Active Receptor Conformation Research Articles

Importance of the second extracellular loop for melatonin MT1 receptor function and absence of melatonin binding in GPR50.

Recent crystal structures of GPCRs have emphasized the previously unappreciated role of the second extracellular (E2) loop in ligand binding and gating and receptor activation. Here, we have assessed the role of the E2 loop in the activation of the melatonin MT1 receptor and in the inactivation of the closely related orphan receptor GPR50. Chimeric MT1 -GPR50 receptors were generated and functionally analysed in terms of 2-[125 I]iodomelatonin binding, Gi /cAMP signalling and β-arrestin2 recruitment. We also used computational molecular dynamics (MD) simulations. MD simulations of 300ns revealed (i) the tight hairpin structure of the E2 loop of the MT1 receptor (ii) the most suitable features for melatonin binding in MT1 receptors and (iii) major predicted rearrangements upon MT1 receptor activation, stabilizing interaction networks between Phe179 or Gln181 in the E2 loop and transmembrane helixes 5 and 6. Functional assays confirmed these predictions, because reciprocal replacement of MT1 and GPR50 residues/domains led to the predicted loss- and gain-of-melatonin action of MT1 receptors and GPR50 respectively. Our work demonstrated the crucial role of the E2 loop for MT1 receptor and GPR50 function by proposing a model in which the E2 loop is important in stabilizing active MT1 receptor conformations and by showing how evolutionary processes appear to have selected for modifications in the E2 loop in order to make GPR50 unresponsive to melatonin. This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

Toward Understanding the Structural Basis of Partial Agonism at the Dopamine D3 Receptor.

Both dopamine D3 receptor (D3R) partial agonists and antagonists have been implicated as potential medications for substance use disorders. In contrast to antagonists, partial agonists may cause fewer side effects since they maintain some dopaminergic tone and may be less disruptive to normal neuronal functions. Here, we report three sets of 4-phenylpiperazine stereoisomers that differ considerably in efficacy: the (R)-enantiomers are antagonists/weak partial agonists, whereas the (S)-enantiomers are much more efficacious. To investigate the structural basis of partial agonism, we performed comparative microsecond-scale molecular dynamics simulations starting from the inactive state of D3R in complex with these enantiomers. Analysis of the simulation results reveals common structural rearrangements near the ligand binding site induced by the bound (S)-enantiomers, but not by the (R)-enantiomers, that are features of partially activated receptor conformations. These receptor models bound with partial agonists may be useful for structure-based design of compounds with tailored efficacy profiles.

Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor.

Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence probe at the end of trans-membrane helix 6 and reconstituted in phospholipid nanodiscs tethered to a microscope slide. Individual receptor molecules are then monitored over time by single-molecule total internal reflection fluorescence microscopy, revealing spontaneous transitions between inactive and active-like conformations. The assay provides information on the equilibrium distribution of inactive and active receptor conformations and the rate constants for conformational exchange. The experiments can be performed in the absence of ligands, revealing the spontaneous conformational transitions responsible for basal signaling activity, or in the presence of agonist or inverse agonist ligands, revealing how the ligands alter the dynamics of the receptor to either stimulate or repress signaling activity. The resulting mechanistic information is useful for the design of improved GPCR-targeting drugs. The single-molecule assay is described in the context of the β2 adrenergic receptor, but can be extended to a variety of GPCRs.

Fluorinated oxysterol analogues: Synthesis, molecular modelling and LXRβ activity

Liver X receptors (LXRs) are nuclear receptors that play central roles in the transcriptional control of lipid metabolism. The ability of LXRs to integrate metabolic and inflammation signalling makes them attractive targets for intervention in human metabolic diseases. Several oxidized metabolites of cholesterol (oxysterols) are endogenous LXR ligands, that modulate their transcriptional responses. While 25R-cholestenoic acid is an agonist of the LXRs, the synthetic analogue 27-norcholestenoic acid that lacks the 25-methyl is an inverse agonist. This change in the activity profile is triggered by a disruption of a key interaction between residues His435 and Trp457 that destabilizes the H11-H12 region of the receptor and favors the binding of corepressors. The introduction of fluorine atoms on the oxysterol side chain can favor both hydrophobic interactions as well as hydrogen bonds with the fluorine atoms and may thus induce changes in the receptor that may lead to changes in the activity profile. To evaluate these effects we have synthesized two fluorinated 27-nor-steroids, analogues of 27-norcholestenoic acid, the 25,25-difluoroacid and the corresponding 26-alcohol. The key step was a Reformatsky reaction on the C-24 cholenaldehyde, with ethyl bromodifluoroacetate under high intensity ultrasound (HIU) irradiation, followed by a Barton-McCombie type deoxygenation. Activity was evaluated in a luciferase reporter assay in the human HEK293T cells co-transfected with full length human LXRβ expression vector. The 25,25-difluoro-27-norcholestenoic acid was an inverse agonist and antagonist similar to its non-fluorinated analogue while its reduced derivative 25,25-difluoro-27-norcholest-5-ene-3β,26-diol was an agonist. Molecular dynamics simulation of the ligand-receptor complexes showed that the difluoroacid disrupted the His435-Trp457 interaction although the resulting conformational changes were different from those induced by the non-fluorinated analogue. In the case of the difluoroalcohol, the fluorine atoms actively participated in the interaction with several residues in the ligand binding pocket leading to a stabilization of the active receptor conformation.

Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies how “biased agonists” activate specific subsets of a given receptor’s signaling profile. However, stabilization of distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function via unique, previously unappreciated mechanisms. Here, utilizing a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor

G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site.

Activation of NMDA receptors and the mechanism of inhibition by ifenprodil.

SUMMARYThe physiology of N-Methyl-D-aspartate (NMDA) receptors in mammals is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino terminal domain (ATD). Recent crystal structures of GluN1/GluN2B NMDA receptors in the presence of agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here, we combine x-ray crystallography, single-particle electron cryomicroscopy, and electrophysiology to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open-conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel.

Structural Basis for Allosteric Coupling Between G Protein and the Agonist‐Binding Pocket in GPCRs

G protein‐coupled receptors (GPCRs) translate extracellular ligand binding into intracellular signaling responses via allosteric communication between two distant binding sites. For many GPCRs, allosteric communication between agonists and G proteins has been observed in the form of G protein‐mediated enhancement of agonist affinity. However, the mechanism by which G proteins allosterically regulate a receptor's orthosteric ligand binding site has remained unclear. Based on recent crystal structures of the β2‐adrenergic receptor (β2AR), we hypothesized that stabilization of an active receptor conformation by nucleotide‐free G protein leads to closing of the receptor's orthosteric site around a bound agonist, thus slowing agonist dissociation and enhancing affinity. Using purified receptors reconstituted into high‐density lipoprotein (rHDL) particles, we performed equilibrium and kinetic radioligand binding assays to characterize the allosteric effects of G protein and G protein‐mimetic nanobodies on the ligand‐binding properties of multiple GPCRs. We also investigated the contribution of individual residues within β2AR in mediating high‐affinity agonist binding. When β2AR was bound to nucleotide‐free Gs heterotrimer, we observed slowed association of the antagonist [3H]DHAP, which was reversible by GDP or GTPγS. A Gs‐mimetic nanobody (Nb80), like the nucleotide‐free G protein, slowed [3H]DHAP association but also slowed [3H]DHAP dissociation. However, this effect was not antagonist‐specific, as Nb80 also inhibited association of the full agonist [3H]formoterol, the partial agonist [3H]CGP‐12177, and the inverse agonist [3H]carvedilol. Mutation of tyrosine 308 to alanine in β2AR abolished the rate‐slowing effects of Nb80 on antagonist and agonist association. Together, these data suggest that transit into or out of the orthosteric site in the active β2AR conformation (stabilized by either Gs or Nb80) is restricted due to closing of the orthosteric site, and that Tyr308 of β2AR is an important residue for controlling access to the orthosteric site. Interestingly, the M2 muscarinic acetylcholine receptor and mu opioid receptor behaved similarly to β2AR when bound to nucleotide‐free G protein or G protein‐mimetic nanobodies, suggesting that G protein‐mediated closing of the orthosteric site may extend to GPCRs beyond β2AR and the amine receptor family.Support or Funding InformationThis work was supported by AHA 13PRE17110027 and NIH GM083118.

