ◽  
pp. 53-61 ◽  
Author(s):  
Ashmeetha Manilall ◽  
B. Andre Stander ◽  
Michael T. Madziva ◽  
Robert P. Millar ◽  
Colleen A. Flanagan
Keyword(s):  
G Protein ◽  
Salt Bridge ◽  
Gnrh Receptor ◽  
G Protein Coupled Receptor ◽  
Receptor Structure ◽  
G Protein Coupled

Effects of Asn87 and Asp318 mutations on ligand binding and signal transduction in the rat GnRH receptor.

1993 ◽  
Vol 139 (3) ◽  
pp. R1-R4 ◽  
Author(s):  
J. V. Cook ◽  
E. Faccenda ◽  
L. Anderson ◽  
G. G, Couper ◽  
K. A. Eidne ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Inositol Phosphate ◽  
Gnrh Receptor ◽  
G Protein Coupled Receptors ◽  
Site Directed Mutagenesis ◽  
Inositol Phosphate Production ◽  
Messenger System ◽  
Gonadotrophin Releasing Hormone ◽  
G Protein Coupled ◽  
Conserved Amino Acids

ABSTRACT The gonadotrophin-releasing hormone (GnRH) receptor is unlike other G-protein coupled receptors in that the highly conserved amino acids, Asp in the second transmembrane region and Asn in the seventh, are interchanged. Site-directed mutagenesis studies mutated these residues back to their normally conserved positions. Two single mutants Asn87Asp & Asp318Asn and one double mutant Asn87Asp Asp318Asn were transiently expressed in COS-1 cells and their effect on binding to GnRH and inositol phosphate production measured. The single mutant Asp318Asn had no effect on ligand binding but abolished GnRH-dependent inositol phosphate production, whereas mutations Asn87Asp and Asn87Asp Asp318Asn show a complete loss of GnRH binding and subsequent inactivation of its second messenger system. Journal of Molecular Endocrinology

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