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Keywords:

  • bacterial toxins;
  • CRH receptor;
  • G-proteins;
  • rat cortex;
  • second messengers

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The wide distribution of corticotrophin-releasing hormone (CRH) receptors in brain and periphery appear to be important in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli. The type 1 receptors are highly expressed throughout the cerebral cortex, a region involved in cognitive function and modulation of stress responses, where they are coupled to the adenylyl cyclase system. Using techniques that analyse receptor-mediated guanine-nucleotide binding protein (G-proteins) activation, we recently demonstrated that expressed type 1α CRH receptors are capable of activating multiple G-proteins, which suggests that CRH can regulate multiple signalling pathways. In an effort to characterize the intracellular signals generated by CRH in the rat cerebral cortex we sought to identify G-proteins activated by CRH in a physiological membrane environment. Rat cerebral cortical membrane suspensions were analysed for the ability of CRH to stimulate incorporation of [α-32P]-GTP-γ-azidoanilide to various G-protein α-chains. Our results show that CRH receptors are coupled to and activate at least five different G-proteins (Gs, Gi, Gq/11, Go and Gz) with subsequent stimulation of at least two intracellular signalling cascades. In addition, the photoaffinity experiments indicated that the CRH receptors preferentially activate the 45 kDa form of the Gsα-protein. This data may help elucidate the intracellular signalling pathways mediating the multiple actions of CRH especially under different physiological conditions.

Abbreviations used
BSA

bovine serum albumin

CRH

corticotropin-releasing hormone

CTX

cholera toxin

DTT

dithiothreitol

ECL

enhanced chemiluminescence

HEK

human embryonic kidney

G-proteins

GTP-binding proteins

IP3

inositol triphosphate

IBMX

3-isobutyl-1-methylxanthine

PBS

phosphate-buffered saline

PKC

protein kinase C

PLC

phospholipase C

PTH

parathyroid hormone

PTX

pertussis-toxin

PVDF

polyvinylidene difluoride

SDS

sodium dodecyl saline

7TMD

seven transmembrane domain

TSH

thyroid-stimulating hormone

Corticotropin-releasing hormone (CRH) is a 41-amino acid peptide (Vale et al. 1981), that is capable of integrating the neuroendocrine, behavioural, autonomic and immune responses to stress (Koob 1985; Besedovsky et al. 1986; Fisher 1989; Irwin 1993). CRH activates transcription of the proopiomelanocortin (POMC) gene and stimulates the release of ACTH and β endorphin from cells in the anterior pituitary gland. In addition, CRH can induce behavioural responses, stimulate thermogenesis, influence reproductive and cardiovascular function and exert both antiand pro-inflammatory effects (Chrousos et al. 1998; Fisher et al. 1982; Karalis et al. 1991). CRH is expressed in a number of brain regions including the hypothalamus, amygdala and cerebral cortex (Swanson et al. 1983), and receptors for CRH are distributed diversely throughout the brain.

The CRH receptor is a member of a specialized subfamily of GTP-binding protein (G-protein) coupled seven transmembrane domain (7TMD) receptors that includes the receptors for calcitonin, vasoactive intestinal peptide, and parathyroid hormone (PTH) (Perrin et al. 1993). Two subtypes of CRH receptors, termed R1 and R2, have been identified in the rat (Perrin et al. 1993; Lovenberg et al. 1995) and are encoded by separate genes. These two receptors share 70% identity. The R1 receptor binds CRH as well as CRH-like peptides (urocortin, urotensin and sauvagine) with equivalent high affinity, but the R2 receptor binds urocortin with higher affinity than the other CRH-like peptides, suggesting that urocortin may be the natural ligand (Vaughan et al. 1995). In the brain, the R1 receptor is found predominantly in the neocortex, cerebellum, olfactory bulb and anterior pituitary whilst the R2 receptor is localized to the subcortical, amygdaloid and hypothalamic regions (Primus et al. 1997); these data suggest that the R1 and R2 receptors might serve different roles in mediating CRH actions. Both the R1 and R2 genes encode multiple splice variants; the R2 gene exhibits even greater diversity through the use of alternative 5′ exons that produce three different receptors (R2α, R2β, R2γ).

Both R1 and R2 CRH receptors are coupled to Gs protein and activate adenylyl cyclase and cAMP production (Chen et al. 1986). Over the last few years it has been recognized that many members of the 7TMD receptor superfamily, including the PTH and the thyroid-stimulating hormone (TSH) receptor (Laugwitz et al. 1996; May and Gay 1997), are coupled to multiple G-proteins and thereby can regulate several signal transduction pathways. We have recently shown that the type 1α CRH receptor stably expressed in HEK293 cells can stimulate multiple G-proteins (Gs, Gi, Gq/11, and Go) (Grammatopoulos et al. 1999). These data are in agreement with other studies showing that intracellular signalling pathways other than adenylyl cyclase are activated by CRH such as the inositol triphosphate (IP3) and PKC pathways in Leydig cells, cultured astrocytes and epidermoid cells (Ulisse et al. 1990; Takuma et al. 1994; Kiang 1997). Also, in LLCPK-1 cells the stably expressed CRH-R1 is weakly coupled to the PLC-IP3 pathway (Nabhan et al. 1995). The rat cerebral cortex contains multiple CRH receptor isoforms (Grammatopoulos and Hillhouse 1998) that may be coupled to both cholera and pertussis toxin-sensitive G-proteins (Grammatopoulos et al. 1994). Accordingly, the present study was carried out to identify the G-proteins that are linked to rat cerebral cortical CRH receptors and to determine the mechanism by which the intracellular signals are generated.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

