Effects of pH and temperature on photoaffinity labeling of Family B G protein-coupled receptors

The efficiency of covalent labeling of a receptor by a photolabile analogue of its natural ligand is dependent on the spatial approximation of the probe and its target. Systematic application of intrinsic photoaffinity labeling to the secretin receptor, a prototypic Family B G protein-coupled receptor, demonstrated reduced efficiency of labeling for amino-terminal and mid-region sites of labeling relative to carboxyl-terminal sites. Reduction of pH from 7.4 to 5.5 and reduction of temperature from 25 °C to 4 °C improved the efficiency of covalent labeling of the receptor with these probes. This correlated with sites of labeling at the interface between the receptor amino terminus and the receptor core, a region containing histidine residues that have their ionization affected in this pH range. Application to the calcitonin receptor, another Family B G protein-coupled receptor, yielded analogous results. These results support the consistent mode of docking peptide ligands to this group of receptors.     

Photoaffinity scanning in the mapping of the peptide receptor interface of class II G protein-coupled receptors.

The family of G protein-coupled receptors constitutes about 50% of the therapeutic drug targets used in clinical medicine today, although the mechanisms of ligand binding, activation and signal transduction for G protein-coupled receptors are not yet well defined. This review discusses ongoing research using the photoaffinity scanning method to map the bimolecular interface between class II G protein-coupled receptors and their ligands. Furthermore the available computer model of class II peptide ligand docking into the receptor, based on the positional constraints imposed by the photoaffinity scanning analyses, will be discussed briefly. The ultimate goal of these efforts is to understand the molecular basis of receptor binding and therefore to generate a template for rational drug design.     

Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM₁ and AM₂ receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM₁ and AM₂ receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM₁ and AM₂ receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.     

Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization compared with wild-type receptors. This distinct phenotype of the fusion proteins can not be mimicked by coexpressing wild-type receptor with (beta)arr2. However, when the wild-type receptor was coexpressed with both (beta)arr2 and G protein-coupled receptor kinase 5, a phenotype similar to that observed for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with (beta)arr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level.     

The Activation Mechanism of Glycoprotein Hormone Receptors with Implications in the Cause and Therapy of Endocrine Diseases

Glycoprotein hormones (GPHs) are the main regulators of the pituitary-thyroid and pituitary-gonadal axes. Selective interaction between GPHs and their cognate G protein-coupled receptors ensure specificity in GPH signaling. The mechanisms of how these hormones activate glycoprotein hormone receptors (GPHRs) or how mutations and autoantibodies can alter receptor function were unclear. Based on the hypothesis that GPHRs contain an internal agonist, we systematically screened peptide libraries derived from the ectodomain for agonistic activity on the receptors. We show that a peptide (p10) derived from a conserved sequence in the C-terminal part of the extracellular N terminus can activate all GPHRs in vitro and in GPHR-expressing tissues. Inactivating mutations in this conserved region or in p10 can inhibit activation of the thyroid-stimulating hormone receptor by autoantibodies. Our data suggest an activation mechanism where, upon extracellular ligand binding, this intramolecular agonist isomerizes and induces structural changes in the 7-transmembrane helix domain, triggering G protein activation. This mechanism can explain the pathophysiology of activating autoantibodies and several mutations causing endocrine dysfunctions such as Graves disease and hypo- and hyperthyroidism. Our findings highlight an evolutionarily conserved activation mechanism of GPHRs and will further promote the development of specific ligands useful to treat Graves disease and other dysfunctions of GPHRs.     

Structural Perspective on Ancient Neuropeptide Y-like System reveals Hallmark Features for Peptide Recognition and Receptor Activation

The neuropeptide Y (NPY) family is a peptide-activated G protein-coupled receptor system conserved across all bilaterians, and is involved in food intake, learning, and behavior. We hypothesized that comparing the NPY system in evolutionarily ancient organisms can reveal structural determinants of peptide recognition and receptor activation conserved in evolution. To test this hypothesis, we investigated the homologous FLP/NPR system of the protostome C. elegans. For three prototypic peptide–receptor complexes representing different ligand types, we integrate extensive functional data into structural models of the receptors. Common features include acidic patches in the extracellular loops (ECLs) of the receptors that cooperatively ‘draw’ the peptide into the binding pocket, which was functionally validated in vivo. A structurally conserved glutamate in the ECL2 anchors the peptides by a conserved salt bridge to the arginine of the RFamide motif. Beyond this conserved interaction, peptide binding show variability enabled by receptor-specific interactions. The family-conserved residue Q^3.32 is a key player for peptide binding and receptor activation. Altered interaction patterns at Q^3.32 may drastically increase the efficacy to activate the receptor.     

