Rhodopsin and the Others: A Historical Perspective on Structural Studies of G Protein-Coupled Receptors

The role of rhodopsin as a structural prototype for the study of the whole superfamily of G protein-coupled receptors (GPCRs) is reviewed in an historical perspective. Discovered at the end of the nineteenth century, fully sequenced since the early 1980s, and with direct three-dimensional information available since the 1990s, rhodopsin has served as a platform to gather indirect information on the structure of the other superfamily members. Recent breakthroughs have elicited the solution of the structures of additional receptors, namely the beta 1- and beta 2-adrenergic receptors and the A(2A) adenosine receptor, now providing an opportunity to gauge the accuracy of homology modeling and molecular docking techniques and to perfect the computational protocol. Notably, in coordination with the solution of the structure of the A(2A) adenosine receptor, the first "critical assessment of GPCR structural modeling and docking" has been organized, the results of which highlighted that the construction of accurate models, although challenging, is certainly achievable. The docking of the ligands and the scoring of the poses clearly emerged as the most difficult components. A further goal in the field is certainly to derive the structure of receptors in their signaling state, possibly in complex with agonists. These advances, coupled with the introduction of more sophisticated modeling algorithms and the increase in computer power, raise the expectation for a substantial boost of the robustness and accuracy of computer-aided drug discovery techniques in the coming years.

Evidence for a Direct and Functional Interaction between the Regulators of G Protein Signaling-2 and Phosphorylated C Terminus of Cholecystokinin-2 Receptor

Signaling of G protein-coupled receptors (GPCRs) is regulated by different mechanisms. One of these involves regulators of G protein signaling (RGS), which are diverse and multifunctional proteins that bind to active G alpha subunits of G proteins and act as GTPase-activating proteins. Little is known about the molecular mechanisms that govern the selective use of RGS proteins in living cells. We first demonstrated that CCK2R-mediated inositol phosphate production, known to be G(q-)dependent, is more sensitive to RGS2 than to RGS4 and is insensitive to RGS8. Both basal and agonist-stimulated activities of the CCK2R are regulated by RGS2. By combining biochemical functional, and in silico structural approaches, we demonstrate that a direct and functional interaction occurs between RGS2 and agonist-stimulated cholecystokinin receptor-2 (CCK2R) and identified the precise residues involved: phosphorylated Ser434 and Thr439 located in the C-terminal tail of CCK2R and Lys62, Lys63, and Gln67, located in the N-terminal domain of RGS2. These findings confirm previous reports that RGS proteins can interact with GPCRs to modulate their signaling and provide a molecular basis for RGS2 recognition by the CCK2R.

Ligand and structure-based methodologies for the prediction of the activity of G protein-coupled receptor ligands

Accurate in silico models for the quantitative prediction of the activity of G protein-coupled receptor (GPCR) ligands would greatly facilitate the process of drug discovery and development. Several methodologies have been developed based on the properties of the ligands, the direct study of the receptor-ligand interactions, or a combination of both approaches. Ligand-based three-dimensional quantitative structure-activity relationships (3D-QSAR) techniques, not requiring knowledge of the receptor structure, have been historically the first to be applied to the prediction of the activity of GPCR ligands. They are generally endowed with robustness and good ranking ability; however they are highly dependent on training sets. Structure-based techniques generally do not provide the level of accuracy necessary to yield meaningful rankings when applied to GPCR homology models. However, they are essentially independent from training sets and have a sufficient level of accuracy to allow an effective discrimination between binders and nonbinders, thus qualifying as viable lead discovery tools. The combination of ligand and structure-based methodologies in the form of receptor-based 3D-QSAR and ligand and structure-based consensus models results in robust and accurate quantitative predictions. The contribution of the structure-based component to these combined approaches is expected to become more substantial and effective in the future, as more sophisticated scoring functions are developed and more detailed structural information on GPCRs is gathered.

Mechanism of Activation of a G Protein-coupled Receptor, the Human Cholecystokinin-2 Receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.

Modeled structure of the whole regulator G-protein signaling-2

There is an increasing interest towards the mechanism by which regulators of G-protein signaling regulate signals of G-protein-coupled receptors. RGS2 is a regulator of Gq protein signaling (RGS), the N-terminal region of which is known to contain determinants for G protein-coupled receptor recognition, but its structure is still unknown. To understand the molecular basis for this recognition, the three-dimensional model of RGS2, including N-terminal region and RGS box, was modeled. For this, RGS4 box structure and data from circular dichroism study of RGS2 N-terminal region were used. Then, membrane-targeting activity of the RGS2 amphipathic helix contained in the N-terminal region was investigated. Furthermore, in cellulo study provided first evidence that an internal sequence within the N-terminal region of RGS2 is involved in RGS2 regulation of cholecystokinin receptor-2 signal. RGS2 modeled structure can now serve to study molecular recognition of RGS2 by signaling molecules. © 2006 Elsevier Inc. All rights reserved.