Ovine-CRH, and human/rat CRH were obtained from Peninsula Laboratories (Merseyside, UK). The α1 and α2 adrenergic agonists were obtained from Tocris Cookson Ltd (Churchill Building, Bristol, UK). Dithiothreitol (DTT), isoproterenol, GTP, cholera and pertussis toxins, 2-(N-morpholino)ethanesulfonic acid (MES), 1,4-dioxane, triethylamine and all other chemicals were purchased from Sigma Chemicals Company Ltd (Poole, Dorset, UK). Waters Sep-Pak C18 columns were obtained from Millipore (UK) Ltd (Watford, Herts, UK). Polyclonal anti-Gα chain antibodies that were raised in rabbits immunized with synthetic peptides [RM/1 (anti-Gsα), AS/7 (anti-Gi1α, Gi2α), GC/2 (anti-Goα), QL (anti-Gq/11α)] and cAMP assay kits were obtained from Dupont-NEN (Hertfordshire, UK). Monoclonal antisera against the N-terminus of Gz α chain was purchased from Calbiochem (Nottingham, UK). Protein-A sepharose beads (CL-4B) were purchased from Pharmacia (Uppsala, Sweden). [α-32P]-GTP and BioTrak [3H]-IP3 assay system were obtained from Amersham International (Little Chalfont, Buckinghamshire, UK). 4-Azidoanilide-HCl, 1-(3-dimethylaminopropyl)-3-ethylenecarbo diimidehydrochloride (NDEC) were purchased from Aldrich Chem. Co (Gillingham, Dorset, UK).

Preparation of cortical membranes

Cerebral cortex was obtained from male Wistar rats derived from inbred colonies. Rats were housed in standard cages (< 6 animals/cage) at a constant temperature with a controlled light–dark cycle. Animals were given continuous unlimited access to food and water and were regularly handled. They were killed by decapitation immediately after being taken out of their cages. Brains and testes were carefully removed, frozen quickly on dry ice and stored at −80°C until use.

Tissues were homogenized in Dulbecco's phosphate-buffered saline containing 10 mm MgCl2, 2 mm EGTA, 1.5 g/L bovine serum albumin (w/v), 0.15 mm bacitracin, 1 mm phenylmethyl sulfonylfluoride pH 7.2 (extraction buffer) at 22°C. The homogenate was centrifuged at 1500 g for 30 min at 4°C. The pellet was discarded and the supernatant spun at 45 000 g for 60 min at 4°C. The resultant pellet was washed, resuspended in extraction buffer and spun at 45 000 g for a further 60 min at 4°C. The final pellet was resuspended in 10 mL of extraction buffer using the homogenizer. The protein concentration of the membrane suspension was determined using the bicinchoninic acid method (Smith et al. 1985) with bovine serum albumin (BSA) as a standard.

Pretreatment of rat cerebral cortical membranes with bacterial toxins

Membranes were treated with cholera-toxin, cholera toxin was pre-activated with 20 mm dithiothreitol (DTT) for 30 min at 37°C. Rat cerebral cortical membranes were incubated with 50 µL of 50 mm Tris-HCl, containing 1 mm ATP, 10 mm thymidine, 0.25 mm GTP, 10 mm MgCl2, 10 µm NAD, 7.4 mg/mL creatine phosphate, 1 mg/mL creatine phosphokinase, pH 7.4 in the presence of cholera toxin (150 µg/mL) or 20 mm DTT for 45 min at 30°C. The reaction was stopped by the addition of 1 mL of ice-cold extraction buffer, followed by centrifugation at 10 000 g for 15 min. The pellet was then washed with extraction buffer and centrifuged at 10 000 gfor 15 min (three times) before re-suspension in the same buffer.

Cerebral cortical membrane preparations were treated with pertussis toxin that had been pre-activated with 50 mm DTT for 30 min at 30°C. Briefly, cerebral cortical membranes were incubated with 500 µL of 50 mm Tris-HCl containing 1 mm ATP, 10 mm thymidine, 0.1 mm GTP, 10 mm MgCl2, 1 µg/mL digitonin, 1 mm NAD, 7.4 mg/mL creatine phosphate + 1 mg/mL creatine phosphokinase, pH 7. in the presence of pertussis toxin (final concentration 25 µg/mL) or 50 mm DTT for 45 min at 30°C. The reaction was stopped by the addition of 1 mL of ice-cold extraction buffer, followed by centrifugation at 12 000 g in a Beckman J20 centrifuge, for 15 min. The pellet was then washed with Extraction Buffer and centrifuged at 12 000 gfor 15 min (three times). The final pellet was resuspended in extraction buffer and homogenized.

cAMP studies

Rat cerebral cortical preparations (50 µg protein) were preincubated with different concentrations of h/rCRH (0.1–1000 nm), in 50 µL of extraction buffer for 30 min at 22°C, prior to the addition of 100 µL of 50 mm Tris-HCl containing 10 mm MgCl2, 1 mm EGTA, 1 g/L BSA, 1 mm ATP, ATP regeneration system (7.4 mg/mL creatine phosphate, 1 mg/mL creatine phosphokinase), 100 µm 3-isobutyl-1-methylxanthine (IBMX), 0.15 mm bacitracin, pH 7.4 at 37°C. The reaction was terminated after 10 min by the addition of 1 mL of 0.1 m imidazole buffer pH 7, followed by heating of the tubes in boiling water for 5 min. The amount of cAMP in the supernatants was determined by radioimmunoassay.

Inositol triphosphate assay

Rat brain membranes (100 µg) were incubated with CRH (100 pm-1 µm) or GTP (10 µm) in 50 µL of extraction buffer for 30 min at room temperature, followed by the addition of 200 µL of IP3 generation buffer containing, 25 mm Tris-acetate buffer pH 7.2, 5 mm Mg acetate, 1 mm DTT, 0.5 mm ATP, 0.1 mm CaCl2, 0.1 mg/mL BSA, 10 µm GTP. Membranes were incubated for 3 min at 37°C, and the reaction was terminated by the addition of 1 m ice-cold trichloroacetic acid, followed by extraction of inositol phosphates and neutralization (Chilvers et al. 1991). IP3 levels were determined by competitive binding assay based on displacement of [3H] IP3 from specific bovine adrenocortical IP3 binding proteins (Palmer et al. 1989). The sensitivity of the assay was 0.11 pmol/well and the precision was: intra-assay coefficient of variation (CV) 12.9% and interassay CV 11.7%.

