Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine.

Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.     

Molecular determinants of odorant receptor function in insects

The olfactory system of Drosophila melanogaster provides a powerful model to study molecular and cellular mechanisms underlying function of a sensory system. In the 1970s Siddiqi and colleagues pioneered the application of genetics to olfactory research and isolated several mutant Drosophila with odorant-specific defects in olfactory behaviour, suggesting that odorants are detected differentially by the olfactory system. Since then basic principles of olfactory system function and development have emerged using Drosophila as a model. Nearly four decades later we can add computational methods to further our understanding of how specific odorants are detected by receptors. Using a comparative approach we identify two categories of short amino acid sequence motifs: ones that are conserved family-wide predominantly in the C-terminal half of most receptors, and ones that are present in receptors that detect a specific odorant, 4-methylphenol, found predominantly in the N-terminal half. The odorant-specific sequence motifs are predictors of phenol detection in Anopheles gambiae and other insects, suggesting they are likely to participate in odorant binding. Conversely, the family-wide motifs are expected to participate in shared functions across all receptors and a mutation in the most conserved motif leads to a reduction in odor response. These findings lay a foundation for investigating functional domains within odorant receptors that can lead to a molecular understanding of odor detection.     

Molecular determinants of glycine receptor subunit assembly.

The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous alpha and beta subunits. Single expression of alpha subunits generates functional homo-oligomeric GlyRs, whereas the beta subunit requires a co-expressed alpha subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (alpha(3)beta(2)). Here, we identified eight amino acid residues within the N-terminal region of the alpha1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the alpha1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR alpha and beta subunits.     

Molecular determinants of FGF-21 activity-synergy and cross-talk with PPARgamma signaling

Fibroblast growth factor (FGF)-21 is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. The precise mechanisms whereby FGF-21 regulates metabolism remain to be determined. Here we describe the early signaling events triggered by FGF-21 treatment of 3T3-L1 adipocytes and reveal a functional interplay between FGF-21 and peroxisome proliferator-activated receptor gamma (PPARgamma) pathways that leads to a marked stimulation of glucose transport. While the early actions of FGF-21 on 3T3-L1 adipocytes involve rapid accumulation of intracellular calcium and phosphorylation of Akt, GSK-3, p70(S6K), SHP-2, MEK1/2, and Stat3, continuous treatment for 72 h induces an increase in PPARgamma protein expression. Moreover, chronic activation of the PPARgamma pathway in 3T3-L1 adipocytes with the PPARgamma agonist and anti-diabetic agent, rosiglitazone (BRL 49653), enhances FGF-21 action to induce tyrosine phosphorylation of FGF receptor-2. Strikingly, treatment of cells with FGF-21 and rosiglitazone in combination leads to a pronounced increase in expression of the GLUT1 glucose transporter and a marked synergy in stimulation of glucose transport. Together these results reveal a novel synergy between two regulators of glucose homeostasis, FGF-21 and PPARgamma, and further define FGF-21 mechanism of action.     

Molecular determinants of ginkgolide binding in the glycine receptor pore

Ginkgolides are potent blockers of the glycine receptor Cl- channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on alpha1, alpha2, alpha1beta and alpha2beta GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the beta subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by beta subunit expression. However, ginkgolide A sensitivity was increased in the alpha2beta GlyR relative to the alpha2 GlyR but not in the alpha1beta GlyR relative to the alpha1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2' and 6' pore-lining positions. The increased ginkgolide A sensitivity of the alpha2beta GlyR was transferred to the alpha1beta GlyR by the G2'A (alpha1 to alpha2 subunit) substitution. In addition, the alpha1 subunit T6'F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2' residues of both alpha1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position.     

