Ligand-mimicking Receptor Variant Discloses Binding and Activation Mode of Prolactin-releasing Peptide

The prolactin-releasing peptide receptor and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the ligand binding mode of peptide G-protein-coupled receptors (GPCRs). By receptor mutagenesis, we identified the conserved aspartate in the upper transmembrane helix 6 (Asp(6.59)) of the receptor as the first position that directly interacts with arginine 19 of the ligand (Arg(19)). Replacement of Asp(6.59) with Arg(19) of PrRP20 led to D6.59R, which turned out to be a constitutively active receptor mutant (CAM). This suggests that the mutated residue at the top of transmembrane helix 6 mimics Arg(19) by interacting with additional binding partners in the receptor. Next, we generated an initial comparative model of this CAM because no ligand docking was required, and we selected the next set of receptor mutants to find the engaged partners of the binding pocket. In an iterative process, we identified two acidic residues and two hydrophobic residues that form the peptide ligand binding pocket. As all residues are localized on top or in the upper part of the transmembrane domains, we clearly can show that the extracellular surface of the receptor is sufficient for full signal transduction for prolactin-releasing peptide, rather than a deep, membrane-embedded binding pocket. This contributes to the knowledge of the binding of peptide ligands to GPCRs and might facilitate the development of GPCR ligands, but it also provides new targeting of CAMs involved in hereditary diseases.     

The function of G-protein coupled receptors and membrane cholesterol: specific or general interaction?

Cholesterol is an essential component of eukaryotic membranes and plays a crucial role in membrane organization, dynamics and function. The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes and constitute ~1–2% of the human genome. GPCRs have emerged as major targets for the development of novel drug candidates in all clinical areas due to their involvement in the generation of multitude of cellular responses. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. This effect could either be due to specific molecular interaction between cholesterol and GPCR, or due to alterations in the membrane physical properties induced by cholesterol. Alternatively, membrane cholesterol could modulate receptor function by occupying the ‘nonannular’ sites around the receptor. In this review, we have highlighted the nature of cholesterol dependence of GPCR function taking a few known examples.     

Theme and variations on kinetics of GPCR activation/deactivation

G protein-coupled receptors (GPCRs) initiate intracellular signaling pathways in response to physiologically and medically important extracellular ligands such as peptide and large glycoprotein hormones, neurotransmitters, sensory stimuli (odorant and taste molecules, light), calcium, l-amino acids, and are the target of many clinical drugs. The conversion of these extracellular stimuli into intracellular signals involves sequential and reversible reactions that initially take place at the plasma membrane. These reactions are mediated not only by dynamic interactions between ligands, receptors and heterotrimeric G proteins, but also by conformational changes associated with the activation/deactivation process of each protein. This review discusses the kinetic characteristics and rate-limiting reactions engaged in signal propagation that are involved in systems as diverse as neurotransmitter and hormonal signaling, and that have been recorded in live cells by Förster resonance energy transfer (FRET) approaches.     

Are specific nonannular cholesterol binding sites present in G-protein coupled receptors?

The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major drug targets in all clinical areas. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. Interestingly, recently reported crystal structures of GPCRs have shown structural evidence of cholesterol binding sites. Two possible mechanisms have been previously suggested by which membrane cholesterol could influence the structure and function of GPCRs (i) through a direct/specific interaction with GPCRs, which could induce a conformational change in the receptor, or (ii) through an indirect way by altering the membrane physical properties in which the receptor is embedded or due to a combination of both. We discuss here a novel mechanism by which membrane cholesterol could affect structure and function of GPCRs and propose that cholesterol binding sites in GPCRs could represent ‘nonannular’ binding sites. Interestingly, previous work from our laboratory has demonstrated that membrane cholesterol is required for the function of the serotonin_1A receptor, which could be due to specific interaction of the receptor with cholesterol. Based on these results, we envisage that there could be specific/nonannular cholesterol binding site(s) in the serotonin_1A receptor. We have analyzed putative cholesterol binding sites from protein databases in the serotonin_1A receptor, a representative GPCR, for which we have previously demonstrated specific requirement of membrane cholesterol for receptor function. Our analysis shows that cholesterol binding sites are inherent characteristic features of serotonin_1A receptors and are conserved over evolution. Progress in deciphering molecular details of the nature of GPCR-cholesterol interaction in the membrane would lead to better insight into our overall understanding of GPCR function in health and disease, thereby enhancing our ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.     