Synthesis of 32P-GTP-γ-azidoanilide (GTP-AA) and photolabelling of Gα subunits

GTP-AA was synthesized by a previously described method (Schwindinger et al. 1998). Rat cortical or testicular membranes (100 µg) were incubated with or without h/rCRH (1 pm−100 nm) or adrenergic agonists (10 µm) for 5min at 30°C before the addition of 5 µCi of 32P-GTP-AA in 120 µL of 50 mm HEPES buffer, pH 7.4, containing 30 mm KCl, 10 mm MgCl2, 1 mm benzamidine, 5 µm GDP, 0.1 mm EDTA, in a darkened room. After incubation for 3 min at 30°C membranes were collected by centrifugation and resuspended in 100 µL of the above buffer containing 2 mm glutathione, placed on ice and exposed to UV light (254 nm) at a distance of 5 cm for 5 min.

G-protein immunoprecipitation

32P-GTP-AA-labelled G-proteins were precipitated by centrifugation and solubilized in 120 µL of 2% SDS. Then 360 µL of 10 mm Tris-HCl buffer, pH 7.4, containing 1% (v/v) Triton X-100, 1% (v/v) deoxycholate, 0.5% (w/v) SDS, 150 mm NaCl, 1 mm DTT, 1 mM EDTA, 0.2 mm PMSF, 10 µg/mL aprotinin were added and insoluble material was removed by centrifugation. Solubilized membranes were divided into 100 µL aliquots and each aliquot was incubated with 10 µL of undiluted G-protein antiserum at 4°C for 2 h under constant rotation. Then 50 µL of protein A Sepharose beads (10% w/v in the above buffer) were added and the incubation was continued at 4°C overnight under constant rotation. The beads were collected by centrifugation, washed twice with 1 mL of a 50-mm Tris-HCl buffer, pH 7.4, containing 10% NP-40, 0.5% SDS, 600 mm NaCl and then were further washed twice with 1 mL of a 100-mm Tris-HCl buffer, pH 7.4, containing 300 mm NaCl, 10 mm EDTA and dried under vacuum in a Speed-Vac microconcentrator. The immune complexes were dissociated from protein A by reconstitution in Laemmli's buffer (100 µL) and boiling for 5 min. Samples were then subjected to gel electrophoresis using discontinuous SDS-PAGE slab gels (10% running; 5% stacking). Molecular weight markers dissolved in solubilization buffer were also electrophoresed. The gels were then stained with Coomassie Blue, dried using a slab gel dryer and exposed to Fuji X-ray film at − 70°C for 2–5 days.

In some experiments G-protein immunoprecipitates were subject to Western blot analysis using different G-protein antibodies for detection.

Immunoblotting

Cerebral cortical membranes (50 µg of protein) were resuspended in Laemmli's buffer (100 µL) and boiled for 5 min. Samples were then subjected to gel electrophoresis using discontinuous SDS-PAGE slab gels (10% running; 5% stacking). Molecular weight markers dissolved in solubilization buffer were also electrophoresed. In some experiments G-protein immunoprecipitates were also subjected to immunoblotting. The resolved proteins were transferred to polyvinylidene difluoride (PVDF) membrane at 100 mA for 90 min. The membrane was then blocked with 5% non-fat dry milk at room temperature for 30 min and subsequently incubated at 4°C for 2 h with G-protein subtype-specific antisera. Polyvinylidene difluoride (PVDF)membranes were washed twice with phosphate-buffered saline (PBS)-Tween 20 (0.05%), incubated with goat antirabbit antibody conjugated with horseradish peroxidase for 1 h. Following washing twice with PBS-Tween 20 (0.05%), immunoreactivity was detected by enhanced chemiluminescence (ECL).

Statistical analysis

Data are shown as the mean ± SEM of each measurement. Comparison between group means was performed by analysis of variance (anova). p < 0.05 was considered significant. The relative density of the bands was measured by optical density scanning using the software Scion Image-Beta 3b for Windows (Scion Corporation, Frederick, MD).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Activation of Gsα and adenylate cyclase system by CRH

Upon receptor stimulation, the nonhydrolysable GTP analogue, [α-32P]-GTP-AA binds to the GTP-binding site of activated Gα-proteins. Immunoprecipitation with specific Gα chain antibodies was used to identify agonist-dependent activation of G-proteins. Optimal labelling of Gα chains with GTP-AA requires receptor activation of heterotrimeric G-proteins with release of bound GDP. The binding of GTP-AA is dependent upon GDP concentration, GTP affinity of the α chain and agonist-incubation time (Offermanns et al. 1991). Therefore, the conditions for labelling Gα chains were established empirically. Our results showed that basal GTP-AA labelling of Gsα was GDP-concentration dependent, and optimum labelling was obtained in the presence of 5–10 µm GDP (Fig. 1a). CRH-induced labelling with GTP-AA was time-dependent with an optimal CRH-incubation time of 5 min (Fig. 1b). The specificity of labelling of Gsα protein was assessed by inclusion of GTP (100 nm−10 µm) in the incubation medium, which greatly reduced CRH-induced labelling. By contrast, ATP at concentrations up to 10 µm had no effect on agonist-induced labelling of Gsα-protein (Fig. 1c). The optimal conditions for CRH-induced GTP-AA labelling of Gα chains were determined for all five G-proteins, and were found to be comparable but not identical to those of Gsα-protein (data not shown). Therefore, appropriate experimental conditions were chosen in order to achieve labelling of all Gα-proteins by at least 80–90% of maximum.

image

Figure 1. Determination of optimal conditions for CRH-induced labelling of Gsα-proteins from rat cerebral cortical membranes. Effect of (a) GDP concentration, or (b) CRH stimulation time or (c) presence of GTP/ATP on 32P-GTP-AA photolabelling of Gsα-proteins. Membranes (100 µg) were incubated with or without CRH (100 nm) for the indicated period at 30°C before the addition of 5 µCi of 32P-GTP-AA in buffer containing different concentrations of GDP in the presence or absence of GTP (10 nm or 10 µm) or ATP (10 µm). After incubation for 3 min at 30°C membranes were collected, placed on ice and exposed to UV light (254 nm) at a distance of 5 cm for 5 min 32P-GTP-AA-labelled Gsα-subunits were immunoprecipitated and were subjected to SDS-PAGE. The gels were stained with Coomassie Blue, dried and exposed to Fuji X-ray film at − 70°C for 2–5 days with intensifying screens. Identical results were obtained from three independent experiments.

