scholarly journals Changes to Gonadotropin-Releasing Hormone (GnRH) Receptor Extracellular Loops Differentially Affect GnRH Analog Binding and Activation: Evidence for Distinct Ligand-Stabilized Receptor Conformations

Endocrinology ◽  
2008 ◽  
Vol 149 (6) ◽  
pp. 3118-3129 ◽  
Author(s):  
Kevin D. G. Pfleger ◽  
Adam J. Pawson ◽  
Robert P. Millar
Keyword(s):  
Ligand Binding ◽  
Receptor Activation ◽  
Gnrh Receptor ◽  
Binding Modes ◽  
Extracellular Loop ◽  
Gnrh Analog ◽  
Chimeric Receptors ◽  
Gnrh Receptors ◽  
Extracellular Loops ◽  
Loop Conformation

GnRH and its structural variants bind to GnRH receptors from different species with different affinities and specificities. By investigating chimeric receptors that combine regions of mammalian and nonmammalian GnRH receptors, a greater understanding of how different domains influence ligand binding and receptor activation can be achieved. Using human-catfish and human-chicken chimeric receptors, we demonstrate the importance of extracellular loop conformation for ligand binding and agonist potency, providing further evidence for GnRH and GnRH II stabilization of distinct active receptor conformations. We demonstrate examples of GnRH receptor gain-of-function mutations that apparently improve agonist potency independently of affinity, implicating a role for extracellular loops in stabilizing the inactive receptor conformation. We also show that entire extracellular loop substitution can overcome the detrimental effects of localized mutations, thereby demonstrating the importance of considering the conformation of entire domains when drawing conclusions from point-mutation studies. Finally, we present evidence implicating the configuration of extracellular loops 2 and 3 in combination differentiating GnRH analog binding modes. Because there are two endogenous forms of GnRH ligand but only one functional form of full-length GnRH receptor in humans, understanding how GnRH and GnRH II can elicit distinct functional effects through the same receptor is likely to provide important insights into how these ligands can have differential effects in both physiological and pathological situations.

scholarly journals Autocrine Regulation of Gonadotropin-Releasing Hormone Secretion in Cultured Hypothalamic Neurons

Endocrinology ◽  
1999 ◽  
Vol 140 (3) ◽  
pp. 1423-1431 ◽  
Author(s):  
Lazar Z. Krsmanovic ◽  
Antonio J. Martinez-Fuentes ◽  
Krishan K. Arora ◽  
Nadia Mores ◽  
Carlos E. Navarro ◽  
...  
Keyword(s):  
Hormone Secretion ◽  
Receptor Activation ◽  
Gnrh Receptor ◽  
Hypothalamic Neurons ◽  
Gnrh Receptors ◽  
Pulsatile Gnrh ◽  
Gnrh Neurons ◽  
Gnrh Release ◽  
Hypothalamic Cells

Abstract Episodic hormone secretion is a characteristic feature of the hypothalamo-pituitary-gonadal system, in which the profile of gonadotropin release from pituitary gonadotrophs reflects the pulsatile secretory activity of GnRH-producing neurons in the hypothalamus. Pulsatile release of GnRH is also evident in vitro during perifusion of immortalized GnRH neurons (GT1–7 cells) and cultured fetal hypothalamic cells, which continue to produce bioactive GnRH for up to 2 months. Such cultures, as well as hypothalamic tissue from adult rats, express GnRH receptors as evidenced by the presence of high-affinity GnRH binding sites and GnRH receptor transcripts. Furthermore, individual GnRH neurons coexpress GnRH and GnRH receptors as revealed by double immunostaining of hypothalamic cultures. In static cultures of hypothalamic neurons and GT1–7 cells, treatment with the GnRH receptor antagonist, [d-pGlu1, d-Phe2, d-Trp3,6]GnRH caused a prominent increase in GnRH release. In perifused hypothalamic cells and GT1–7 cells, treatment with the GnRH receptor agonist, des-Gly10-[d-Ala6]GnRH N-ethylamide, reduced the frequency and increased the amplitude of pulsatile GnRH release, as previously observed in GT1–7 cells. In contrast, exposure to the GnRH antagonist analogs abolished pulsatile secretion and caused a sustained and progressive increase in GnRH release. These findings have demonstrated that GnRH receptors are expressed in hypothalamic GnRH neurons, and that receptor activation is required for pulsatile GnRH release in vitro. The effects of GnRH agonist and antagonist analogs on neuropeptide release are consistent with the operation of an ultrashort-loop autocrine feedback mechanism that exerts both positive and negative actions that are necessary for the integrated control of GnRH secretion from the hypothalamus.

