Functional characterization and evolution of PTH/PTHrP receptors: insights from the chicken

ABSTRACT: BACKGROUND: The parathyroid hormone (PTH)-family consists of a group of structurally related factors that regulate calcium and bone homeostasis and are also involved in development of organs such as the heart, mammary gland and immune system. They interact with specific members of family 2 B1 G-protein coupled receptors (GPCRs), which have been characterised in teleosts and mammals. Two PTH/PTHrP receptors, PTH1R and PTH2R exist in mammals and in teleost fish a further receptor PTH3R has also been identified. Recently in chicken, PTHfamily members involved in calcium transport were characterized and specific PTHRs are suggested to exist although they have not yet been isolated or functionally characterized. The aim of this study is to further explore the evolution and function of the vertebrate PTH/PTHrP system through the isolation, phylogenetic analysis and functional characterization of the chicken receptors. RESULTS: Two PTHRs were isolated in chicken and sequence comparison and phylogenetic analysis indicate that the chicken receptors correspond to PTH1R and PTH3R, which emerged prior to the teleost/tetrapod divergence since they are present in cartilaginous fish. The vertebrate PTH2R receptor and its ligand TIP39 have been lost from bird genomes. Chicken PTH1R and PTH3R have a divergent and widespread tissue expression and are also evident in very early embryonic stages of development. Receptor stimulation studies using HEK293 cells stably expressing the chicken PTH1R and PTH3R and monitoring cAMP production revealed they are activated by chicken 1-34 N-terminal PTH-family peptides in a dose dependent manner. PTH-L and PTHrP were the most effective peptides in activating PTH1R (EC50 = 7.7 nM and EC50 = 22.7 nM, respectively). In contrast, PTH-L (100 nM) produced a small cAMP accumulation on activation of PTH3R but PTHrP and PTH (EC50 = 2.5 nM and EC50 = 22.1 nM, respectively) readily activated the receptor. PTHrP also stimulated intracellular Ca2+ accumulation on activation of PTH1R but not PTH3R. CONCLUSION: Two PTHR homologues of the vertebrate PTH1R and PTH3R were isolated and functionally characterized in chicken. Their distinct pattern of expression during embryo development and in adult tissues, together with their ligand preference, suggests that they have acquired specific functions, which have contributed to their maintenance in the genome. PTH2R and its activating ligand, TIP39, are absent from bird genomes. Nonetheless identification of putative PTH2R and TIP39 in the genome of an ancient agnathan, lamprey, suggests the PTH/PTHrP ligand and receptor family was already present in an early basal paraphyletic group of vertebrates and during the vertebrate radiation diverged via gene/genome duplication and deletion events. Knowledge of the role PTH/PTHrP system in early vertebrates will help to establish evolution of function.     

Optical and electrical recording of neural activity evoked by graded contrast visual stimulus

Background  

Brain activity has been investigated by several methods with different principles, notably optical ones. Each method may offer information on distinct physiological or pathological aspects of brain function. The ideal instrument to measure brain activity should include complementary techniques and integrate the resultant information. As a "low cost" approach towards this objective, we combined the well-grounded electroencephalography technique with the newer near infrared spectroscopy methods to investigate human visual function.     

Heterotrimeric G proteins demonstrate differential sensitivity to β-arrestin dependent desensitization

G15 is a heterotrimeric G protein of the Gq/11 family. In this study, we describe its exceptional poor sensitivity to the general regulatory mechanism of G protein-coupled receptor (GPCR) desensitization.Enhancing β2 adrenergic receptor desensitization by arrestin overexpression, did not affect signalling to G15. Similarly, increased levels of arrestin did not affect G15 signalling triggered by the activation of V2 vasopressin and δ opioid receptors.Furthermore, co-immunoprecipitation experiments showed that G15 α subunit (as opposed to Gαq and Gαs) is recruited to a V2 vasopressin receptor mutant that is constitutively desensitized by β-arrestin. Interestingly, co-expression of Gα15 partially rescued cell surface localization and signalling capabilities of the same mutant receptor and reduced β2 adrenergic receptor internalization.Taken together, these findings provide evidence for a novel mechanism whereby GPCR desensitization can be bypassed and G15 can support sustained signalling in cells chronically exposed to hormones or neurotransmitters.     

The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor

Nucleotides and cysteinyl-leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G-protein-coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi-coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist-response profile, as determined by [(35)S]GTPgammaS binding, was different from that of already known CysLT and P2Y receptors, with EC(50) values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.     

DESIGN: computerized optimization of experimental design for estimating Kd and Bmax in ligand binding experiments. I. Homologous and heterologous binding to one or two classes of sites

We have developed a versatile computer program for optimization of ligand binding experiments (e.g., radioreceptor assay system for hormones, drugs, etc.). This optimization algorithm is based on an overall measure of precision of the parameter estimates (D-optimality). The program DESIGN uses an exact mathematical model of the equilibrium ligand binding system with up to two ligands binding to any number of classes of binding sites. The program produces a minimal list of the optimal ligand concentrations for use in the binding experiment. This potentially reduces the time and cost necessary to perform a binding experiment. The program allows comparison of any proposed experimental design with the D-optimal design or with assay protocols in current use. The level of nonspecific binding is regarded as an unknown parameter of the system, along with the affinity constant (Kd) and binding capacity (Bmax). Selected parameters can be fixed at constant values and thereby excluded from the optimization algorithm. Emphasis may be placed on improving the precision of a single parameter or on improving the precision of all the parameters simultaneously. We present optimal designs for several of the more commonly used assay protocols (saturation binding with a single labeled ligand, competition or displacement curve, one or two classes of binding sites), and evaluate the robustness of these designs to changes in parameter values of the underlying models. We also derive the theoretical D-optimal design for the saturation binding experiment with a homogeneous receptor class.     