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CRH-induced activation of adenylyl cyclase was accompanied by activation of Gs-protein, as shown by increase of radiolabelling of the 45 kDa Gsα isoform (Fig. 2a). Based on GTP-AA labelling, CRH was able to activate the Gs-protein at a threshold concentration of 100 pm with maximal activation at a concentration of 10–100 nm. By contrast, CRH-dependent increases in cAMP showed a threshold of 1 nm CRH, while the maximal response (205 ± 9% increase of basal) occurred at a concentration of 100 nm. At maximal CRH concentration (100 nm), GTP-AA incorporation was also observed in bands with apparent molecular weights of 52 and 68 kDa which likely represent the larger isoforms of Gsα-protein and XLGsα (Kehlenbach et al. 1994), respectively (Fig. 2b). Analysis of the Gsα immunoreactivity by Western blot revealed that both the 45 and 52 kDa isoforms of Gsα were present in these membranes with the 52 kDa isoform the most prominent and that both isoforms could be immunoprecipitated by the Gsα-protein antibody used (Fig. 2c). In addition, a 68-kDa band was present which corresponds to the XLGsα isoform.

image

Figure 2. (a) CRH-induced cAMP release from rat cerebral cortical membranes in the presence of different concentrations of h/rCRH. Rat cerebral cortical preparations (50 µg protein) were preincubated with different concentrations of h/rCRH (0.1–1000 nm), in 50 µL of Extraction buffer for 30 min at 22°C, prior to the addition of 100 µL of cAMP assay buffer at 37°C. The reaction was terminated after 10 min and the amount of cAMP in the supernatants was determined by radioimmunoassay. Results are expressed as the mean ± SEM of four estimations from six independent experiments. *p < 0.05 compared to basal. Inset: CRH dose-dependent binding of 32P-GTP-AA to Gsα-proteins immunoprecipitated from rat cerebral cortical membranes. (b) Identification of Gsα-proteins photolabeled with 32P-GTP-AA from rat cerebral cortical membranes in the presence of CRH. Membranes were incubated with CRH (100 nm), for 5min at 30°C before the addition of 5 µCi of 32P-GTP-AA for 3 min at 30°C. Following centrifugation, membranes were placed on ice and exposed to UV light at a distance of 5 cm for 5 min 32P-GTP-AA-labelled G-proteins were immunoprecipitated and were resolved on SDS-PAGE gels followed by autoradiography. Identical results were obtained from three independent experiments. (c) Western blot analysis of Gsα-protein isoforms from rat cerebral cortical membranes. Cerebral cortical membranes (50 µg of protein) were resuspended in Laemmli's buffer (100 µL) (left panel) or were solubilized and incubated with Gsα-protein antiserum in order to immunoprecipitate the Gsα-subunits (right panel) and were then subjected to gel electrophoresis using discontinuous SDS-PAGE slab gels (10% running; 5% stacking). The resolved proteins were transferred to PVDF membrane at 100 mA for 90 min. The membrane was then blocked with 5% non-fat dry milk at room temperature for 30 min and subsequently incubated at 4°C for 2 h with Gsα-protein specific antisera. PVDF membranes were washed twice with PBS-Tween 20 (0.05%), incubated with goat antirabbit antibody conjugated with horseradish peroxidase for 1 h. Following washing twice with PBS-Tween 20 (0.05%), immunoreactivity was detected by enhanced chemiluminescence (ECL). Identical results were obtained from three independent experiments.

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Activation of multiple G-proteins by the CRH receptor

Using GTP-AA labelling we sought additional G-protein subtypes that are coupled to the rat cerebral cortex CRH receptor. We found that the CRH receptor is also coupled to at least four G-proteins: the Gq/11, Gi1/2, Go, and Gz subtypes (Fig. 3a). Both Gq and Go could be activated at low CRH concentrations with a threshold of about 100 pm and maximal 32P-GTP-AA incorporation was obtained at a concentration range of 10–100 nm (350% for Gq/11 and 470% for Go increase over basal). Labelling of the Gi1/2- and Gz-protein subtypes required a 10-fold greater CRH concentration (1 nm), and maximal GTP-AA incorporation was found at a concentration of 100 nm (450% and 400% increase over basal for Gi and Gz, respectively) (Fig. 3b). Urocortin, a mammalian CRH-like peptide, was able to stimulate the same G-proteins as CRH with similar potency except for Gq/11, where urocortin was found to be 30–40% more potent than CRH (Fig. 4a; Table 1). CRH-induced activation of all G-proteins was completely inhibited by 500 nm astressin, a CRH receptor antagonist (Fig. 4b) indicating that G-protein activation was mediated specifically via CRH receptors.

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Figure 3. (a) Autoradiograph of CRH-induced photolabelling (with 32P-GTP-AA) of various Gα-proteins from rat cerebral cortical membranes. Membranes were incubated with 32P-GTP-AA and different concentrations of CRH (0.01–100 nm), followed by UV crosslinking and immunoprecipitation of the Gα-subunits using specific antibodies. Proteins were resolved on SDS-PAGE gels, followed by autoradiography. Identical results were obtained from three independent experiments. (b) Quantification of agonist-induced photolabelling of specific Gα-subunits using densitometry scanning. For each G-protein data are expressed as –fold increase of 32P-GTP-AA photolabelling above basal, in which the 32P-GTP-AA labelling in untreated membranes was defined as 1.0. Results are expressed as the mean ± SEM of three estimations from three independent experiments. *p < 0.05 compared to basal.

