Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding.

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPγS), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTPγS binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the µ-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Déficit de GTPCH 1 autosómico recesivo: importancia del análisis de los neurotransmisores en el líquido cefalorraquídeo / Autosomal recessive GTPCH 1 deficiency: the importance of the analysis of neurotransmitters in cerebrospinal fluid

Introducción. El déficit de la enzima trifosfato de guanosina ciclohidrolasa 1 (GTPCH 1) origina una disminución de la síntesis de la tetrahidrobiopterina (BH4), cofactor indispensable en la síntesis de la tirosina, la dopamina y la serotonina. Es una enfermedad poco frecuente que produce un retraso o regresión psicomotora y trastornos del movimiento, y en la que el tratamiento puede mejorar o incluso corregir la clínica. Caso clínico. Niña afecta de déficit de GTPCH con herencia autosómica recesiva, diagnosticada a los 14 meses con estudio del líquido cefalorraquídeo con déficit de pterinas, HVA y 5-HIAA, test de sobrecarga de fenilalanina y estudio genético positivos. La clínica comenzó a los 5 meses con temblor cefálico y de las extremidades superiores, en reposo e intencional, intermitente, que desapareció en un mes. El desarrollo psicomotor era normal, destacaba una hipotonía axial leve en la exploración y las pruebas complementarias realizadas fueron normales. Posteriormente presentó regresión psicomotora con pérdida del sostén cefálico, disminución de los movimientos activos, dificultad para la manipulación bimanual, hipomimia e hipotonía global grave, lo que motivó el estudio de una encefalopatía progresiva. Tras el diagnóstico de déficit de GTPCH, inició tratamiento sustitutivo con levodopa/carbidopa, OH triptófano y BH4, con muy buena evolución tanto motora como cognitiva. Actualmente, la paciente tiene 5 años, presenta un desarrollo psicomotor adecuado a su edad, cursa tercer curso de educación infantil y ha alcanzado el nivel de su clase. Conclusión. Hay que destacar en este caso la mejoría tan satisfactoria, tanto motora como cognitiva, tras iniciar el tratamiento sustitutivo, ya que el nivel cognitivo suele quedar afectado en muchos casos (AU)

Introduction. A deficiency of the enzyme guanosine triphosphate cyclohydrolase I (GTPCH 1) causes a reduction in the synthesis of tetrahydrobiopterin (BH4), a cofactor that is essential in the synthesis of tyrosine, dopamine and serotonin. It is an infrequent disease that produces psychomotor delay or regression and movement disorders, although treatment can improve or even correct the clinical signs and symptoms. Case report. We report the case of a girl with autosomal recessive GTPCH deficiency, who was diagnosed at 14 months by means of an analysis of the cerebrospinal fluid with pterin, HVA and 5-HIAA deficiency, and positive phenylalanine overload test and genetic study. The clinical features began at the age of 5 months with intermittent upper limb and brain tremors, both at rest and intentional, that disappeared after a month. Psychomotor development was normal, mild axial hypotonia being found in the examination while the complementary tests that were performed were normal. The patient later presented psychomotor regression with loss of head control, diminished active movements, difficulty in bimanual manipulation, hypomimia and severe global hypotonia, which was the reason for the study of a progressive encephalopathy. Following the diagnosis of GTPCH deficiency, replacement therapy was established with levodopa/carbidopa, OH tryptophan and BH4, with excellent progress made in motor and cognitive functioning. Today, the patient is 5 years old, has an adequate psychomotor development for her age, is in the third year of preschool education and has caught up with the level of the rest of her classmates. Conclusion. In this case attention must be drawn to the extremely satisfactory motor and cognitive improvement of the patient after starting replacement therapy, as in many cases the cognitive level is usually affected on a permanent basis (AU)

Engineering a GPCR-ligand pair that simulates the activation of D(2L) by Dopamine.