A Comprehensive Description of the Homo and Heterodimerization Mechanism of the Chemokine Receptors CCR5 and CXCR4

Signal transduction across cellular membranes is controlled by G protein-coupled receptors (GPCRs). In this family of transmembrane proteins the binding of extracellular physiological ligands stabilise the active conformation of receptors, leading to cellular response. It is widely accepted that members of the largest GPCR family self-assemble as dimers or higher-order but the functional consequences of the dimerization was described only for few receptors. The chemokines receptors are GPCRs implicated in many physiological processes, including the functioning and maintenance of the immune system. These receptors represent prime targets for therapeutic intervention in a wide spectrum of inflammatory and autoimmune diseases, heart diseases, and HIV. The CXCR4 and CCR5 receptors are two of the manly studied playing crucial roles in different pathologies. It was recently shown that inhibition of the CCR5-CXCR4 heterodimer formation reduces atherosclerosis in a hyperlipidemic mouse model. Furthermore the entry of HIV-1 virus into host cells requires CXCR4 and CCR5 in addition to its main receptor CD4. In this scenario the use of computational techniques able to describe complex biological processes such as protein dimerization acquires a great importance. Combining coarse-grained (CG) molecular dynamics and well-tempered metadynamics (MetaD) we are able to describe the mechanism of dimer formation, capturing multiple association and dissociation events allowing to compute a detailed free energy landscape of the process. CG-MetaD is an enhanced sampling method particularly suitable to describe processes with very slow rates of interconversion among the possible states of the system (i.e. protein dimerization). This approach provides an accurate and comprehensive description of the dimerization free energy landscape, thereby revealing critical motions and important structural-dynamical features (i.e. transient complex, role of lipids) involved in the homo and heterodimer formation of the CCR5 and CXCR4 receptors.

Comparative MD Simulations Indicate a Dual Role for Arg1323.50 in Dopamine-Dependent D2R Activation

Residue Arg3.50 belongs to the highly conserved DRY-motif of class A GPCRs, which is located at the bottom of TM3. On the one hand, Arg3.50 has been reported to help stabilize the inactive state of GPCRs, but on the other hand has also been shown to be crucial for stabilizing active receptor conformations and mediating receptor-G protein coupling. The combined results of these studies suggest that the exact function of Arg3.50 is likely to be receptor-dependent and must be characterized independently for every GPCR. Consequently, we now present comparative molecular-dynamics simulations that use our recently described inactive-state and Gα-bound active-state homology models of the dopamine D2 receptor (D2R), which are either bound to dopamine or ligand-free, performed to identify the function of Arg1323.50 in D2R. Our results are consistent with a dynamic model of D2R activation in which Arg1323.50 adopts a dual role, both by stabilizing the inactive-state receptor conformation and enhancing dopamine-dependent D2R-G protein coupling.

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Seven-transmembrane receptors (7TMRs) have evolved in prokaryotes and eukaryotes over hundreds of millions of years. Comparative structural analysis suggests that these receptors may share a remote evolutionary origin, despite their lack of sequence similarity. Here we used structure-based computations to compare 221 7TMRs from all domains of life. Unexpectedly, we discovered that these receptors contain spatially conserved networks of buried ionizable groups. In microbial 7TMRs these networks are used to pump ions across the cell membrane in response to light. In animal 7TMRs, which include light- and ligand-activated G protein-coupled receptors (GPCRs), homologous networks were found to be characteristic of activated receptor conformations. These networks are likely relevant to receptor function because they connect the ligand-binding pocket of the receptor to the nucleotide-binding pocket of the G protein. We propose that agonist and G protein binding facilitate the formation of these electrostatic networks and promote important structural rearrangements such as the displacement of transmembrane helix-6. We anticipate that robust classification of activated GPCR structures will aid the identification of ligands that target activated GPCR structural states.

Structural Basis for Allosteric Enhancement of Agonist Affinity by G Proteins

G protein‐coupled receptors (GPCRs) are allosteric machines that translate extracellular ligand binding into intracellular signaling events. Many GPCRs display higher affinity for agonists in the presence of G proteins, but the mechanism for this allosteric enhancement of agonist binding is unclear. Based on recent structural data, we hypothesized that in an active receptor conformation, the orthosteric binding site constricts around its ligand, thus slowing dissociation and enhancing affinity. Using purified receptors, we characterized the effects of G protein and G protein‐mimetic nanobodies on the ligand‐binding properties of multiple GPCRs. When β2‐adrenergic receptor (β2AR) was bound to nucleotide‐free Gs heterotrimer, we observed slowed association of the antagonist [3H]DHAP. A Gs‐mimetic nanobody (Nb80) slowed both [3H]DHAP association and dissociation. However, this effect was not antagonist‐specific, as Nb80 also inhibited association of radiolabeled agonists. Together, these data suggest that transit into or out of the orthosteric site in the active β2AR conformation (bound either to Gs or Nb80) is restricted due to closure of the orthosteric site. The M2 muscarinic acetylcholine receptor and mu opioid receptor behaved similarly to β2AR, suggesting that G protein‐mediated closing of the orthosteric site may extend to GPCRs beyond β2AR and the amine receptor family.