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image

Figure 4. (a) Comparison of CRH and urocortin-induced photolabelling with 32P-GTP-AA of Gsα-proteins from rat cerebral cortical membranes. Membranes were incubated with CRH or urocortin (100 nm), for 5min at 30°C before the addition of 5 µCi of 32P-GTP-AA for 3 min at 30°C. Following UV crosslinking for 5 min, 32P-GTP-AA-labelled G-proteins were immunoprecipitated and were resolved on SDS-PAGE gels followed by autoradiography. Identical results were obtained from three independent experiments. (b) Effect of astressin on basal and CRH-induced photolabelling with 32P-GTP-AA of Gsα- and Goα-proteins from rat cerebral cortical membranes. Membranes were incubated with CRH (100 nm) in the presence or absence of astressin (500 nm), for 5min at 30°C before the addition of 5 µCi of 32P-GTP-AA for 3 min at 30°C. Following UV crosslinking for 5 min, 32P-GTP-AA-labelled G-proteins were immunoprecipitated and were resolved on SDS-PAGE gels followed by autoradiography. Similar results were obtained for the other G-proteins. Identical results were obtained from four independent experiments.

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Table 1.  Comparison of CRH and urocortin-induced maximal activation of G-proteins in rat cortical membranes
 Fold increase above basal
G-protein subtypeCRHUrocortin
  1. Cerebral cortical membranes (50 µg protein) were incubated with 32P- GTP-AA in the presence or absence of CRH or Urocortin (100 nm), followed by UV crosslinking and immunoprecipitation of the various Gα-subunits using specific antibodies. Proteins were resolved on SDS-PAGE gels, followed by autoradiography. Results are expressed as the mean ± SEM of four independent experiments. *p < 0.05 compared with CRH.

Gs5.0 ± 0.54.55 ± 0.7
Gq3.7 ± 0.34.9 ± 0.65*
Gi3.3 ± 0.53.1 ± 0.25
Gz3.9 ± 0.63.7 ± 0.5
Go4.5 ± 0.53.5 ± 0.9

Activation of Gq/11 is predicted to stimulate phospholipase C and increase generation of inositol triphosphates. We found that CRH treatment of rat cortical membranes stimulated significant dose-dependent production of IP3, with a concentration threshold of 10 nm and maximum response at 500 nm (maximum 290 ± 21% of basal) (Fig. 5).

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Figure 5. CRH-induced inositol triphosphate accumulation from rat cerebral cortical membranes. Membranes (100 µg) were incubated with CRH (100 pm−1 µm) for 30 min at room temperature, followed by the addition of 200 µL of IP3 generation buffer and further incubation for 3 min at 37°C. This was followed by extraction of inositol phosphates and neutralization. IP3 levels were determined by competitive binding assay. Results are expressed as the mean ± SEM of four estimations from three independent experiments. *p < 0.05 compared to basal.

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As controls for the techniques in labelling activated G-proteins, we used a series of α- and β-adrenergic receptor agonists (isoproterenol, α1 and α2 agonists-Fig. 6). Consistent with previous data, the α1-specific agonist 3-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-1,5-dimethylpyrimido [5,4-β]indole-2,4-dione (10 µm) could selectively activate only Gq-proteins. Furthermore, the α2-specific agonist UK-14,304 [5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline, 10 µm] could selectively activate only Gi1/2 and Go-proteins. Lastly, isoproterenol (β-agonist-10 µm) could only stimulate labelling of Gs-proteins.

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Figure 6. Autoradiogragh of α- and β-adrenergic agonist-induced photolabelling with 32P-GTP-AA of various Gα-proteins from rat cerebral cortical membranes. Membranes were incubated with 32P-GTP-AA and agonists (10 µm), followed by UV crosslinking and immunoprecipitation of the Gα-subunits using specific antibodies. Proteins were resolved on SDS-PAGE gels, followed by autoradiography.

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The specificity of the immunoprecipitating properties of some of the antibodies used for G-protein detection were assessed by using two different strategies: first, we compared the migration positions of CRH-induced GTP-AA photoaffinity labelled G-proteins with those ADP-ribosylated using 32P-NAD with cholera (for Gs-protein) and pertussis (for Gi/Go-protein) toxins to demonstrate that the same protein band was radiolabelled and immunoprecipitated by the specific antibodies. Results showed that cholera toxin-labelled bands which were immunoprecipitated by specific Gsα-protein antibodies comigrated with 32P-GTP-AA labelled proteins with apparent molecular weight of 45 and 52 kDa suggesting that these protein bands corresponded to Gsα-protein (Fig. 7a). Similarly, pertussis toxin-labelled bands which were immunoprecipitated by specific Giα- or Goα-protein antibodies comigrated with 32P-GTP-AA labelled protein with apparent molecular weight of 40 kDa suggesting that these proteins corresponded to Giα-/Goα-proteins. Consistent with these results, CRH could stimulate 32P-GTP-AA binding to either Gsα- or Giα-/Goα-proteins.

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Figure 7. (a) Identification of 32P-GTP-AA-labelled rat cerebral cortical membrane G-proteins, by alignment of 32P-GTP-AA-labelled proteins in the presence or absence of 100 nm CRH with proteins labelled with 32P-NAD with either cholera (CTX) (for Gs-protein) or pertussis (PTX) (for Gi and Go-proteins) toxins. Labelling was performed as described under Materials and methods and was followed by immunoprecipitation using specific antibodies. Proteins were resolved on SDS-PAGE gels, followed by autoradiography. Identical results were obtained from three independent experiments. (b) Effect of pertussis-toxin pretreatment (PTX) on CRH-induced photolabelling with 32P-GTP-AA of various Gα-proteins from rat cerebral cortical membranes. Membranes were pretreated with pertussis toxin (final concentration 25 µg/mL) for 45 min at 30°C, washed three times before the addition of CRH (100 nm) and 32P-GTP-AA. This was followed by UV crosslinking and immunoprecipitation of the Gα-subunits using specific antibodies. Proteins were resolved on SDS-PAGE gels, followed by autoradiography and densitometry scanning for quantification of agonist-induced photolabelling of specific Gα-subunits. Identical results were obtained from six independent experiments.