In the past decade, engineered G-protein-coupled receptors activated solely by synthetic ligands (RASSLs) have been implemented as a new means to study neurotransmission, which is controlled by G-protein-coupled receptors in vitro and in vivo. In this study, we report an engineered dopamine receptor D(2L) F390(6.52)W, which is the first identified RASSL for the dopamine receptor family. The mutant receptor is characterized by a disrupted ligand binding and complete loss of efficacy for the endogenous ligand, dopamine, which is putatively due to a sterically induced perturbation of H-bonding with conserved serine residues in TM5. Based on this model, we rationally developed an aminoindane-derived set of agonists. Because these agonists forgo analogous H-bonding functionalities, their binding energy does not depend on the respective interactions. Binding affinity and potency were optimized by ligand modifications bearing molecular appendages that obviously interact with a secondary recognition site provided by four hydrophobic residues in TM2 and TM3. Thus, the ferrocenyl carboxamide 5b (FAUC 185) was identified as a synthetic agonist that is able to stimulate the mutant receptor in a manner similar to that by which endogenous dopamine activates the D(2L) wild-type receptor. The engineered dopamine receptor D(2L) F390(6.52)W in combination with FAUC 185 (5b) provides a new tool to probe GPCR functions selectively in specific cell populations in vitro and in vivo.

Structural motifs of importance for the constitutive activity of the orphan 7TM receptor EBI2: analysis of receptor activation in the absence of an agonist.

The Epstein-Barr induced receptor 2 (EBI2) is a lymphocyte-expressed orphan seven transmembrane-spanning (7TM) receptor that signals constitutively through Galphai, as shown, for instance by guanosine 5'-O-(3-thio)triphosphate incorporation. Two regions of importance for the constitutive activity were identified by a systematic mutational analysis of 29 residues in EBI2. The cAMP response element-binding protein transcription factor was used as a measure of receptor activity and was correlated to the receptor surface expression. PheVI:13 (Phe257), and the neighboring CysVI:12 (Cys256), in the conserved CW/FxP motif in TM 6, acted as negative regulators as Ala substitutions at these positions increased the constitutive activity 5.7- and 2.3-fold, respectively, compared with EBI2 wild type (wt). In contrast, ArgII:20 (Arg87) in TM-2 acted as a positive regulator, as substitution to Ala, but not to Lys, decreased the constitutive activity more than 7-fold compared with wt EBI2. IleIII:03 (Ile106) is located only 4 A from ArgII:20, and a favorable electrostatic interaction with ArgII:20 was created by introduction of Glu in III:03, given that the activity increased to 4.4-fold of that wt EBI2. It is noteworthy that swapping these charges by introduction of Glu in II:20 and Arg in III:03 resulted in a 2.7-fold increase compared with wt EBI2, thereby rescuing the two signaling-deficient single mutations, which exhibited a 3.8- to 4.5-fold decrease in constitutive activity. The uncovering of these molecular mechanisms for EBI2 activation is important from a drug development point of view, in that it may facilitate the rational design and development of small-molecule inverse agonists against EBI2 of putative importance as antiviral- or immune modulatory therapy.

Agonist activity of naloxone benzoylhydrazone at recombinant and native opioid receptors.

