Pharmacological characterization of JNJ-40068782, a new potent, selective, and systemically active positive allosteric modulator of the mGlu2 receptor and its radioligand [3H]JNJ-40068782.

Modulation of the metabotropic glutamate type 2 (mGlu2) receptor is considered a promising target for the treatment of central nervous system diseases such as schizophrenia. Here, we describe the pharmacological properties of the novel mGlu2 receptor positive allosteric modulator (PAM) 3-cyano-1-cyclopropylmethyl-4-(4-phenyl-piperidin-1-yl)-pyridine-2(1H)-one (JNJ-40068782) and its radioligand [(3)H]JNJ-40068782. In guanosine 5'-O-(3-[(35)S]thio)triphosphate binding, JNJ-40068782 produced a leftward and upward shift in the glutamate concentration-effect curve at human recombinant mGlu2 receptors. The EC50 of JNJ-40068782 for potentiation of an EC20-equivalent concentration of glutamate was 143 nM. Although JNJ-40068782 did not affect binding of the orthosteric antagonist [(3)H]2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY-341495), it did potentiate the binding of the agonist [(3)H](2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine (DCG-IV), demonstrating that it can allosterically affect binding at the agonist recognition site. The binding of [(3)H]JNJ-40068782 to human recombinant mGlu2 receptors in Chinese hamster ovary cells and rat brain receptors was saturable with a KD of ∼10 nM. In rat brain, the anatomic distribution of [(3)H]JNJ-40068782 was consistent with mGlu2 expression previously described and was most abundant in cortex and hippocampus. The ability of structurally unrelated PAMs to displace [(3)H]JNJ-40068782 suggests that PAMs may bind to common determinants within the same site. It is noteworthy that agonists also increased the binding affinity of [(3)H]JNJ-40068782. JNJ-40068782 influenced rat sleep-wake organization by decreasing rapid eye movement sleep with a lowest active dose of 3 mg/kg PO. In mice, JNJ-40068782 reversed phencyclidine-induced hyperlocomotion with an ED50 of 5.7 mg/kg s.c. Collectively, the present data demonstrate that JNJ-40068782 has utility in investigating the potential of mGlu2 modulation for the treatment of diseases characterized by disturbed glutamatergic signaling and highlight the value of [(3)H]JNJ-40068782 in exploring allosteric binding.

Structure-activity relationships of 2-arylamido-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide derivatives as cannabinoid receptor agonists and their analgesic action.

SAR studies were performed on a series of 2-arylamido-5,7-dihydro-4H-thieno[2,3-c]pyran-3-carboxamide derivatives as cannabinoid receptor agonists. Starting from a HTS hit both potency and selectivity could be improved. Modifications to the thiophene fusion and C-3 amides were studied. A representative compound 3t produced analgesia when dosed orally in inflammatory pain models of writhing and carrageenan-induced allodynia.

Molecular mechanisms of corticotropin-releasing factor receptor-induced calcium signaling.

The molecular mechanisms governing calcium signal transduction of corticotropin-releasing factor (CRF) receptors CRF(1) and CRF(2(a)) stably expressed in human embryonic kidney (HEK) 293 cells were investigated. Calcium signaling strictly depended on intracellular calcium sources, and this is the first study to establish a prominent contribution of the three major G-protein families to CRF receptor-mediated calcium signaling. Overexpression of Galpha(q/11) and Galpha(16) led to leftward shifts of the agonist concentration-response curves. Blockade of Galpha(q/11) proteins by the small interfering RNA (siRNA) technology partially reduced agonist-mediated calcium responses in CRF(1)- and CRF(2(a))-expressing HEK293 cells, thereby proving a contribution of the G(q) protein family. A small but significant inhibition of calcium signaling was recorded by pharmacological inhibition of G(i/o) proteins with pertussis toxin treatment. This effect was mediated by direct binding of Gbetagamma subunits to phospholipase C. G(i/o) inhibition also elevated cAMP responses in CRF receptor-overexpressing HEK293 cells and in Y79 retinoblastoma cells endogenously expressing human CRF(1) and CRF(2(a)) receptors, thereby demonstrating natural coupling of G(i) proteins to both CRF receptors. The strongest reduction of CRF receptor-mediated calcium mobilization was noted when blocking the G(s) signaling protein either by cholera toxin or by siRNA. It is noteworthy that simultaneous inhibition of two G-proteins shed light on the additive effects of G(s) and G(q) on the calcium signaling and, hence, that they act in parallel. On the other hand, G(i) coupling required prior G(s) activation.