Structural determinants of natriuretic peptide receptor specificity and degeneracy

Cardiovascular homeostasis and blood pressure regulation are reliant, in part, on interactions between natriuretic peptide (NP) hormones and natriuretic peptide receptors (NPR). The C-type NPR (NPR-C) is responsible for clearance of NP hormones from the circulation, and displays a cross-reactivity for all NP hormones (ANP, BNP, and CNP), in contrast to other NPRs, which are more restricted in their specificity. In order to elucidate the structural determinants for the binding specificity and cross-reactivity of NPR-C with NP hormones, we have determined the crystal structures of the complexes of NPR-C with atrial natriuretic peptide (ANP), and with brain natriuretic peptide (BNP). A structural comparison of these complexes, with the previous structure of the NPR-C/CNP complex, reveals that NPR-C uses a conformationally inflexible surface to bind three different, highly flexible, NP ligands. The complex structures support a mechanism of rigid promiscuity rather than conformational plasticity by the receptor. While ANP and BNP appear to adopt similar receptor-bound conformations, the CNP structure diverges, yet shares sets of common receptor contacts with the other ligands. The degenerate versus selective hormone recognition properties of different NPRs appears to derive largely from two cavities on the receptor surfaces, pocket I and pocket II, that serve as anchoring sites for hormone side-chains and modulate receptor selectivity.     

PREDICT modeling and in-silico screening for G-protein coupled receptors

G-protein coupled receptors (GPCRs) are a major group of drug targets for which only one x-ray structure is known (the nondrugable rhodopsin), limiting the application of structure-based drug discovery to GPCRs. In this paper we present the details of PREDICT, a new algorithmic approach for modeling the 3D structure of GPCRs without relying on homology to rhodopsin. PREDICT, which focuses on the transmembrane domain of GPCRs, starts from the primary sequence of the receptor, simultaneously optimizing multiple 'decoy' conformations of the protein in order to find its most stable structure, culminating in a virtual receptor-ligand complex. In this paper we present a comprehensive analysis of three PREDICT models for the dopamine D2, neurokinin NK1, and neuropeptide Y Y1 receptors. A shorter discussion of the CCR3 receptor model is also included. All models were found to be in good agreement with a large body of experimental data. The quality of the PREDICT models, at least for drug discovery purposes, was evaluated by their successful utilization in in-silico screening. Virtual screening using all three PREDICT models yielded enrichment factors 9-fold to 44-fold better than random screening. Namely, the PREDICT models can be used to identify active small-molecule ligands embedded in large compound libraries with an efficiency comparable to that obtained using crystal structures for non-GPCR targets.     

Peptide design and structural characterization of a GPCR loop mimetic

G protein-coupled receptors (GPCRs) control fundamental aspects of human physiology and behaviors. Knowledge of their structures, especially for the loop regions, is limited and has principally been obtained from homology models, mutagenesis data, low resolution structural studies, and high resolution studies of peptide models of receptor segments. We developed an alternate methodology for structurally characterizing GPCR loops, using the human S1P(4) first extracellular loop (E1) as a model system. This methodology uses computational peptide designs based on transmembrane domain (TM) model structures in combination with CD and NMR spectroscopy. The characterized peptides contain segments that mimic the self-assembling extracellular ends of TM 2 and TM 3 separated by E1, including residues R3.28(121) and E3.29(122) that are required for sphingosine 1-phosphate (S1P) binding and receptor activation in the S1P(4) receptor. The S1P(4) loop mimetic peptide interacted specifically with an S1P headgroup analog, O-phosphoethanolamine (PEA), as evidenced by PEA-induced perturbation of disulfide cross-linked coiled-coil first extracellular loop mimetic (CCE1a) (1)H and (15)N backbone amide chemical shifts. CCE1a was capable of weakly binding PEA near biologically relevant residues R29 and E30, which correspond to R3.28 and E3.29 in the full-length S1P(4) receptor, confirming that it has adopted a biologically relevant conformation. We propose that the combination of coiled-coil TM replacement and conformational stabilization with an interhelical disulfide bond is a general design strategy that promotes native-like structure for loops derived from GPCRs.     