1. In the present study, we examined the pharmacological activity of the putative kappa3-opioid receptor agonist naloxone benzoylhydrazone (NalBzoH) at recombinant human opioid receptors individually expressed in Chinese hamster ovary (CHO) cells and native opioid receptors present in rat striatum. 2. At the mu-opioid receptor (MOR), NalBzoH stimulated guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding (pEC50=8.59) and inhibited cyclic AMP accumulation (pEC50=8.74) with maximal effects (Emax) corresponding to 55 and 65% of those obtained with the MOR agonist DAMGO, respectively. The MOR antagonist CTAP blocked the stimulatory effects of NalBzoH and DAMGO with similar potencies. 3. At the kappa-opioid receptor (KOR), NalBzoH stimulated [35S]GTPgammaS binding (pEC50=9.70) and inhibited cyclic AMP formation (pEC50=9.45) as effectively as the selective KOR agonist (-)-U-50,488. The NalBzoH effect was blocked by the KOR antagonist nor-binaltorphimine (nor-BNI) (pKi=10.30). 4. In CHO cells expressing the delta-opioid receptor (DOR), NalBzoH increased [35S]GTPgammaS binding (pEC50=8.49) and inhibited cyclic AMP formation (pEC50=8.61) almost as effectively as the DOR agonist DPDPE. Naltrindole (NTI), a selective DOR antagonist, completely blocked the response to NalBzoH (pKi of 10.40). 5. In CHO cells expressing the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), NalBzoH failed to exert agonist effects and antagonized the agonist-induced receptor activation. 6. When compared to other opioid receptor ligands, NalBzoH showed an efficacy that was lower than that of morphine at MOR, but higher at KOR and DOR. 7. In rat striatum, NalBzoH enhanced [35S]GTPgammaS binding and inhibited adenylyl cyclase activity. These effects were antagonized by either CTAP, nor-BNI or NTI, each antagonist blocking a fraction of the NalBzoH response. 8. These data demonstrate that NalBzoH displays agonist activity at MOR, DOR and KOR expressed either in a heterologous cell system or in a native environment.

Ontogenetic development of cannabinoid receptor expression and signal transduction functionality in the human brain.

Previous evidence suggests that the endogenous cannabinoid system emerges relatively early during brain development in the rat. However, the pre- and postnatal pattern of appearance of CB1 cannabinoid receptors in humans has not been analysed in detail. Furthermore, there is a complete lack of information about the functional ability of these proteins to activate signal transduction mechanisms during human development. In the present study we have explored CB1 receptor expression throughout the different areas of the developing human brain by [3H]CP55 940 autoradiography. We have also assessed CB1 functional coupling to G proteins during brain development by agonist-stimulated [35S]GTPgammaS autoradiography in the same cases. Our results indicate a significant density of cannabinoid receptors at 19 weeks' gestation in the same areas that contain these receptors in the adult human brain. Autoradiographic levels of CB1 receptors in these structures seem to increase progressively from early prenatal stages to adulthood. Interestingly, high densities of cannabinoid receptors have also been detected during prenatal development in fibre-enriched areas that are practically devoid of them in the adult brain. In parallel with these data, we have found that brain cannabinoid receptors are functionally coupled to signal transduction mechanisms from early prenatal stages. This early pattern of expression of functionally active cannabinoid receptors, along with the transient and atypical localization of these proteins in white matter areas during the prenatal stages, suggest an specific role of the endocannabinoid system in the events related to human neural development.

A fully automated [35S]GTPgammaS scintillation proximity assay for the high-throughput screening of Gi-linked G protein-coupled receptors.

The diversity of physiological functions mediated by the GPCR superfamily provides a rich source of molecular targets for drug discovery programs. Consequently, a variety of assays have been designed to identify lead molecules based on ligand binding or receptor function. In one of these, the binding of [(35)S]GTPgammaS, a nonhydrolyzable analogue of GTP, to receptor-activated G-protein alpha subunits represents a unique functional assay for GPCRs and is well suited for use with automated HTS. Here we compare [(35)S]GTPgammaS scintillation proximity binding assays for two different G(i)-coupled GPCRs, and describe their implementation with automated high-throughput systems.

[(35)S]GTPgammaS autoradiography reveals a wide distribution of G(i/o)-linked ADP receptors in the nervous system: close similarities with the platelet P2Y(ADP) receptor.