Genomic instability after targeted irradiation of human lymphocytes: Evidence for inter-individual differences under bystander conditions

Environmental (222)radon exposure is a human health concern, and many studies demonstrate that very low doses of high LET alpha-particle irradiation initiate deleterious genetic consequences in both radiated and non-irradiated bystander cells. One consequence, radiation-induced genomic instability (RIGI), is a hallmark of tumorigenesis and is often assessed by measuring delayed chromosomal aberrations We utilised a technique that facilitates transient immobilization of primary lymphocytes for targeted microbeam irradiation and have reported that environmentally relevant doses, e.g. a single He-3(2+) particle traversal to a single cell, are sufficient to Induce RIGI Herein we sought to determine differences in radiation response in lymphocytes isolated from five healthy male donors Primary lymphocytes were irradiated with a single particle per cell nucleus. We found evidence for inter-individual variation in radiation response (Rid, measured as delayed chromosome aberrations) Although this was not highly significant, it was possibly masked by high levels of intra-individual variation While there are many studies showing a link between genetic predisposition and RIGI, there are few studies linking genetic background with bystander effects in normal human lymphocytes In an attempt to investigate inter-individual variation in the induction of bystander effects, primary lymphocytes were irradiated with a single particle under conditions where fractions of the population were traversed We showed a marked genotype-dependent bystander response in one donor after exposure to 15% of the population The findings may also be regarded as a radiation-induced genotype-dependent bystander effect triggering an instability phenotype (C) 2010 Elsevier B.V. All rights reserved.

Identification of GSK625433: A novel clinical candidate for the treatment of hepatitis C.

Hepatitis C is an infection of the liver caused by a pos. single-stranded RNA virus (HCV) which affects 170 million people worldwide. It is responsible for 40-60% of all liver disease and is the major cause of liver transplants in the United States. The HCV NS5B gene encodes the viral RNA-dependent RNA polymerase which is essential for HCV replication. We have previously reported the identification of acylpyrrolidines as potent inhibitors of NS5B; however their activity is attenuated against genotype 1a. The design of improved broader-spectrum compds., capable of effective inhibition of both genotypes 1b and 1a is desirable. An understanding of the binding site and genotype sequence differences was utilized to design compds. with greatly enhanced genotype 1a and 1b potency. Our studies led to the identification of GSK625433, a potent, homochiral inhibitor of these HCV genotypes in both enzyme and sub-genomic replicon cell-based assays. GSK625433 has a good pharmacokinetic profile in pre-clin. animal species, enabling progression to clin. evaluation.

Variation in RTN3 and PPIL2 Genes Does not Influence Platelet Membrane β-Secretase Activity or Susceptibility to Alzheimer’s Disease in the Northern Irish Population

beta-site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of beta-amyloid peptides (A beta), which are proposed to drive the pathological changes found in Alzheimer's disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (beta-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulates BACE1 expression. The present study investigated whether there was any association between genetic variation in RTN3 and PPIL2, and either risk for AD, or levels of platelet beta-secretase activity, in a large Northern Irish case-control sample. Four hundred and sixty-nine patients with a diagnosis of probable AD (NINCDS-ADRDA criteria) and 347 control individuals (MMSE > 28/30) were genotyped. SNPs in both genes were selected by downloading genotype data from the International HapMap Project (Phase II) and tags selected using multimarker approach in Haploview, where r (2) > 0.8 and LOD > 3.0. Non-synonymous SNPs of interest were also included. Genotyping was performed by Sequenom iPLEX and TaqMan technologies. Alleles, genotypes and multi-marker haplotypes were tested for association with AD, and platelet beta-secretase activities were measured for a subset of individuals (n = 231). Eight SNPs in RTN3 and 7 in PPIL2 were genotyped. We found no significant associations between allele, genotype or haplotype frequencies and risk of AD. Further, there was no effect of genotype on platelet membrane beta-secretase activity. We conclude that common or potentially functional genetic variation in these BACE1 interacting proteins does not affect platelet membrane beta-secretase activity or contribute to risk of AD in this population.