CRF1 Receptor Signaling via the ERK1/2-MAP and Akt Kinase Cascades: Roles of Src, EGF Receptor, and PI3-Kinase Mechanisms.

In the present study, we determined the cellular regulators of ERK1/2 and Akt signaling pathways in response to human CRF1 receptor (CRF1R) activation in transfected COS-7 cells. We found that Pertussis Toxin (PTX) treatment or sequestering Gßγ reduced CRF1R-mediated activation of ERK1/2, suggesting the involvement of a Gi-linked cascade. Neither Gs/PKA nor Gq/PKC were associated with ERK1/2 activation. Besides, CRF induced EGF receptor (EGFR) phosphorylation at Tyr1068, and selective inhibition of EGFR kinase activity by AG1478 strongly inhibited the CRF1R-mediated phosphorylation of ERK1/2, indicating the participation of EGFR transactivation. Furthermore, CRF-induced ERK1/2 phosphorylation was not altered by pretreatment with batimastat, GM6001, or an HB-EGF antibody indicating that metalloproteinase processing of HB-EGF ligands is not required for the CRF-mediated EGFR transactivation. We also observed that CRF induced Src and PYK2 phosphorylation in a Gßγ-dependent manner. Additionally, using the specific Src kinase inhibitor PP2 and the dominant-negative-SrcYF-KM, it was revealed that CRF-stimulated ERK1/2 phosphorylation depends on Src activation. PP2 also blocked the effect of CRF on Src and EGFR (Tyr845) phosphorylation, further demonstrating the centrality of Src. We identified the formation of a protein complex consisting of CRF1R, Src, and EGFR facilitates EGFR transactivation and CRF1R-mediated signaling. CRF stimulated Akt phosphorylation, which was dependent on Gi/ßγ subunits, and Src activation, however, was only slightly dependent on EGFR transactivation. Moreover, PI3K inhibitors were able to inhibit not only the CRF-induced phosphorylation of Akt, as expected, but also ERK1/2 activation by CRF suggesting a PI3K dependency in the CRF1R ERK signaling. Finally, CRF-stimulated ERK1/2 activation was similar in the wild-type CRF1R and the phosphorylation-deficient CRF1R-Δ386 mutant, which has impaired agonist-dependent ß-arrestin-2 recruitment; however, this situation may have resulted from the low ß-arrestin expression in the COS-7 cells. When ß-arrestin-2 was overexpressed in COS-7 cells, CRF-stimulated ERK1/2 phosphorylation was markedly upregulated. These findings indicate that on the base of a constitutive CRF1R/EGFR interaction, the Gi/ßγ subunits upstream activation of Src, PYK2, PI3K, and transactivation of the EGFR are required for CRF1R signaling via the ERK1/2-MAP kinase pathway. In contrast, Akt activation via CRF1R is mediated by the Src/PI3K pathway with little contribution of EGFR transactivation.

Nociceptive and behavioural sensitisation by protein kinase Cepsilon signalling in the CNS.