Electrostatically-driven fast association and perdeuteration allow detection of transferred cross-relaxation for G protein-coupled receptor ligands with equilibrium dissociation constants in the high-to-low nanomolar range

The mechanism of signal transduction mediated by G protein-coupled receptors is a subject of intense research in pharmacological and structural biology. Ligand association to the receptor constitutes a critical event in the activation process. Solution-state NMR can be amenable to high-resolution structure determination of agonist molecules in their receptor-bound state by detecting dipolar interactions in a transferred mode, even with equilibrium dissociation constants below the micromolar range. This is possible in the case of an inherent ultra-fast diffusive association of charged ligands onto a highly charged extracellular surface, and by slowing down the ¹H–¹H cross-relaxation by perdeuterating the receptor. Here, we demonstrate this for two fatty acid molecules in interaction with the leukotriene BLT2 receptor, for which both ligands display a submicromolar affinity.     

Inhibition of chemoattractant N-formyl peptide receptor trafficking by active arrestins

Recent studies have highlighted the emergence of a class of G protein-coupled receptors that are internalized in an arrestin-independent manner. In addition to demonstrating that the N-formyl peptide receptor belongs in this family, we have recently shown that recycling of the receptor requires the presence of arrestins. To further elucidate mechanisms of arrestin-dependent regulation of G protein-coupled receptor processing, we examined the effects of altering the receptor-arrestin complex on ternary complex formation and cellular trafficking of the N-formyl peptide receptor by studying two active arrestin-2 mutants (truncated arrestin-2 [1-382], and arrestin-2 I386A, V387A, F388A). Complexes between the N-formyl peptide receptor and active arrestins exhibited higher affinity in vitro than the complex between the N-formyl peptide receptor and wild-type arrestin and furthermore were observed in vivo by colocalization studies using confocal microscopy. To assess the effects of these altered interactions on receptor trafficking, we demonstrated that active, but not wild-type, arrestin expression retards N-formyl peptide receptor internalization. Furthermore, expression of arrestin-2 I386A/V387A/F388A but not arrestin-2 [1-382] inhibited recycling of the N-formyl peptide receptor, reflecting an expanded role for arrestins in G protein-coupled receptor processing and trafficking. Whereas the extent of N-formyl peptide receptor phosphorylation had no effect on the inhibition of internalization, N-formyl peptide receptor recycling was restored when the receptor was only partially phosphorylated. These results indicate not only that a functional interaction between receptor and arrestin is required for recycling of certain G protein-coupled receptors, such as the N-formyl peptide receptor, but that the pattern of receptor phosphorylation further regulates this process.     

Signal transduction profiling using label-free biosensors

Knowledge of the way in which ligands modulate cellular responses via membrane-associated receptors is of central importance to drug discovery and elucidation of signal transduction pathways. Biophysical label-free methods can be used to characterize ligand and drug candidate interactions with neurotransmitters, cytokine receptors, tyrosine kinase receptors, ligand- and voltage-gated ion channels, G protein-coupled receptors (GPCRs), and antibody receptors. Ligand or drug candidate screening typically involves selecting ligands or subsets of a compound library for analysis, transfecting a cell line overexpressing the target receptor, then monitoring one or two downstream reporters of receptor activation such as Ca²⁺, cAMP, inositol phosphate, etc. Inevitably, this process leads to a data set predicated by these selections. In contrast, label-free screening techniques allow a holistic, pathway-independent screening strategy to provide a functional or phenotypic readout of receptor activation. Detection techniques that measure changes in cell conductance, viscoelastic properties, refractive index, and other optical parameters that are modulated as a consequence of receptor activation are reviewed.     