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In a different strategy designed specifically for the pertussis-toxin sensitive G-proteins, we used the pertussis-toxin ability to inactivate the PTX-sensitive G-proteins, Gi1/2 and Go, prior to CRH-induced labelling with 32P-GTP-AA. As predicted, PTX-pretreated membranes showed impaired CRH-induced incorporation of 32P-GTP-AA only to the PTX-sensitive G-proteins Gi1/2, and Go (Fig. 7b). In contrast, PTX treatment had no effect on the incorporation of GTP-AA to the PTX-resistant proteins Gsα, or Gq/11α (data not shown).

The specificity of the immunoprecipitating properties of the antibodies used for G-protein detection was assessed by experiments where G-protein immunoprecipitates were subjected to immunoblotting with other G-protein antibodies to verify the absence of coprecipitated amounts of other G-proteins (Fig. 8). Results showed that only the anti Gz α-chain antibody could detect the precipitated protein by immunoblotting, whilst antibodies against Gs, Go, Gq/11 and Gi1/2 α-chains did not recognize the immunoprecipitated proteins (Fig. 8). Identical results were obtained for all G-protein antibodies used in this study (data not shown).

image

Figure 8. Western blot analysis of Gα-protein isoforms immunoprecipitaed with Gzα antiserum from rat cerebral cortical membranes. Cerebral cortical membranes (50 µg of protein) were solubilized and incubated with Gzα-protein antiserum in order to immunoprecipitate the Gsα-subunits (right panel) and were then subjected to gel electrophoresis using discontinuous SDS-PAGE slab gels (10% running; 5% stacking). The resolved proteins were transferred to PVDF membrane at 100 mA for 90 min. The membrane was then blocked with 5% non-fat dry milk at room temperature for 30 min and subsequently incubated at 4°C for 2 h with various Gα-protein specific antisera. PVDF membranes were washed twice with PBS-Tween 20 (0.05%), incubated with goat antirabbit antibody conjugated with horseradish peroxidase for 1 h. Following washing twice with PBS-Tween 20 (0.05%), immunoreactivity was detected by enhanced chemiluminescence (ECL). Identical results were obtained from two independent experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Considerable evidence suggests that CRH action in the brain is mediated via stimulation of the adenylyl cyclase system. The full complement of G-proteins and second messengers that can be activated by CRH have yet to be identified. This study, however, provides direct evidence that in the rat cerebral cortex, stimulation of endogenous CRH receptors leads to activation of multiple of G-protein subtypes with subsequent stimulation of at least two intracellular signalling cascades.

To identify G-proteins coupled to endogenous CRH receptors we used G-protein antisera to immunoprecipitate α subunits after incubation of membranes with 32P-GTP-AA and CRH. The reaction conditions for agonist-specific labelling of Gs-protein were dependent upon time and the concentration of CRH and GDP. Activation of Gs-proteins correlated with stimulation of adenylyl cyclase activity and generation of cAMP. Interestingly, incorporation of 32P-GTP-AA to Gs-protein occurred at a 10-fold lower concentration of CRH than required for stimulation of adenylyl cyclase. Although this discrepancy might be due to experimental conditions or to the sensitivity of the cAMP assay, it is similar to the kinetic relationship between receptor binding and activation of adenylyl cyclases that has led to the concept of spare receptors. Whether our findings imply that a threshold number of Gs molecules are required to induce activation of adenylyl cyclase or that some CRH-coupled Gs molecules are not able to activate adenylyl cyclase (e.g. due to differences in membrane distribution) cannot be determined from these experiments. Immunoblot analysis showed that cerebral cortical membrane homogenates contain substantially more of the 52 KDa isoform of Gsα than of the short form of Gs. Surprisingly our 32P-GTP-AA photoaffinity experiments indicated that the CRH receptors preferentially activate the 45 kDa form of the Gsα-protein. However, this appears to be a tissue specific phenomenon since using the same labelling procedure in human myometrium, CRH activated both 45 and 52 kDa forms with equal potency (Grammatopoulos & Hillhouse, unpublished observations).

Our results also demonstrated for the first time that the CRH receptors in the rat cerebral cortex are coupled to multiple G-proteins, including Gs, Gq/11, Gi1/2, Go and Gz. This finding is in agreement with our previous experiments which demonstrated that the rat cerebral cortex CRH receptors are coupled to cholera and pertussis toxin-sensitive G-proteins (Grammatopoulos et al. 1994), a finding that is true for many members of the 7TMD receptors family. This property of the CRH receptor has also been demonstrated in HEK293 cells stably expressing the CRH receptor type 1α (May and Gay 1997). Activation of all the G-proteins occurred at subnanomolar concentrations of CRH except the Gi1/2- and Gz-proteins, which suggests that the CRH-R is relatively weakly coupled to these G-proteins. Western blot analysis showed that all types of G-proteins were present in the cerebral cortical membrane homogenates (data not shown). CRH-induced activation of these proteins could be blocked by the peptide CRH receptor antagonist astressin, whilst urocortin, the other CRH-R1 ligand, had comparable G-protein activation profile and potency. Urocortin was more potent than CRH in its ability to activate Gq-proteins, and the functional importance of this interaction requires further investigation.

We confirmed the specificity of our techniques by using agonists known to activate specific G-proteins such as α- and β-adrenergic receptor agonists. Furthermore, PTX-pretreatment of the rat cortical membranes abolished the CRH-R-induced activation of the Gi and Go-proteins, consistent with the view that PTX-stimulated ADP-ribosylation retains the G-protein in its heterotrimeric GDP-bound inactive form, therefore reducing the incorporation of GTP (or GTP-AA) (Moss and Vaughan 1988). Moreover, PTX-pretreatment had no effect on the 32P-GTP-AA labelling of Gsα or Gq/11α, α chains that are not PTX-substrates.