In Silico Characterization of Ligand Binding Modes in the Human Histamine H4 Receptor and their Impact on Receptor Activation

ChemBioChem ◽  
2010 ◽  
Vol 11 (13) ◽  
pp. 1850-1855 ◽  
Author(s):  
Tim Werner ◽  
Kerstin Sander ◽  
Yusuf Tanrikulu ◽  
Tim Kottke ◽  
Ewgenij Proschak ◽  
...  
Keyword(s):  
Ligand Binding ◽  
In Silico ◽  
Receptor Activation ◽  
Binding Modes ◽  
Histamine H4 Receptor ◽  
H4 Receptor ◽  
In Silico Characterization ◽  
Human Histamine

Extracellular loops and ligand binding to a subfamily of Family A G-protein-coupled receptors

2007 ◽  
Vol 35 (4) ◽  
pp. 717-720 ◽  
Author(s):  
M. Wheatley ◽  
J. Simms ◽  
S.R. Hawtin ◽  
V.J. Wesley ◽  
D. Wootten ◽  
...  
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Tertiary Structure ◽  
Large Family ◽  
Peptide Hormone ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Ligand Selectivity ◽  
Extracellular Loops ◽  
G Protein Coupled

GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins which mediate their effects by coupling to G-proteins. The V1aR (V1a vasopressin receptor) is a member of a family of related GPCRs that are activated by vasopressin {AVP ([Arg8]vasopressin)}, OT (oxytocin) and related peptides. These receptors are members of a subfamily of Family A GPCRs called the neurohypophysial peptide hormone receptor family. GPCRs exhibit a conserved tertiary structure comprising a bundle of seven TM (transmembrane) helices linked by alternating ECLs (extracellular loops) and ICLs (intracellular loops). The cluster of TM helices is functionally important for ligand binding, and, furthermore, activation of GPCRs involves movement of these TM helices. Consequently, it might be assumed that the extracellular face of GPCRs is composed of peptide linkers that merely connect important TM helices. However, using a systematic mutagenesis approach and focusing on the N-terminus and the second ECL of the V1aR, we have established that these extracellular domains fulfil a range of important roles with respect to GPCR signalling, including agonist binding, ligand selectivity and receptor activation.

scholarly journals Functional characterization and kinetic studies of an ancestral lamprey GnRH-III selective type II GnRH receptor from the sea lamprey, Petromyzon marinus

2006 ◽  
Vol 36 (3) ◽  
pp. 601-610 ◽  
Author(s):  
M R Silver ◽  
S A Sower
Keyword(s):  
Intact Cell ◽  
Functional Characterization ◽  
Kinetic Studies ◽  
Receptor Activation ◽  
Gnrh Receptor ◽  
Type I ◽  
Type Ii ◽  
Binding Assays ◽  
Gnrh Receptors ◽  
Series Of Experiments