Investigation of Growth Hormone Releasing Hormone Receptor Structure and Activity Using Yeast Expression Technologies

Abstract

Growth hormone releasing hormone (GHRH) is the positive regulator of growth hormone synthesis and secretion in the anterior pituitary. The peptide confers activity by binding to a seven transmembrane domain G protein-coupled receptor. Signal transduction proceeds through subsequent Gas stimulation of adenylyl cyclase. To investigate ligand/receptor and receptor/G protein associations, the human GHRH receptor was expressed in a modified S. cerevisiae strain which allows for facile measurement of receptor activity by cell prototrophy mediated by a reporter gene coupled to the yeast pheromone response pathway. GHRH-dependent signal activation in this system required the substitution of yeast Gα protein with proteins containing C-terminal regions of Gαs. A D60G variant (analogous to the little mouse mutation) of the receptor failed to respond to agonist. In parallel studies, GHRH₂₉ and the N-terminal extracellular region of the receptor were expressed as Gal4 fusion proteins in a 2-hybrid assay. A specific interaction between these proteins was readily observed. The D60G mutation was engineered into the receptor fusion protein. This protein failed to interact with the ligand fusion, confirming the specificity of the association between unmodified proteins. These two yeast expression technologies should prove invaluable in additional structure/activity analyses of this ligand/receptor pair as well as other peptide ligands and receptors.     

Development of a Membrane-anchored Chemerin Receptor Agonist as a Novel Modulator of Allergic Airway Inflammation and Neuropathic Pain

The chemerin receptor (CMKLR1) is a G protein-coupled receptor found on select immune, epithelial, and dorsal root ganglion/spinal cord neuronal cells. CMKLR1 is primarily coupled to the inhibitory G protein, Gα_i, and has been shown to modulate the resolution of inflammation and neuropathic pain. CMKLR1 is activated by both lipid and peptide agonists, resolvin E1 and chemerin, respectively. Notably, these ligands have short half-lives. To expedite the development of long acting, stable chemerin analogs as candidate therapeutics, we used membrane-tethered ligand technology. Membrane-tethered ligands are recombinant proteins comprised of an extracellular peptide ligand, a linker sequence, and an anchoring transmembrane domain. Using this technology, we established that a 9-amino acid-tethered chemerin fragment (amino acids 149–157) activates both mouse and human CMKLR1 with efficacy exceeding that of the full-length peptide (amino acids 21–157). To enable in vivo delivery of a corresponding soluble membrane anchored ligand, we generated lipidated analogs of the 9-amino acid fragment. Pharmacological assessment revealed high potency and wash resistance (an index of membrane anchoring). When tested in vivo, a chemerin SMAL decreased allergic airway inflammation and attenuated neuropathic pain in mice. This compound provides a prototype membrane-anchored peptide for the treatment of inflammatory disease. A parallel approach may be applied to developing therapeutics targeting other peptide hormone G protein-coupled receptors.     

前列腺素核受体系统信号转导及基因表达调控

脂肪酸和前列腺素等脂代谢的产物不仅通过膜受体起作用,也可以通过与核受体结合来调节基因表达.前列腺素I₂(PGI₂)既可以与G蛋白偶联的细胞表面IP受体起作用,也可以通过核受体过氧化物酶体增殖因子活化受体（PPARs）发挥生物学功能.前列腺素E₂(PGE₂)的受体（EPs）不仅仅在质膜上有,最近在核膜上也发现了EPs受体.前列腺素核受体介导的信号转导途径与膜受体介导的信号途径不同,对于基因转录的调控机制也不同.     

Juxtamembranous Region of the Amino Terminus of the Family B G Protein-coupled Calcitonin Receptor Plays a Critical Role in Small-molecule Agonist Action