Molecular determinants of P2Y2 nucleotide receptor function

In the mammalian nervous system, P2 nucleotide receptors mediate neurotransmission, release of proinflammatory cytokines, and reactive astrogliosis. Extracellular nucleotides activate multiple P2 receptors in neurons and glial cells, including G protein-coupled P2Y receptors and P2X receptors, which are ligand-gated ion channels. In glial cells, the P2Y2 receptor subtype, distinguished by its ability to be equipotently activated by ATP and UTP, is coupled to pro-inflammatory signaling pathways. In situ hybridization studies with rodent brain slices indicate that P2Y2 receptors are expressed primarily in the hippocampus and cerebellum. Astrocytes express several P2 receptor subtypes, including P2Y2 receptors whose activation stimulates cell proliferation and migration. P2Y2 receptors, via an RGD (Arg-Gly-Asp) motif in their first extracellular loop, bind to alphavbeta3/beta5 integrins, whereupon P2Y2 receptor activation stimulates integrin signaling pathways that regulate cytoskeletal reorganization and cell motility. The C-terminus of the P2Y2 receptor contains two Src-homology-3 (SH3)-binding domains that upon receptor activation, promote association with Src and transactivation of growth factor receptors. Together, our results indicate that P2Y2 receptors complex with both integrins and growth factor receptors to activate multiple signaling pathways. Thus, P2Y2 receptors present novel targets to control reactive astrogliosis in neurodegenerative diseases.     

Molecular determinants of the alpha-2D adrenergic receptor subtype

The alpha-2 adrenergic receptor in the bovine pineal gland and the rodent homologues of the human alpha-2-C10 receptor express alpha-2D subtype pharmacological characteristics. The alpha-2 adrenergic receptor in the chicken pineal expresses characteristics similar to the alpha-2A subtype found in human and pig. The rodent receptors (alpha-2D) contain a serine residue at position 201 whereas the human and porcine receptors (alpha-2A) have a cysteine at this position. Our results indicate that the bovine pineal receptor has a serine at position 201, supporting the alpha-2D classification. However, the chicken pineal receptor also contains a serine at position 201 suggesting that other amino acids may be responsible for the differences in pharmacological characteristics.     

Molecular determinants of the granulocyte-macrophage colony-stimulating factor receptor complex assembly

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is composed of two chains that belong to the superfamily of cytokine receptors typified by the growth hormone receptor. A common structural element found in cytokine receptors is a module of two fibronectin-like domains, each characterized by seven beta-strands denoted A-G and A'-G', respectively. The alpha-chain (GMRalpha) confers low affinity GM-CSF binding (K(d) = 1-5 nM), whereas the beta-chain (beta(c)) does not bind GM-CSF by itself but confers high affinity binding when associated with alpha (K(d) = 40-100 pM). In the present study, we define the molecular determinants required for ligand recognition and for stabilization of the complex through a convergence of several approaches, including the construction of chimeric receptors, the molecular dynamics of our three-dimensional model of the GM.GMR complex, and site-directed mutagenesis. The functional importance of individual residues was then investigated through ligand binding studies at equilibrium and through determination of the kinetic constants of the GM.GMR complex. Critical to this tripartite complex is the establishment of four noncovalent bonds, three that determine the nature of the ligand recognition process involving residues Arg(280) and Tyr(226) of the alpha-chain and residue Tyr(365) of the beta-chain, since mutations of either one of these residues resulted in a significant decrease in the association rate. Finally, residue Tyr(365) of beta(c) serves a dual function in that it cooperates with another residue of beta(c), Tyr(421) to stabilize the complex since mutation of Tyr(365) and Tyr(421) result in a drastic increase in the dissociation rate (Koff). Interestingly, these four residues are located at the B'-C' and F'-G' loops of GMRalpha and of beta(c), thus establishing a functional symmetry within an apparently asymmetrical heterodimeric structure.     

Molecular and spatial constraints on NB-LRR receptor signaling

In plants, a large polymorphic family of intracellular NB-LRR receptors lies at the heart of robust resistance to diverse pathogens and mechanisms by which these versatile molecular switches operate in effector-triggered immunity are beginning to emerge. We outline recent advances in our understanding of NB-LRR receptor signaling leading to disease resistance. Themes covered are (i) NB-LRR molecular constraining forces and their intimate relationship with receptor activation in different parts of the cell, (ii) cooperativity between NB-LRR proteins and the formation of higher order NB-LRR signaling complexes, and (iii) the spatial separation of different resistance branches within cells. Finally, we examine evidence for dynamic signaling across cell compartments in coordinating diverse immune outputs.     

Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling

To identify structural determinants of ligand binding in the glucagon receptor, eight receptor chimeras and additional receptor point mutants were prepared and studied. Amino acid residues 103-117 and 126-137 in the extracellular N-terminal tail and residues 206-219 and 220-231 in the first extracellular loop of the glucagon receptor were replaced with the corresponding segments of the glucagon-like peptide-1 receptor or the secretin receptor. Specific segments of both the N-terminal tail and the first extracellular loop of the glucagon receptor are required for hormone binding. The 206-219 segment of the first loop appears to be important for both glucagon binding and receptor activation. Functional studies with a synthetic chimeric peptide consisting of the N-terminal 14 residues of glucagon and the C-terminal 17 residues of glucagon-like peptide 1 suggest that hormone binding specificity may involve this segment of the first loop. The binding selectivity may arise in part from aspartic acid residues in this segment. Mutation of R-202 located at the junction between the second transmembrane helix and the first loop resulted in a mutant receptor that failed to bind glucagon or signal. We conclude that high-affinity glucagon binding requires multiple contacts with residues in the N-terminal tail and first extracellular loop domain of the glucagon receptor, with hormone specificity arising primarily from the amino acid 206-219 segment. The data suggest a model whereby glucagon first interacts with the N-terminal domain of the receptor followed by more specific interactions between the N-terminal half of the peptide and the first extracellular loop of the receptor, leading to activation.     

Molecular determinants that convert hormone sensitive lipase into gibberellin receptor

Gibberellins (GAs) are tetracyclic, diterpenoid plant hormones, essential for many developmental processes in higher plants. Plants perceive GA through a nuclear-localized GA receptor, GA INSENSITIVE DWARF1 (GID1). From sequence similarity, it is suggested that GID1 evolved from a hormone-sensitive lipase (HSL), and recent x-ray crystallography of the GA-GID1 complex has given insights into how GID1 recognizes GA. Analyses of the GA signaling pathway in several plant species further suggest that the GID1-mediated GA signaling pathway emerged in the vascular plant lineage and since then regulation of GA recognition specificity seems to have been fine tuned to strictly regulate the on-off GA signal.     

Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated by low nanomolar ivermectin concentrations. The crystal structure of the Caenorhabditis elegans α GluClR complexed with ivermectin has recently been published. Here, we probed ivermectin sensitivity determinants on the α1 GlyR using site-directed mutagenesis and electrophysiology. Based on a mutagenesis screen of transmembrane residues, we identified Ala288 and Pro230 as crucial sensitivity determinants. A comparison of the actions of selamectin and ivermectin suggested the benzofuran C05-OH was required for high efficacy. When taken together with docking simulations, these results supported a GlyR ivermectin binding orientation similar to that seen in the GluClR crystal structure. However, whereas the crystal structure shows that ivermectin interacts with the α GluClR via H-bonds with Leu218, Ser260, and Thr285 (α GluClR numbering), our data indicate that H-bonds with residues homologous to Ser260 and Thr285 are not important for high ivermectin sensitivity or direct agonist efficacy in A288G α1 GlyRs or three other GluClRs. Our data also suggest that van der Waals interactions between the ivermectin disaccharide and GlyR M2-M3 loop residues are unimportant for high ivermectin sensitivity. Thus, although our results corroborate the ivermectin binding orientation as revealed by the crystal structure, they demonstrate that some of the binding interactions revealed by this structure do not pertain to other highly ivermectin-sensitive Cys-loop receptors.     

Molecular determinants of Pb2+ interaction with NMDA receptor channels

Lead (Pb2+) is a potent neurotoxin that acts as a non-competitive, voltage-independent antagonist of the NMDA receptor (NR) channel. Pb2+ action partially overlaps with that of zinc (Zn2+), but precise coincidence with Zn2+ binding site is debated. We investigated the site of Pb2+ interaction in NR channels expressed in Xenopus laevis oocytes from the clones zeta1, epsilon1 or epsilon2 and mutated epsilon1 or epsilon2 forms. For each epsilon subunit we chose two mutations that have been identified as 'strong mutations' for Zn2+ binding and examined the effect of Pb2+ on channels that contained those mutations. In epsilon1-containing channels, mutations D102A and H128A caused a decrease of Pb2+ inhibition with a 10-fold (D102A) and four-fold (H128A) shift of IC50. In epsilon2-containing channels, the most effective mutation in removing Pb2+ inhibition was H127A, with a five-fold increase of IC50, while D101A was virtually ineffective. Other mutations, D104A, T103A, and T233A, were less effective. The double mutation D101AH127A, while reducing Zn2+ inhibition by nearly nine-fold, caused a minor (less than two-fold) shift in Pb2+ IC50. Competition experiments showed that increasing doses of Zn2+ reduced the apparent affinity for Pb2+ in epsilon1-containing receptors, but not in epsilon2-containing receptors. In addition the effect of Pb2+ on epsilon2-containing channels was additive with that of ifenprodil, with no competition for the site. Although none of the mutations that we have tested abolished the block by Pb2+, our results indicate that the action of this toxic metal on NR channels is more dependent on the receptor composition than previously thought, because Zn2+ is able to displace Pb2+ from its binding site in epsilon1-containing channels, but not in epsilon2-containing channels.     