The recently cloned lamprey GnRH receptor was shown to have several unique features, including the longest intracellular C-terminal tail (120 amino acids (aa)) of any previously described GnRH receptor. In the current study, a series of experiments were performed examining cAMP responses, binding kinetics, whole cell competitive binding assays and internalization studies of the lamprey GnRH receptor using a series of three C-terminal tail truncations (80 aa, 40 aa and 0 aa) to better describe the functional significance of this unique vertebrate GnRH receptor. Activation of the lamprey GnRH receptor was shown to stimulate cAMP production in a dose-dependant manner when treated with either lamprey GnRH-I (LogEC50 −6.57±0.15) or lamprey GnRH-III (LogEC50 −8.29±0.09). Truncation analysis indicated that the membrane proximal 40 aa of the lamprey GnRH receptor C-terminal tail contain a motif required for cAMP accumulation. Saturation binding assays using the wild type and truncated lamprey GnRH receptors revealed that all of three truncated lamprey GnRH receptors were capable of binding lamprey GnRH-I. Competitive, intact cell-binding assays suggested that the lamprey GnRH receptor is lamprey GnRH-III selective, based on the observed pharmacological profile: lamprey GnRH-III (Inhibitory constant (Ki) 0.708±0.245 nM)=chicken GnRH-II (Ki 0.765±0.160 nM) > mammalian GnRH (Ki 12.9±1.96 nM) > dAla6Pro9NEt mammalian GnRH (Ki 21.6±9.68 nM) > lamprey GnRH-I (Ki 118.0±23.6). Finally, the lamprey GnRH receptor was shown to undergo rapid ligand-dependant internalization, which was significantly diminished in the tail-less truncated form. We have shown from our current and our previous structural studies that this unique lamprey GnRH receptor shares several characteristics of both type I and type II GnRH receptors which suggests that this receptor has retained ancestral characteristics that can provide insight into the function and evolution of the vertebrate GnRH receptor family.

Reversibly Sampling Conformations and Binding Modes Using Molecular Darting

2020 ◽  
Author(s):  
Samuel C. Gill ◽  
David Mobley
Keyword(s):  
Monte Carlo ◽  
Ligand Binding ◽  
Binding Sites ◽  
Degrees Of Freedom ◽  
Dynamics Simulation ◽  
Hiv Integrase ◽  
Binding Modes ◽  
System A ◽  
Multiple Binding ◽  
Internal Degrees Of Freedom

<div>Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called Molecular Darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves.</div><div>We apply this technique to a simple dipeptide system, a ligand binding to T4 Lysozyme L99A, and ligand binding to HIV integrase in order to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal, and rotational/translational degrees of freedom in these systems.</div>

Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes Using Nonequilibrium Candidate Monte Carlo

2017 ◽  
Author(s):  
Samuel Gill ◽  
Nathan M. Lim ◽  
Patrick Grinaway ◽  
Ariën S. Rustenburg ◽  
Josh Fass ◽  
...  
Keyword(s):  
Monte Carlo ◽  
Ligand Binding ◽  
Binding Mode ◽  
Free Energy Calculations ◽  
Dynamics Simulation ◽  
Binding Modes ◽  
Enhanced Sampling ◽  
Recent Success ◽  
Multiple Binding ◽  
Proper Sampling

<div>Accurately predicting protein-ligand binding is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes.</div><div><br></div><div>In this technique the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.</div>

Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes Using Nonequilibrium Candidate Monte Carlo

2018 ◽  
Author(s):  
Samuel Gill ◽  
Nathan M. Lim ◽  
Patrick Grinaway ◽  
Ariën S. Rustenburg ◽  
Josh Fass ◽  
...  
Keyword(s):  
Monte Carlo ◽  
Ligand Binding ◽  
Binding Mode ◽  
Free Energy Calculations ◽  
Dynamics Simulation ◽  
Binding Modes ◽  
Enhanced Sampling ◽  
Recent Success ◽  
Multiple Binding ◽  
Proper Sampling

<div>Accurately predicting protein-ligand binding is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes.</div><div><br></div><div>In this technique the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.</div>

N‐glycosylation of the human neuropeptide QRFP receptor QRFPR is essential for ligand binding and receptor activation

2021 ◽  
Author(s):  
Weiwei Wang ◽  
Yanan Tian ◽  
Xiaoliu Shi ◽  
Qiang Ma ◽  
Yue Xu ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Receptor Activation
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