Unlocking the secrets of the gatekeeper: Methods for stabilizing and crystallizing GPCRs

G-protein coupled receptors (GPCRs) are integral membrane cell surface receptors with key roles in mediating the cellular responses to a wide range of biologically relevant molecules including hormones, neurotransmitters and importantly the majority of currently available drugs. The first high-resolution, X-ray crystallographic structure of a GPCR, that of rhodopsin, was obtained in 2000. It took a further seven years for the next structure, that of the β₂ adrenergic receptor. Remarkably, at the time of writing, there have been an astonishing 18 further independent high-resolution GPCR structures published in the last five years (overall total of 68 structures in different conformations or bound to different ligands). Of particular note is the recent structure of the β₂ adrenergic receptor in complex with its cognate heterotrimeric G-protein revealing for the first time molecular details of the interaction between a GPCR and the complete G-protein. Together these structures have provided unprecedented detail into the mechanism of action of these incredibly important proteins. This review describes several key methodological advances that have made such extraordinarily fast progress possible.     

Structure of the Human Angiotensin II Type 1 (AT1) Receptor Bound to Angiotensin II from Multiple Chemoselective Photoprobe Contacts Reveals a Unique Peptide Binding Mode

Breakthroughs in G protein-coupled receptor structure determination based on crystallography have been mainly obtained from receptors occupied in their transmembrane domain core by low molecular weight ligands, and we have only recently begun to elucidate how the extracellular surface of G protein-coupled receptors (GPCRs) allows for the binding of larger peptide molecules. In the present study, we used a unique chemoselective photoaffinity labeling strategy, the methionine proximity assay, to directly identify at physiological conditions a total of 38 discrete ligand/receptor contact residues that form the extracellular peptide-binding site of an activated GPCR, the angiotensin II type 1 receptor. This experimental data set was used in homology modeling to guide the positioning of the angiotensin II (AngII) peptide within several GPCR crystal structure templates. We found that the CXC chemokine receptor type 4 accommodated the results better than the other templates evaluated; ligand/receptor contact residues were spatially grouped into defined interaction clusters with AngII. In the resulting receptor structure, a β-hairpin fold in extracellular loop 2 in conjunction with two extracellular disulfide bridges appeared to open and shape the entrance of the ligand-binding site. The bound AngII adopted a somewhat vertical binding mode, allowing concomitant contacts across the extracellular surface and deep within the transmembrane domain core of the receptor. We propose that such a dualistic nature of GPCR interaction could be well suited for diffusible linear peptide ligands and a common feature of other peptidergic class A GPCRs.     

Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data.

Most neuropeptide and protein hormone receptors belong to the large superfamily of G-protein-coupled receptors (GPCRs). These cell membrane proteins steer many important processes such as development, reproduction, homeostasis and behaviour when activated by their corresponding ligands. The first insect genome, that of the fruitfly Drosophila melanogaster, was sequenced in 2000, and about 200 GPCRs have been annnotated in this model insect. About 50 of these receptors were predicted to have neuropeptides or protein hormones as their ligands. Since 2000, the cDNAs of most of these candidate receptors have been cloned and for many receptors the endogenous ligand has been identified. In this review, we will give an update about the current knowledge of all Drosophila neuropeptide and protein hormone receptors, and discuss their phylogenetic relationships.     