Previous studies had demonstrated that CRH receptors in rat Leydig cells are coupled to PTX-insensitive G-proteins and activate phospholipase C but not adenylyl cyclase (Ulisse et al. 1990). Our 32P-GTP-AA labelling experiments confirmed these earlier observations, and showed that CRH could stimulate increased GTP-AA incorporation in Gq/11- and Gi-, but not Gs-proteins. By contrast, hCG, acting through the LH receptor, induced 32P-GTP-AA labelling of Gsα. In these cells the CRH receptor does not appear to couple to adenylyl cyclase, a phenomenon that is similar to that found in human placenta and fetal membranes (Karteris et al. 2000).

Here we showed that in rat cerebral cortical membranes activation of Gq/11, Gi and Go G-proteins by CRH could stimulate phospholipase C with subsequent generation of inositol triphosphates suggesting the existence of an alternative second messenger pathway by which CRH can exert its actions. It is also possible that CRH-induced activation of Gi-proteins may lead to liberation of βγ subunits which in turn can activate types II/IV/VII adenylyl cyclase (Ahmed and Heppel 1997) all of which are present in the cerebral cortex (Hellevuo et al. 1996; Mons et al. 1998). Such synergistic interactions between Gs and Gi-dependent signalling pathways in the stimulation of type II adenylate cyclase have been well-documented (Tsu and Wong 1996) and would explain the partial inhibition of the basal and CRH-stimulated adenylate cyclase activity observed following PTX treatment of cerebral cortical membranes (Grammatopoulos et al. 1994).

Also, our experiments demonstrated for the first time that the CRH receptor can activate Go- and Gz-proteins. These G-proteins have been implicated in the inhibition of adenylase cyclase and modulation of cation channels (Harris-Warrick et al. 1988; Fields and Casey 1997); however, thus far the physiological consequences of their activation remain unknown.