Comparison of the effect of lorglumide on pancreatic growth stimulated by camostate in rat and hamster

We conducted a 14 day experiment in which we administered camostate (a trypsin inhibitor) and cholecystokinin alone or in combination with lorglumide, a cholecystokinin receptor antagonist, to both rats and hamsters. Plasma cholecystokinin levels were 21.7 +/- 3.2 pM and 19.6 +/- 2.5 pM with camostate, 16.3 +/- 2.4 pM and 14.8 +/- 2.2 pM with exogenous cholecystokinin, and 3.7 +/- 0.4 pM and 4.2 +/- 1.0 pM in control experiments in rats and hamsters, respectively. Both cholecystokinin and camostate were found to promote pancreatic growth in rats (18 +/- 4 and 111 +/- 7%, respectively) and hamsters (76 +/- 18 and 61 +/- 12%, respectively). Although lorglumide caused a decrease of this effect of camostate in both rats (78 +/- 5%) and hamsters (25 +/- 10%), it only became significant in rats. We therefore conclude that there are important interspecies differences in the role cholecystokinin plays in mediating the trophic effects of trypsin inhibitors on the pancreas.     

Map position of Aldox (Aldehyde-oxidase) and Adh (Alcohol dehydrogenase) loci on autosome II of Musca domestica L.

The Aldox and Adh structural loci of Musca domestica L. belong to autosome II. They code for the enzymes aldehyde oxidase and alcohol dehydrogenase. Both these enzymes have allelic variants with specific electrophoretic mobility, which, on cellogel, are seen as single bands. The Aldox and Adh loci encompass a large map interval, which includes the morphological markers ar, cm, and car. The recombination frequencies between these five loci indicate the alignment Aldox-ar-cm-car-Adh.

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Localisation chez Musca domestica L. des gènes Aldox et Adh codant les enzymes ald\;ehyde oxydase (AO) et alcool déshydrogénase (ADH)

Résumé Chez la mouche, les enzymes AO et ADH, observées par électrophorèse sur cellogel, présentent toutes 2 des formes alléliques exprimées par des bandes ayant une mobilité anodique propre.Les gènes structuraux Aldox et Adh, codant ces formes, sont liés entre eux et situés sur le chromosome 2. Ils se recombinent avec une fréquence élevée d'interchange; ils sont donc séparés par un intervalle important dans lequel sont compris les caractères visibles ar, cm, car. La fréquence des recombinaisons entre caractères visibles et gènes enzymatiques indique l'ordre suivant sur ce segment du deuxième chromosome de la mouche: Aldox, ar, cm, car, Adh.

     

Towards a reporter system to identify regulators of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis

The plant signaling hormones salicylic acid (SA) and jasmonic acid (JA) are regulators of inducible defenses that are activated upon pathogen or insect attack. Cross-talk between SA- and JA-dependent signaling pathways allows a plant to finely tune its response to the attacker encountered. In Arabidopsis, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway. SA-mediated suppression of the JA-responsive PDF1.2 promoter was exploited for setting up a genetic screen aiming at the isolation of signal transduction mutants that are impaired in this cross-talk mechanism. The PDF1.2 promoter was fused to the herbicide resistance gene BAR to allow for life/death screening of a population of mutagenized transgenic plants. Non-mutant plants should survive herbicide treatment when methyl jasmonate (MeJA) is applied, but suppression of the JA response by SA should be lethal in combination with the herbicide. Conversely, crucial SA/JA cross-talk mutants should survive the combination treatment. SA effectively suppressed the expression of the PDF1.2::BAR transgene. However, suppression of the BAR gene did not result in suppression of herbicide resistance. Hence, a screening method based on quantitative differences in the expression of a reporter gene may be better suited to identify SA/JA cross-talk mutants. Here, we demonstrate that the PDF1.2::GUS reporter will be excellently suited in this respect.Key words: plant defense, salicylic acid, jasmonic acid, cross-talk, mutant screen, Arabidopsis     

Full and Partial Agonists of Thromboxane Prostanoid Receptor Unveil Fine Tuning of Receptor Superactive Conformation and G Protein Activation

The intrahelical salt bridge between E/D^3.49 and R^3.50 within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E^3.49/6.30 in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow “resistant” to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.     

Different peripheral and central antagonistic activity of new glutaramic acid derivatives on satiety induced by cholecystokinin in rats

New glutaramic acid derivatives with cholecystokinin antagonistic activity were evaluated for their capacity to inhibit the satiety effect induced in the rat by intraperitoneal (i.p.) injection of cholecystokinin octapeptide (CCK-8). The most active compound, CR 1409, is about 4000 times more potent than proglumide when injected peripherally (i.p.). This compound competitively inhibits the action of CCK-8 at the receptor responsible for the satiety effect. In contrast, CR 1409, i.p. or intracerebroventricularly (i.c.v.) injected does not exhibit antagonistic effects when CCK-8 is administered i.c.v., confirming the existence of at least two different populations of CCK receptors.