Structural Dynamics and Energetics Underlying Allosteric Inactivation of a GPCR: Insights Gained from Site-Directed Fluorescence Labeling (SDFL) Studies of the Cannabinoid Receptor CB1

Biased signaling, signaling through non G-protein linked pathways, occurs in some GPCRs through mechanisms that are still not clear. Here we investigated how an allosteric ligand for the cannabinoid receptor, CB1, could induce such behavior, with the aim of identifying structural changes in the receptor and gaining mechanistic insights underlying these phenomena. The ligand, Org 27569, induces very unusual properties in CB1 - it increases agonist binding to the receptor, yet simultaneously decreases G-protein signaling while increasing biased signaling through beta-arrestin mediated pathways. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique and separate site than traditional antagonists, and can act alone, without need for agonist co-binding. Similar analysis of constitutively activating and inactivating mutations in CB1, which shift the equilibrium between inactive (R) and active (R∗) receptor conformations, indicate that Org acts by modulating the amount of active R∗ species present. To determine the effect of Org on the structure of CB1, we carried out site-directed fluorescence labeling (SDFL) studies. The SDFL results clearly show that Org induces structural changes in CB1 that are different than those caused by either antagonists or antagonists. While Org27569 blocks agonist-induced movements at TM6 (consistent with its ability to block G-protein signaling), it also enhances agonist-induced movements at H8, providing a clue to its ability to induce biased signaling. Together, these results help map the conformational dynamics involved in allosteric modulation and biased signaling in CB1.

Conformational Dynamics of a G Protein-Coupled Receptor at the Single-Molecule Level

G protein-coupled receptors (GPCRs) are the integral membrane proteins that detect extracellular ligands and mediate signal transduction. Binding of ligands promotes transitions from inactive to active receptor conformations, which are recognized by intracellular effectors. Chemically distinct ligands may trigger different signaling responses by altering dynamics of the receptor. We developed a single-molecule fluorescence system to observe conformational switching of the β2-adrenergic receptor in real-time within a native-like membrane environment. The receptor was covalently labeled with a photostable and environmentally-responsive Cy3 fluorophore at the cytoplasmic end of either transmembrane (TM) helix VI or helix VII. Individual receptor molecules were incorporated in phospholipid nanodiscs, tethered to a microscope cover slip and visualized over time by total internal reflection fluorescence microscopy. We observed spontaneous transitions of TM helices VI and VII between inactive and active conformations, even in the absence of any ligands. Studies of ligands that span a comprehensive range of pharmacological efficacies showed that full agonists shorten the time spent in the inactive conformations and prolong the time in the active conformations, leading to an increased population of active species, while an inverse agonist prolonged the time spent in inactive conformations. These observations provide new insights into the mechanism of GPCR activation and the molecular basis for the variable pharmacological efficacies of different drug molecules.

Auto-inhibition at a ligand-gated ion channel: a cross-talk between orthosteric and allosteric sites.

A ligand is believed to produce either positive or negative responses, or to block both of them. However, an indole compound was found to promote both positive and negative effects at the 5-HT3 AB receptor, which displays a low level of spontaneous activity. The present study attempted to delineate the mechanisms underlying this phenomenon. The spontaneously active V291S 5-HT3 A receptor was used to explore the properties of 5-hydroxyindole (5-HoI) and 5-methoxyindole (5-MoI), structural analogues of 5-HT, either alone or in combination with orthosteric probes. Two types of efficacy switching were initiated by altering ligand structure and concentration. At lower concentrations, a subtle structural change at position 5 of the indole molecule resulted in opposite effects. 5-HoI apparently elicited partial allosteric inverse agonism, whereas 5-MoI induced allosteric agonism. Interestingly, at a higher concentration, these indoles produced distinct auto-inhibition, manifested as a switch from positive to negative effects. 5-HoI induced a transition from orthosteric agonism to allosteric inverse agonism, whereas 5-MoI produced a shift from allosteric agonism to orthosteric inverse agonism. The auto-inhibition appears to involve communication between orthosteric and allosteric sites of the active receptor conformation and/or between inactive and active conformations. An additive effect of orthosteric and allosteric inverse agonism and insensitivity of allosteric agonism to orthosteric antagonism were also demonstrated. Together, the results suggest that the moiety at position 5 of the indole structure is a critical determinant of a ligand's properties at the 5-HT3 A receptor, providing new insights into understanding ligand-receptor interactions.