No G(i)-linked P2Y receptors have been cloned to date but the presence of such receptors is thought to be restricted to platelets and certain clonal cell lines. Using the functional approach of [(35)S]guanosine 5'-[gamma-thio]-triphosphate autoradiography, we uncovered the widespread presence of such receptors in the CNS. Under conditions in which the prominent signal due to tonic adenosine receptor activity is masked, ADP and ATP stimulated G-protein activity in multiple grey and white matter regions. Localization in the grey matter suggests inhibitory auto-/heteroreceptor function. In the white matter, activated G proteins appeared as 'hot spots' (presumed oligodendrocyte progenitors) with scattered distribution along the main fibre tracts. Responses to ATP were diminished under conditions that inhibited degradation, suggesting that prior conversion to ADP explained agonist action. Uracil nucleotides were ineffective but 2-methylthio-ADP activated G proteins approximately 500-fold more potently than ADP, although both were similarly degraded. Throughout the brain, ADP-dependent G-protein activity was reversed by 2-hexylthio-AdoOC(O)Asp(2), a non-phosphate ATP analogue, whereas selective P2Y(1) receptor antagonists proved ineffective. A similar receptor was also disclosed from the adrenal medulla. These data witness a hitherto unrecognized abundance of G(i/o)-linked ADP receptors in the nervous system. Biochemical and pharmacological behaviour suggests striking similarities to the elusive platelet P2Y(ADP) receptor.

Opioids activate G proteins in REM sleep-related brain stem nuclei of rat.

Mu opioid receptors within the pontine reticular formation contribute to opioid-induced rapid eye movement (REM) sleep inhibition. Mu receptors are coupled to guanine nucleotide binding (G) proteins and this study tested the hypothesis that the micro opioid agonist [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) would activate G proteins in rat brain stem nuclei known to regulate REM sleep. In vitro autoradiography of DAMGO-stimulated [35S]GTPgammaS binding showed that, compared with basal [35S]GTPgammaS binding, DAMGO significantly increased G protein activation in the nucleus pontis oralis (56.2%), nucleus pontis caudalis (57.3%), laterodorsal tegmental nucleus (75.8%), pedunculopontine tegmental nucleus (72.4%), nucleus locus coeruleus (77.2%) and dorsal raphe nucleus (73.4%). DAMGO stimulation of [35S]GTPgammaS binding in nuclei regulating REM sleep suggests that opioid-induced REM sleep inhibition involves activation of G proteins.

Intercellular guanosine-5'-0-(3-thiotriphosphate) blocks chemotactic motility of Dictyostelium discoideum amoebae.

Starving amoebae of the cellular slime mold Dictyostelium discoideum react chemotactically towards the attractant cAMP. In this study, the effect of nonhydrolyzable analogs of GTP and GDP on the chemotactic behavior was analyzed with light microscopic techniques. Guanosine-5'-0-(2-thiotriphosphate) (GTP gamma S) or guanosine-5'-0-(2-thiodiphosphate) (GDP beta S) was scrape-loaded into the cytoplasm of cells, together with a fluorescent marker. Stimulation with a cAMP-filled glass capillary revealed a reduced capacity of loaded cells to migrate towards the capillary tip. Most cells still protruded filopods in the direction of the capillary tip, but full extension of pseudopods was inhibited in a dose-dependent and reversible manner. This indicates that in the presence of the analogs, chemotactic sensing still occurs, and that a more distal step of the cascade of events leading to the formation of the pseudopod is impaired. In cells loaded with the analogs together with the calcium indicator fura-2, stimulation with 10 microM cAMP led to a transient change in the intracellular free calcium concentration ([Ca2+]i), which was detectable in 28% of the cells. Furthermore, large vacuoles were found containing high amounts of calcium. On the other hand, clamping of [Ca2+]i at low levels with 1,2-bis(2-aminophenoxy) ethane N,N,N',N'-tetraacetic acid (BAPTA) also inhibited motility, with neither filopods nor pseudopods formed. The data suggest that chemotactic migratory activity involves GTP-dependent processes that participate in the regulation of the Ca2+ homeostasis of the cell and in the regulation of membrane traffic that contributes to the directed locomotion.