Despite the apparent homology in the protein kinase C (PKC) family, it has become clear that slight structural differences are sufficient to have unique signalling properties for each individual isoform. For PKCepsilon in depth investigation of these aspects revealed unique actions in the CNS and lead to development of specific modulators with clinical perspective. In this review, we describe to which extent PKCepsilon is distinct from other isoforms on the level of tissue expression and protein structure. As this kinase is highly expressed in the brain, we outline three main aspects of PKCepsilon signalling in the CNS. First, its ability to alter the permeability of N-type Ca2+ channels in dorsal root ganglia has been shown to enhance nociception. Secondly, PKCepsilon increases anxiety by diminishing GABA(A)R-induced inhibitory post-synaptic currents in the prefrontal cortex. Another important aspect of the latter inhibition is the reduced sensitivity of GABA(A) receptors to ethanol, a mechanism potentially contributing to abuse. A third signalling cascade improves cognitive functions by facilitating cholinergic signalling in the hippocampus. Collectively, these findings point to a physical and behavioural sensitising role for this kinase.

Expression, binding, and signaling properties of CRF2(a) receptors endogenously expressed in human retinoblastoma Y79 cells: passage-dependent regulation of functional receptors.

Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF(1)-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of approximately 100 pmoles/10(5) cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of approximately 25-30 pmoles/10(5) cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the approximately 300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 +/- 1.6 nmol/L), whereas the CRF(1)-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 microL. When the CRF(1)-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 +/- 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 +/- 0.2 microL). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2a receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture.

Therapeutic potential of group III metabotropic glutamate receptors.

Metabotropic glutamate (mGlu) receptors have received much attention, driven by a strong belief in the potential of these modulatory glutamate receptors as drug targets. So far, major drug discovery programs have largely focused on group I (mGlu1 and 5) and II (mGlu2 and 3) mGlu receptors, which have been implicated in neuropathological and various psychiatric disorders. The four group III representatives (mGlu4, mGlu6, mGlu7 and mGlu8) are less understood, mainly due to the paucity of specific compounds. Recent advances in the identification of selective or specific compounds, and the generation of transgenic animals have, however, revealed important insights into the potential role of group III receptors in the pathophysiology of neurological and mood disorders. Activation of the mGlu4 receptor seems to be beneficial for treating Parkinson-like symptoms and a potential role in the treatment of mood disorders is slowly growing. Similarly, genetic inactivation studies and usage of relatively selective agonists strongly support the involvement of the mGlu8 receptor for anxiety disorders. In contrast, controversial data have been obtained for the mGlu7 receptor. While mGlu7 receptor-deficient animals show an anxiolytic profile in several in vivo readouts, the first selective allosteric agonist AMN082 has also been reported to improve anxiety-like behaviour despite activating the stress axis. The least investigated receptor remains the mGlu6 receptor, which is mostly based on its predominant expression in the retina.

Blocking melanin-concentrating hormone MCH1 receptor affects rat sleep-wake architecture.

Melanin-concentrating hormone (MCH) is a hypothalamic peptide that centrally regulates food intake, energy balance and emotion. Interestingly, MCH and melanin-concentrating hormone MCH(1) receptors are distributed in brain areas known to regulate vigilance states. Effects of subcutaneous administration of two selective melanin-concentrating hormone MCH(1) receptor antagonists, labeled A and B were examined over a broad dose range (1, 3, 10, 20, 40 mg/kg) on rat sleep-wake architecture. Both compounds have a nanomolar antagonist activity at recombinant human melanin-concentrating hormone MCH(1) receptor (IC(50)=44.1+/-6.1 nM and 26.6+/-5.4 nM, respectively) and potently inhibited the MCH-induced mobilization of [Ca(2+)] (IC(50) 29.1+/-8.1 nM and 10.5+/-4.1 nM, respectively). The selectivity of both compounds was further confirmed on a panel of receptors, transporters and channels. In vivo, both compounds dose-dependently decreased deep sleep primarily by decreasing the mean duration of episodes during the first 4 h post-administration. In parallel, REM sleep and intermediate stage sleep were decreased while active and passive waking increased. Deep sleep and REM sleep onset latencies were significantly prolonged at higher doses. No homeostatic rebound of deep sleep was observed, while a tendency for recovery of REM sleep was found during subsequent dark phase. Together, the results support a role of the melanin-concentrating hormone MCH(1) receptor in the regulation of deep slow-wave sleep-REM sleep cycle. Therapeutic application of melanin-concentrating hormone MCH(1) receptor-inhibiting agents should take into account the significant decreases in deep sleep without recovery as these may interfere with sleep dependent memory consolidation.