Molecular determinants of ligand binding to the human melanocortin-4 receptor

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.     

Molecular determinants of β-carboline inhibition of the glycine receptor

β-Carbolines are potent modulators of GABA type A receptors and they have recently been shown to inhibit glycine receptors in a subunit-specific manner. The present study screened four structurally similar β-carbolines, 1,2,3,4-tetrahydronorharmane, norharmane, harmane and 6-methoxyharmalan, at recombinantly expressed α1, α1β, α2 and α3 glycine receptors with the aims of identifying structural elements of both the receptor and the compounds that are important for binding and subunit specificity. The four compounds exhibited only weak subunit specificity, rendering them unsuitable as pharmacological probes. Because they displayed competitive antagonist activity, we investigated the roles of known glycine binding residues in coordinating the four compounds. The structural similarity of the compounds, coupled with the differential effects of C-loop mutations (T204A, F207Y) on compound potency, implied direct interactions between variable β-carboline groups and mutated residues. Mutant cycle analysis employing harmane and norharmane revealed a strong pairwise interaction between the harmane methyl group and the C-loop in the region T204 and F207. These results which define the orientation of the bound β-carbolines were supported by molecular docking simulations. The information may also be relevant to understanding the mechanism β-carboline of binding to GABA type A receptors where they are potent pharmacological probes.     

Biaryl amide glucagon receptor antagonists

Biaryl amides derived from a reported series of ureas 1 were evaluated and found to be potent human glucagon receptor antagonists. The benzofuran analogue 6i was administered in Sprague-Dawley rats and blocked the effects of an exogenous glucagon challenge.     

Molecular determinants and feedback circuits regulating type 2 CRH receptor signal integration

In most target tissues, the adenylyl cyclase/cAMP/PKA, the extracellular signal regulated kinase and the protein kinase B/Akt are the main pathways employed by the type 2 corticotropin-releasing hormone receptor to mediate the biological actions of urocortins (Ucns) and CRH. To decipher the molecular determinants of CRH-R2 signaling, we studied the signaling pathways in HEK293 cells overexpressing recombinant human CRH-R2β receptors. Use of specific kinase inhibitors showed that the CRH-R2β cognate agonist, Ucn 2, activated extracellular signal regulated kinase in a phosphoinositide 3-kinase and cyclic adenosine monophosphate/PKA-dependent manner with contribution from Epac activation. Ucn 2 also induced PKA-dependent association between AKAP250 and CRH-R2β that appeared to be necessary for extracellular signal regulated kinase activation. PKB/Akt activation was also mediated via pertussis toxin-sensitive G-proteins and PI3-K activation but did not require cAMP/PKA, Epac or protein kinase C for optimal activation. Potential feedback mechanisms that target the CRH-R2β itself and modulate receptor trafficking and endocytosis were also investigated. Indeed, our results suggested that inhibition of either PKA or extracellular signal regulated kinase pathway accelerates CRH-R2β endocytosis. Furthermore, Ucn 2-activated extracellular signal regulated kinase appeared to target β-arrestin1 and modulate, through phosphorylation at Ser412, β-arrestin1 translocation to the plasma membrane and CRH-R2β internalization kinetics. Loss of this “negative feedback” mechanism through inhibition of the extracellular signal regulated kinase activity resulted in significant attenuation of Ucn 2-induced cAMP response, whereas Akt phosphorylation was not affected by altered receptor endocytosis. These findings reveal a complex interplay between the signaling molecules that allow “fine-tuning” of CRH-R2β functional responses and regulate signal integration.