In summary, we have demonstrated that multiple G-proteins are coupled to CRH receptors in the rat cerebral cortex. Because these investigations were carried out in a physiological membrane system, the G-protein/CRH receptor interactions described are believed to be representative of natural associations between these proteins. Our previous isoelectric focusing data suggested that the rat cerebral cortex contains at least three different CRH receptor types (Grammatopoulos and Hillhouse 1998) which belong to the type R1 CRH receptor family (Grammatopoulos, Phull & Hillhouse, unpublished observations) and might represent differentially glycosylated or other post-translational modification products of the CRH-R1. It is possible that the different CRH-R1 isoforms are coupled to different G-proteins. Although it is possible that post-translational modification can modify coupling of the receptor to specific G-proteins, at present we do not know whether each of the three CRH-R1 receptor isoforms are coupled to unique G-proteins and or all receptor isoforms are able to activate several G-proteins and the mechanism of coupling specificity is currently under investigation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by a Wellcome Trust Career Development Award to DG and a Walsgrave Hospitals NHS Trust R & D award Research and Teaching Development Fund to EWH. EWH holds the WPH Charitable Trust Chair of Medicine. The authors would like to thank Dr WF Schwindinger (Johns Hopkins University, Baltimore, MD, USA) for providing the method for [α-32P]GTP-γ-azidoanilide synthesis and purification and his useful comments and advice.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Ahmed A. H. & Heppel L. A. (1997) Evidence for a role of G protein beta gamma subunits in the enhancement of cAMP accumulation and DNA synthesis by adenosine in human cells. J. Cell. Physiol. 170, 263271.DOI: 10.1002/(sici)1097-4652(199703)170:3<263::aid-jcp7>3.0.co;2-m
  • Besedovsky H., Del Ray A., Sorkin E. & Dinarello C. (1986) Immunoregulatory feedback between interleukin-1 and glucocorticoids. Science 233, 652654.
  • Chen F., Bilezikjian L., Perrin M., Rivier J. & Vale W. (1986) Corticotropin releasing factor receptor-mediated stimulation of adenylate cyclase activity in the rat brain. Brain Res. 381, 4957.
  • Chilvers E. R., Batty I. H., Challiss R. A., Barnes P. J. & Nahorski S. R. (1991) Determination of mass changes in phosphatidylinositol 4,5-bisphosphate and evidence for agonist-stimulated metabolism of inositol 1,4,5-trisphosphate in airway smooth muscle. Biochem. J. 15, 373379.
  • Chrousos G. P., Torpy D. J. & Gold P. W. (1998) Interactions between the hypothalamic-pituitary-adrenal axis and the female reproductive system: clinical implications. Ann. Intern. Med. 129, 229240.
  • Fields T. A. & Casey P. J. (1997) Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins. Biochem. J. 321, 561571.
  • Fisher L. A. (1989) Corticotropin-releasing factor: endocrine and autonomic integration of responses to stress. Trends Pharmacol. Sci. 10, 189193.
  • Fisher L. A., Rivier J., Rivier C., Spiess J., Vale W. W. & Brown M. R. (1982) Corticotropin-releasing factor (CRF): central effects on mean arterial pressure and heart rate in rats, Endocrinology 110, 22222224.
  • Grammatopoulos D. & Hillhouse E. W. (1998) Solubilization and biochemical characterization of the human myometrial corticotrophin-releasing hormone receptor. Mol Cell. Endocrinol. 138, 185198.DOI: 10.1016/s0303-7207(97)00238-4
  • Grammatopoulos D., Kanellopoulou A. & Hillhouse E. W. (1994) The rat cerebral cortical CRF-41 receptor-the effects of cholera and pertussis toxins. J. Endocrinol. 140, P188.
  • Grammatopoulos D., Dai Y., Randeva H. S., Levine M. A., Karteris E., Easton A. J. & Hillhouse E. W. (1999) A novel spliced variant of the type 1 corticotropin-releasing hormone (CRH) receptor with a deletion in the 7th transmembrane domain present in the human pregnant term myometrium and fetal membranes Molec. Endocrinology 13, 21892202.
  • Harris-Warrick R. M., Hammond C., Paupardin-Tritsch D., Homburger V., Rouot B., Bockaert J. & Gerschenfeld H. M. (1988) An alpha 40 subunit of a GTP-binding protein immunologically related to Go mediates a dopamine-induced decrease of Ca2+ current in snail neurons. Neuron 1, 2732.
  • Hellevuo K., Hoffman P. L. & Tabakoff B. (1996) Adenylyl cyclases: mRNA and characteristics of enzyme activity in three areas of brain. J. Neurochem. 67, 177185.
  • Irwin M. (1993) Brain corticotropin-releasing hormone- and interleukin-1 beta-induced suppression of specific antibody production. Endocrinology 133, 13521360.
  • Karalis K., Sano H., Listwak S. & Chrousos G. (1991) Autocrine or paracrine inflammatory actions of corticotropin-releasing hormone in vivo. Science 254, 421423.
  • Karteris E., Grammatopoulos D., Randeva H. S. & Hillhouse E. W. (2000) Signal transduction characteristics of the CRH receptor in the feto-placenta unit. J. Clin. Endocrinol. Metab. 85, 19891996.
  • Kehlenbach R. H., Matthey J. & Huttner W. B. (1994) XL alpha s is a new type of G protein. Nature 372, 804809.
  • Kiang J. G. (1997) Corticotropin-releasing factor-like peptides increase cytosolic [Ca2+] in human epidermoid A-431 cells. Eur. J. Pharmacol. 329, 237244.DOI: 10.1016/s0014-2999(97)00165-9
  • Koob G. R. (1985) Stress, corticotropin-releasing factor and behaviour. Perspect. Behav. Med. 2, 3952.
  • Laugwitz K. L., Allgeier A., Offermanns S., Spicher K., Van Sande J., Dumont J. E. & Schultz G. (1996) The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families. Proc. Natl Acad. Sci. USA 93, 116120. DOI: 10.1073/pnas.93.1.116
  • Lovenberg T. W., Liaw C. W., Grigoriadis D. E., Clevenger W., Chalmers D. T., De Souza E. B. & Oltersdorf T. (1995) Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain. Proc Natl Acad. Sci. USA 92, 836840.
  • May L. G. & Gay C.V. (1997) Multiple G-protein involvement in parathyroid hormone regulation of acid production by osteoclasts. J. Cell. Biochem. 64, 161170.DOI: 10.1002/(sici)1097-4644(199701)64:1<161::aid-jcb18>3.0.co;2-o
  • Mons N., Yoshimura M., Ikeda H., Hoffman P. L. & Tabakoff B. (1998) Immunological assessment of the distribution of type VII adenylyl cyclase in brain. Brain Res. 788, 251261.DOI: 10.1016/s0006-8993(98)00005-5
  • Moss J. & Vaughan M. (1988) ADP-ribosylation of guanyl nucleotide-binding regulatory proteins by bacterial toxins. Adv. Enzymol. 61, 303379.
  • Nabhan C., Xiong Y., Xie L. Y. & Abou-Samra A. B. (1995) The alternatively spliced type II cortictropin-releasing factor receptor, stably expressed in LLCPK-1 cells, is not well coupled to the G protein (s). Biochem. Biophys. Res. Commun. 212, 10151021.DOI: 10.1006/bbrc.1995.2071
  • Offermanns S., Schultz G. & Rosenthal W. (1991) Identification of receptor-activated G-proteins with photoreactive GTP analog [α-32P]GTP azidoanilide. Meth. Enzymol. 195, 286299.
  • Palmer S., Hughes K. T., Lee D. Y. & Wakelam M. J. (1989) Development of a novel, Ins (1,4,5), P3-specific binding assay. Its use to determine the intracellular concentration of Ins (1,4,5), P3 in unstimulated and vasopressin–stimulated rat hepatocytes. Cell Signal. 1, 14756.
  • Perrin M., Donaldson C., Chen R., Lewis K. A. & Vale W. W. (1993) Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor. Endocrinology 133, 30583061.
  • Primus R. J., Yevich E., Baltazar C. & Gallager D. W. (1997) Autoradiographic localization of CRF1 and CRF2 binding sites in adult rat brain. Neurophychopharmacol. 17, 308316.
  • Schwindinger W. F., Fredericks J., Watkins L., Robinson H., Bathon J. M., Pines M., Suva L. J. & Levine M. A. (1998) Coupling of the PTH/PTHrP receptor to multiple G-proteins: direct demonstration of receptor activation of Gs, Gq/11, and Gi1 by [α-32P]GTP-γ-azidoanilide photoaffinity labeling. Endocrine 8, 201209.
  • Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provensano M. D., Fujimoto E. K., Goeke N. M., Olson B. J. & Klenk D. C. (1985) Measurment of protein using bicinchoninic acid. Anal. Biochem. 150, 7685.
  • Swanson L. W., Sawchenko P. E., Rivier J. & Vale W. (1983) Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinol. 36, 165186.
  • Takuma K., Matsuda T., Yoshikawa T., Kitanaka J., Gotoh M., Hayata K. & Baba A. (1994) Corticotropin-releasing factor stimulates Ca2+ influx in cultured rat astrocytes. Biochem. Biophys. Res. Commun. 199, 11031107.DOI: 10.1006/bbrc.1994.1344
  • Tsu R. C. & Wong Y. H. (1996) Gi-mediated stimulation of type II adenylyl cyclase is augmented by Gq-coupled receptor activation and phorbol ester treatment. J. Neurosci. 16, 13171323.
  • Ulisse S., Fabbri A., Tinajero J. C. & Dufau M. L. (1990) A novel mechanism of action of corticotropin-releasing factor in rat Leydig cells. J. Biol. Chem. 265, 19641971.
  • Vale W., Spiess J., Rivier C. L. & Rivier J. (1981) Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and β-endorphin. Science 213, 13941397.
  • Vaughan J., Donaldson C., Lewis K., Sutton S., Chan R., Turnbull A. V., Lovejoy D., Rivier C., Rivier J., Sawchenko P. E. & Vale W. (1995) Urocortin, a mamalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 378, 287292.
Footnotes
  1. 1DG and HR should both be considered first authors by virtue of their equivalent and unique contributions to this work.