Urocortins of the South African clawed frog, Xenopus laevis: conservation of structure and function in tetrapod evolution.

Several corticotropin-releasing factor (CRF) family genes have been identified in vertebrates. Mammals have four paralogous genes that encode CRF or the urocortins 1, 2, and 3. In teleost fishes, a CRF, urotensin I (a fish ortholog of mammalian urocortin 1) and urocortin 3 have been identified, suggesting that at least three of the four mammalian lineages arose in a common ancestor of modern bony fishes and tetrapods. Here we report the isolation of genes orthologous to mammalian urocortin 1 and urocortin 3 from the South African clawed frog, Xenopus laevis. We characterize the pharmacology of the frog peptides and show that X. laevis urocortin 1 binds to and activates the frog CRF1 and CRF2 receptors at picomolar concentrations. Similar to mammals, frog urocortin 3 is selective for the CRF2 receptor. Only frog urocortin 1 binds to the CRF-binding protein, although with significantly lower affinity than frog CRF. Both urocortin genes are expressed in brain, pituitary, heart, and kidney of juvenile frogs; urocortin 1 is also expressed in skin. We also identified novel urocortin sequences in the genomes of pufferfish, zebrafish, chicken, and dog. Phylogenetic analysis supports the view that four paralogous lineages of CRF-like peptides arose before the divergence of the actinopterygian and sarcopterygian fishes. Our findings show that the functional relationships among CRF ligands and binding proteins, and their anorexigenic actions mediated by the CRF2 receptor, arose early in vertebrate evolution.

The CRF peptide family and their receptors: yet more partners discovered.

Abnormal signaling at corticotropin-releasing factor CRF1 and CRF2 receptors might contribute to the pathophysiology of stress-related disorders such as anxiety, depression and eating disorders, in addition to cardiac and inflammatory disorders. Recently, molecular characterization of CRF1 and CRF2 receptors and the cloning of novel ligands--urocortin, stresscopin-related peptide/urocortin II, and stresscopin/urocortin III--have revealed a far-reaching physiological importance for the family of CRF peptides. Although the physiological roles of the CRF2 receptor remain to be defined, the preclinical and clinical development of specific small-molecule antagonists of the CRF1 receptor opens new avenues for the treatment of psychiatric and neurological disorders.

Establishment of robust functional assays for the characterization of neuropeptide Y (NPY) receptors: identification of 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile as selective NPY type 5 receptor antagonist.

The human Neuropeptide Y (NPY) receptors 1 (hY1), 2 (hY2), 4 (hY4), and the mouse type 5 (mY5) receptor were expressed in human embryonic kidney 293 (HEK293) cells. The receptors bound a radioiodinated NPY ligand with high affinity and various NPY analogs competed for binding in a receptor selective-manner. Similarly, cAMP-inhibition and GTPgammaS binding assays were established. The four NPY receptors were further tested in the fluorimetric imaging plate reader (FLIPR) format, a cellular high-throughput assay, in the absence and presence of chimeric G proteins, Gqo5, Gqi5 and Gqi9. The receptors stimulated transient calcium release only in the presence of chimeric G proteins. While hY1, hY2 and hY4 receptors coupled to Gqo5, Gqi5 and Gqi9, the mY5 receptor stimulated transient calcium release only when co-expressed with Gqi9. Using an in silico screening approach we identified a small molecule 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile (compound 1), which bound to the mY5 receptor with high affinity (Ki=32.1+/-1.8 nM), competitively antagonized NPY-mediated GTPgammaS binding and calcium stimulation with high potency, and had no affinity for other NPY receptors. These data show that NPY receptors can be functionally coupled to the FLIPR readout, allowing for high throughput compound testing and identification of novel molecules.