The Family B G protein-coupled calcitonin receptor is an important drug target. The aim of this work was to elucidate the molecular mechanism of action of small-molecule agonist ligands acting at this receptor, comparing it with the action mechanism of the receptor''s natural peptide ligand. cAMP responses to four non-peptidyl ligands and calcitonin were studied in COS-1 cells expressing wild-type and chimeric calcitonin-secretin receptors. All compounds were full agonists at the calcitonin receptor with no activity at the secretin receptor. Only chimeric constructs including the calcitonin receptor amino terminus exhibited responses to any of these ligands. We progressively truncated this domain and tested constructs for cAMP responses. Although calcitonin was able to activate the calcitonin receptor fully with the first 58 residues absent, its potency was 3 orders of magnitude lower than that at the wild-type receptor. After truncation of 114 residues, there was no response to calcitonin. In contrast, small-molecule ligands were fully active at receptors having up to 149 amino-terminal residues absent. Those compounds finally became inactive after truncation of 153 residues. Deletion and/or alanine replacement of the region of the calcitonin receptor between residues 150 and 153 resulted in marked reduction in cAMP responses to these compounds, with some compound-specific differences observed, supporting a critical role for this region. Binding studies further supported distinct sites of action of small molecules relative to that of calcitonin. These findings focus attention on the potential importance of the juxtamembranous region of the amino terminus of the Family B calcitonin receptor for agonist drug action.Calcitonin (CT),³ a 32-amino acid peptide secreted by the thyroid gland in response to elevations in blood calcium levels, acts on bone and kidney to maintain calcium homeostasis (1, 2). CT is widely used therapeutically for the treatment of bone-related disorders such as osteoporosis, hypercalcemia of malignancy, and Paget disease (1, 2). This peptide must be administered parenterally, whereas small-molecule agonist ligands that can be administered orally have substantial clinical advantage, particularly for long-term treatment.CT acts on the CT receptor, a Family B G protein-coupled receptor (GPCR). Members of this family include receptors for secretin, vasoactive intestinal polypeptide, parathyroid hormone, corticotrophin-releasing factor, glucagon, glucagon-like peptide 1, CT, and CT gene-related peptide (3), with each having a long extracellular amino-terminal domain containing six conserved cysteine residues that form three intradomain disulfide bonds. This region has been shown to play a critical role in natural peptide ligand binding and receptor activation (4–11).Development of small-molecule ligands for Family B GPCRs represents an area of great interest. For the CT receptor, several such ligands have been developed (12–14). However, the mechanisms of their actions at their receptors remain unclear. Most recently, synthesis of a series of pyrazolopyridine CT receptor ligands described as partial agonists has been reported (15). Here, we explore the structural basis for their action at the CT receptor. Using CT-secretin receptor chimeric analysis, the amino terminus of the CT receptor was identified as a critical region for the action of these small-molecule ligands but with determinants that are distinct from those interacting with the natural ligand, CT. Truncation, deletion, and alanine replacement mutations of the CT receptor identified that three specific receptor residues, Tyr¹⁵⁰, Leu¹⁵¹, and Ile¹⁵³, within the juxtamembranous region of the amino-terminal domain of the CT receptor, play important roles in the actions of these small-molecule agonists. This study represents the first identification of this region as being critical for the action of small-molecule drugs acting at Family B GPCRs.     

The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.     

Evaluation of novel derivatisation reagents for the analysis of oxysterols

Oxysterols are oxidised forms of cholesterol that are intermediates in the synthesis of bile acids and steroid hormones. They are also ligands to nuclear and G protein-coupled receptors. Analysis of oxysterols in biological systems is challenging due to their low abundance coupled with their lack of a strong chromophore and poor ionisation characteristics in mass spectrometry (MS). We have previously used enzyme-assisted derivatisation for sterol analysis (EADSA) to identify and quantitate oxysterols in biological samples. This technique relies on tagging sterols with the Girard P reagent to introduce a charged quaternary ammonium group. Here, we have compared several modified Girard-like reagents and show that the permanent charge is vital for efficient MSⁿ fragmentation. However, we find that the reagent can be extended to include sites for potential stable isotope labels without a loss of performance.     

DOCKING LIGANDS TO VASOPRESSIN AND OXYTOCIN RECEPTORS VIA GENETIC ALGORITHM

ABSTRACT

The aim of the study was to computer-dock selected ligands to neurophyseal receptors in order to identify amino acid residues responsible for ligand–receptor interactions. To this aim, reliable oxytocin receptor (OTR) and arginine-vasopressin receptor (V1aR/V2R) models were built. The OTR-selective agonist [Thr⁴,Gly⁷]OT, the OTR-selective cyclohexapeptide antagonist L-366,948 and OT itself were docked via genetic algorithm to OTR, V1aR, and V2R and relaxed using a constrained simulated annealing protocol. For the analysis of receptor/ligand interactions a subset of initial conformations was chosen using energetic and steric criteria. All three ligands seem to prefer similar modes of binding to the receptors, manifested by repetitive residues of the receptors which directly interact with the ligands. Taking into account that many aspects of mechanisms of G protein-coupled receptor (GPCR) action are still unsolved, the results obtained with the docking simulations may propose future experimental research, especially in site-directed mutagenesis analysis and searching for key amino acid residues responsible for drug activities.     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp¹, Leu²]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp¹, Leu²]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK_1/2 phosphorylation and, to a lesser extent, IP₁ production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.