The expanding repertoire of receptor activity modifying protein (RAMP) function

Receptor activity modifying proteins (RAMPs) associate with G-protein-coupled receptors (GPCRs) at the plasma membrane and together bind a variety of peptide ligands, serving as a communication interface between the extracellular and intracellular environments. The collection of RAMP-interacting GPCRs continues to expand and now consists of GPCRs from families A, B and C, suggesting that RAMP activity is extremely prevalent. RAMP association with GPCRs can regulate GPCR function by altering ligand binding, receptor trafficking and desensitization, and downstream signaling pathways. Here, we elaborate on these RAMP-dependent mechanisms of GPCR regulation, which provide opportunities for pharmacological intervention.     

M871—A Novel Peptide Antagonist Selectively Recognizing the Galanin Receptor Type 2

Galanin and its three receptors have been linked to a wide variety of physiological processes and are distributed in both the central and peripheral nervous systems. Further knowledge of the properties of galanin-activated signaling systems can best be obtained by the availability of peptide and non-peptide ligands that are selective for the different receptor subtypes. The current study describes binding and signaling data for the chimeric peptide, galanin-(2–13)-Glu-His-(Pro)₃-(Ala-Leu)₂-Ala-amide (M871). This compound binds to the galanin receptor type 2 with more than 30-fold higher affinity than to the galanin receptor type 1 and exhibits antagonist actions at galanin receptor type 2, blocking increased release of inositol phosphate produced by galanin in CHO cells. This peptide opens new possibilities for the study of galanin receptor physiology.     

Structural Features of β2 Adrenergic Receptor: Crystal Structures and Beyond

The beta2-adrenergic receptor (β2AR) family, which is the largest family of cell surface receptors in humans. Extra attention has been focused on the human GPCRs because they have been studied as important protein targets for pharmaceutical drug development. In fact, approximately 40% of marketed drugs directly work on GPCRs. GPCRs respond to various extracellular stimuli, such as sensory signals, neurotransmitters, chemokines, and hormones, to induce structural changes at the cytoplasmic surface, activating downstream signaling pathways, primarily through interactions with heterotrimeric G proteins or through G-protein independent pathways, such as arrestin. Most GPCRs, except for rhodhopsin, which contains covalently linked 11 cis-retinal, bind to diffusible ligands, having various conformational states between inactive and active structures. The first human GPCR structure was determined using an inverse agonist bound β2AR in 2007 and since then, more than 20 distinct GPCR structures have been solved. However, most GPCR structures were solved as inactive forms, and an agonist bound fully active structure is still hard to obtain. In a structural point of view, β2AR is relatively well studied since its fully active structure as a complex with G protein as well as several inactive structures are available. The structural comparison of inactive and active states gives an important clue in understanding the activation mechanism of β2AR. In this review, structural features of inactive and active states of β2AR, the interaction of β2AR with heterotrimeric G protein, and the comparison with β1AR will be discussed.     

Evolutionary Sequence Modeling for Discovery of Peptide Hormones

There are currently a large number of “orphan” G-protein-coupled receptors (GPCRs) whose endogenous ligands (peptide hormones) are unknown. Identification of these peptide hormones is a difficult and important problem. We describe a computational framework that models spatial structure along the genomic sequence simultaneously with the temporal evolutionary path structure across species and show how such models can be used to discover new functional molecules, in particular peptide hormones, via cross-genomic sequence comparisons. The computational framework incorporates a priori high-level knowledge of structural and evolutionary constraints into a hierarchical grammar of evolutionary probabilistic models. This computational method was used for identifying novel prohormones and the processed peptide sites by producing sequence alignments across many species at the functional-element level. Experimental results with an initial implementation of the algorithm were used to identify potential prohormones by comparing the human and non-human proteins in the Swiss-Prot database of known annotated proteins. In this proof of concept, we identified 45 out of 54 prohormones with only 44 false positives. The comparison of known and hypothetical human and mouse proteins resulted in the identification of a novel putative prohormone with at least four potential neuropeptides. Finally, in order to validate the computational methodology, we present the basic molecular biological characterization of the novel putative peptide hormone, including its identification and regional localization in the brain. This species comparison, HMM-based computational approach succeeded in identifying a previously undiscovered neuropeptide from whole genome protein sequences. This novel putative peptide hormone is found in discreet brain regions as well as other organs. The success of this approach will have a great impact on our understanding of GPCRs and associated pathways and help to identify new targets for drug development.     