Stimulation of neuropeptide Y-mediated calcium responses in human SMS-KAN neuroblastoma cells endogenously expressing Y2 receptors by co-expression of chimeric G proteins.

Human SMS-KAN neuroblastoma cells endogenously express the neuropeptide Y (NPY) type 2 (Y(2)) receptor. Although ligand binding and GTPgammaS binding studies supported high functional Y(2) receptor expression, only weak coupling to the natural second messenger cyclic AMP was observed. The main reason was the low responsiveness of SMS-KAN cells to forskolin, a direct activator of adenylyl cyclases. In order to obtain a cell-based functional assay for the Y(2) receptor in SMS-KAN cells, the transient calcium (Ca(2+)) mobilization assay in the fluorimetric imaging plate reader (FLIPR) format was established by stably expressing a chimeric G protein Gq(i9). This manipulation resulted in robust mobilization of Ca(2+) after challenge with various NPY-related agonists in a 384-well format. The sensitivity of the FLIPR readout was in the low nanomolar range for NPY agonists and comparable to that of the recombinant Y(2) receptor. The selective Y(2) antagonist BIIE0246 competitively inhibited NPY-mediated Ca(2+) transients in SMS-KAN/Gq(i9) cells with a pA(2) value of 7.39+/-0.1. This is the first evidence that an endogenously expressed G protein-coupled receptor couples to an overexpressed chimeric G protein, thereby functionally responding in the FLIPR readout.

Structure-activity studies of orexin a and orexin B at the human orexin 1 and orexin 2 receptors led to orexin 2 receptor selective and orexin 1 receptor preferring ligands.

The neuropeptides orexin A and B (also known as hypocretins) play an important role in many physiological and behavioral activities. Orexins are ligands of two closely related G-protein-coupled receptors, that are the named orexin 1 and orexin 2 receptors. To clearly identify the minimal ligand sequences required for receptor activation, we synthesized and analyzed different centrally, C- and N-terminally truncated analogues of orexins A and B. Furthermore, we used the shortest active analogue to screen for important amino acid residues by l-alanine and l-proline replacement scans. For orexin A, only full-length peptides were able to show the same activity as orexin A, but interestingly, reduced orexin A and natural orexin A, which contains the two disulfide bonds, had the same activity. The shortest highly active orexin B analogue was orexin B 6-28. In addition, we identified orexin A 2-33 as the first analogue with orexin 1 receptor preference and orexin B 10-28, [A27]orexin B 6-28, and [P11]orexin B 6-28 as being highly potent orexin 2 receptor selective (>1000-fold) peptides.

Novel hexahydrospiro[piperidine-4,1'-pyrrolo[3,4-c]pyrroles]: highly selective small-molecule nociceptin/orphanin FQ receptor agonists.

Novel hexahydrospiro[piperidine-4,1'-pyrrolo[3,4-c]pyrroles that act as potent and selective orphanin FQ/nociceptin (N/OFQ) receptor (NOP) agonists were identified. The best compound, (+)-5a, potently inhibited 3H-N/OFQ binding to the NOP receptor (K(i) = 0.49 nM) but was >1000-fold less potent in binding to MOP, KOP, and DOP opiate receptors. Further, (+)-5a potently stimulated GTP gamma S binding to NOP membranes (EC50 = 65 nM) and inhibited forskolin-mediated cAMP accumulation in NOP-expressing cells (EC50 = 9.1 nM) with a potency comparable to that of the natural peptide agonist N/OFQ. These results indicate that (+)-5a is a highly selective and potent small-molecule full agonist of the NOP receptor.

Cell-type specific calcium signaling by corticotropin-releasing factor type 1 (CRF1) and 2a (CRF2(a)) receptors: phospholipase C-mediated responses in human embryonic kidney 293 but not SK-N-MC neuroblastoma cells.