A conformation-equilibrium model captures ligand-ligand interactions and ligand-biased signalling by G-protein coupled receptors.

G-protein coupled receptors (GPCRs) are a versatile, important class of cell-surface receptors. GPCRs occur in different conformations that exist in a dynamic ligand-sensitive equilibrium. These conformations vary in their affinities for intracellular signalling proteins and initiate signalling via different intracellular routes. The binding of extracellular ligands and allosteric ligand-ligand interactions shift conformation equilibria to cause biased signalling. Here, we present a mathematical model that describes the effects of ligands on the conformation equilibria of GPCRs. Our extended Monod-Wyman-Changeux model describes the receptor as shifting between active and inactive receptor conformations under the influence of extracellular ligands. For each receptor conformation, the intracellular domain of the receptor can attain alternative domain conformations that differ in their affinity for intracellular signalling proteins. At the extracellular domain, the model can accommodate different mechanisms for allosteric ligand-ligand interactions that induce shifts in receptor and domain conformation equilibria. We use the model to study ligand-biased signalling and how ligand affinity, ligand sensitivity and maximal signalling output depend on allosteric ligand-ligand interactions.

Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism.

In our previous work, using a fluorescent adenosine-A3 receptor (A3AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high-affinity labeling of the active receptor (R*) conformation. In the current study, we used a fluorescent A3AR antagonist (CA200645) to study the binding characteristics of antagonist-occupied inactive receptor (R) conformations in membrane microdomains of individual cells. FCS analysis of CA200645-occupied A3ARs revealed 2 species, τD2 and τD3, that diffused at 2.29 ± 0.35 and 0.09 ± 0.03 μm2/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A3AR exhibited a single diffusing species (0.105 μm2/s). The binding of CA200645 to τD3 was antagonized by nanomolar concentrations of the A3 antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A3AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting τD3 characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with τD2 diffusion. These data show that FCS analysis of ligand-occupied receptors provides a unique means of monitoring ligand A3AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interface.—Corriden, R., Kilpatrick, L. E., Kellam, B., Briddon, S. J., Hill, S. J. Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence for receptor dimerization and allosterism.

Structural insights into the molecular mechanism of vitamin D receptor activation by lithocholic acid involving a new mode of ligand recognition.

The vitamin D receptor (VDR), an endocrine nuclear receptor for 1α,25-dihydroxyvitamin D3, acts also as a bile acid sensor by binding lithocholic acid (LCA). The crystal structure of the zebrafish VDR ligand binding domain in complex with LCA and the SRC-2 coactivator peptide reveals the binding of two LCA molecules by VDR. One LCA binds to the canonical ligand-binding pocket, and the second one, which is not fully buried, is anchored to a site located on the VDR surface. Despite the low affinity of the alternative site, the binding of the second molecule promotes stabilization of the active receptor conformation. Biological activity assays, structural analysis, and molecular dynamics simulations indicate that the recognition of two ligand molecules is crucial for VDR agonism by LCA. The unique binding mode of LCA provides clues for the development of new chemical compounds that target alternative binding sites for therapeutic applications.

The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.

In vitro modification of substituted cysteines as tool to study receptor functionality and structure–activity relationships

Mutagenic investigations of expressed membrane proteins are routine, but the variety of modifications is limited by the twenty canonical amino acids. We describe an easy and effective cysteine substitution mutagenesis method to modify and investigate distinct amino acids in vitro. The approach combines the substituted cysteine accessibility method (SCAM) with a functional signal transduction readout system using different thiol-specific reagents. We applied this approach to the prolactin-releasing peptide receptor (PrRPR) to facilitate biochemical structure–activity relationship studies of eight crucial positions. Especially for D6.59C, the treatment with the positively charged methanethiosulfonate (MTS) ethylammonium led to an induced basal activity, whereas the coupling of the negatively charged MTS ethylsulfonate nearly reconstituted full activity, obviously by mimicking the wild-type charged side chain. At E5.26C, W5.28C, Y5.38C, and Q7.35C, accessibility was observed but hindered transfer into the active receptor conformation. Accordingly, the combination of SCAM and signaling assay is feasible and can be adapted to other G-protein-coupled receptors (GPCRs). This method circumvents the laborious way of inserting non-proteinogenic amino acids to investigate activity and ligand binding, with rising numbers of MTS reagents allowing selective side chain modification. This method pinpoints to residues being accessible but also presents potential molecular positions to investigate the global conformation.