Thematic Minireview Series: New Directions in G Protein-coupled Receptor Pharmacology

Over the past half-century, The Journal of Biological Chemistry has been the venue for many landmark publications on the topic of G protein-coupled receptors (GPCRs, also known as seven-transmembrane receptors). The GPCR superfamily in humans is composed of about 800 members, and is the target of about one-third of all pharmaceuticals. Most of these drugs target a very small subset of GPCRs, and do so by mimicking or competing with endogenous hormones and neurotransmitters. This thematic minireview series examines some emerging trends in GPCR drug discovery. The first article describes efforts to systematically interrogate the human “GPCR-ome,” including more than 150 uncharacterized “orphan” receptors. The second article describes recent efforts to target alternative receptor binding sites with drugs that act as allosteric modulators of orthosteric ligands. The third article describes how the recent expansion of GPCR structures is providing new opportunities for computer-guided drug discovery. Collectively, these three articles provide a roadmap for the most important emerging trends in GPCR pharmacology.     

A crystal clear solution for determining G-protein-coupled receptor structures

G-protein-coupled receptors (GPCRs) are medically important membrane proteins that are targeted by over 30% of small molecule drugs. At the time of writing, 15 unique GPCR structures have been determined, with 77 structures deposited in the PDB database, which offers new opportunities for drug development and for understanding the molecular mechanisms of GPCR activation. Many different factors have contributed to this success, but if there is one single factor that can be singled out as the foundation for producing well-diffracting GPCR crystals, it is the stabilisation of the detergent-solubilised receptor-ligand complex. This review will focus predominantly on one of the successful strategies for the stabilisation of GPCRs, namely the thermostabilisation of GPCRs using systematic mutagenesis coupled with thermostability assays. Structures of thermostabilised GPCRs bound to a wide variety of ligands have been determined, which has led to an understanding of ligand specificity; why some ligands act as agonists as opposed to partial or inverse agonists; and the structural basis for receptor activation.     

Site-directed mutagenesis and PBAN activation of the Helicoverpa zea PBAN-receptor

Pheromone biosynthesis-activating neuropeptide (PBAN) and pyrokinins belong to a family of insect peptide hormones that have a common FXPRLamide C-terminal ending. The G-protein-coupled receptors (GPCRs) for this peptide family were first identified from a moth and Drosophila with sequence similarity to neuromedin U receptors from vertebrates. We have characterized the PBAN-receptor (PBAN-R or PR) active binding domains using chimeric GPCRs and proposed that extracellular loop 3 is critical for ligand selection. Here, we characterized the 3rd extracellular domain of PBAN-R through site-directed point mutations. Results are discussed in context of the structural features required for receptor activation using receptor activation experiments and in silico computational modeling. This research will help in characterizing these receptors towards a goal of finding agonists and/or antagonists for PBAN/pyrokinin receptors.     

Homology modeling of dopamine d(2) and d(3) receptors: molecular dynamics refinement and docking evaluation

Dopamine (DA) receptors, a class of G-protein coupled receptors (GPCRs), have been targeted for drug development for the treatment of neurological, psychiatric and ocular disorders. The lack of structural information about GPCRs and their ligand complexes has prompted the development of homology models of these proteins aimed at structure-based drug design. Crystal structure of human dopamine D(3) (hD(3)) receptor has been recently solved. Based on the hD(3) receptor crystal structure we generated dopamine D(2) and D(3) receptor models and refined them with molecular dynamics (MD) protocol. Refined structures, obtained from the MD simulations in membrane environment, were subsequently used in molecular docking studies in order to investigate potential sites of interaction. The structure of hD(3) and hD(2L) receptors was differentiated by means of MD simulations and D(3) selective ligands were discriminated, in terms of binding energy, by docking calculation. Robust correlation of computed and experimental K(i) was obtained for hD(3) and hD(2L) receptor ligands. In conclusion, the present computational approach seems suitable to build and refine structure models of homologous dopamine receptors that may be of value for structure-based drug discovery of selective dopaminergic ligands.