The human corticotropin-releasing factor (hCRF) receptors CRF1 and CRF2(a) couple to the Gs protein. It has been postulated that CRF receptors may also signal through phospholipase C (PLC). To test this hypothesis, binding and signaling properties were determined for both receptor subtypes stably expressed in human embryonic kidney 293 (HEK293) and human SK-N-MC neuroblastoma cells. CRF receptors were highly expressed and strongly coupled to Gs in HEK293 and SK-N-MC cells. However, when the calcium mobilization pathway was investigated, marked differences were observed. In SK-N-MC cells, neither CRF receptor stimulated calcium mobilization in the fluorometric imaging plate reader (FLIPR) assay, whereas activation of orexin type 1 and 2 receptors stably expressed in SK-N-MC cells revealed robust calcium responses. In contrast, intracellular calcium was strongly mobilized by agonist stimulation of hCRF1 and hCRF2(a) receptors in HEK293 cells. In HEK293 cells, potency rank orders for calcium and cAMP responses were identical for both receptors, despite a rightward shift of the dose-response curves. Complete inhibition of calcium signaling of both hCRF1 and hCRF2(a) receptors was observed in the presence of the PLC inhibitor U-73,122 whereas ryanodine, an inhibitor of calcium release channels and the protein kinase A inhibitor Rp-cAMPS were ineffective. Finally, CRF agonists produced a small but significant stimulation of inositol 1,4,5-triphosphate (IP3) accumulation in hCRF1-and hCRF2(a)-transfected HEK293 cells. These data clearly show that phospholipase C-mediated signaling of CRF receptors is dependent upon the cellular background and that in HEK293 cells human CRF receptors robustly respond in the FLIPR format.

Secondary structure of antisauvagine analogues is important for CRF receptor antagonism: development of antagonists with increased potency and receptor selectivity.

Antisauvagine-30 (aSVG) is the only high-affinity antagonist for the corticotropin-releasing factor (CRF) type 2 (CRF(2)) receptor. A structure-activity relationship study was performed to pinpoint residues conferring aSVG's selectivity. The aSVG-analogues being N-terminally extended by one or two residues or containing the Ala(22)Arg(23)Ala(24) (ARA-motif) of CRF, were synthesized. Additionally, a lactam bridge between positions 29 and 32 was introduced. The modified peptides were analyzed for alpha-helicity properties, binding affinities and antagonistic potencies at the rat CRF(1) and mouse CRF(2B) receptors. While N-terminal prolongation and replacement of D-Phe(11) by Tyr(11) increased the affinity for the CRF(2) receptor, the introduction of the ARA motif resulted in a loss of CRF(2) receptor selectivity. These data show that aSVG(10-40) analogues are more potent CRF(2) receptor antagonists than aSVG(11-40) peptides, while introduction of the ARA-motif or a cyclic constraint between residues 29 and 32 favors binding to the CRF(1) receptor.

Binding differences of human and amphibian corticotropin-releasing factor type 1 (CRF(1)) receptors: identification of amino acids mediating high-affinity astressin binding and functional antagonism.

The corticotropin-releasing factor (CRF) type 1 receptors (CRF(1)) from human (hCRF(1)) and Xenopus (xCRF(1)) differ from one another by their agonist- and antagonist-binding preference. While the agonist-binding site of the xCRF(1) receptor has been mapped, the amino acids that mediate binding of the potent peptide antagonist astressin are unknown. By constructing receptor chimeras followed by site-directed mutagenesis, the astressin-binding site of the xCRF(1) receptor was located between residues 76 and 83. This region partially overlaps with the agonist-selective domain of the xCRF(1) receptor (residues 76-89). Mutagenesis of the amphibian residues Gln(76), Gly(81) and Val(83) to the human sequence (Arg(76)Asn(81)Gly(83)) generated a receptor mutant that bound astressin with even higher affinity than the native hCRF(1) receptor. An amino acid doublet (Glu(70)Tyr(71)) that is conserved in the xCRF(1) and hCRF(2(a)) receptor after incorporation into the hCRF(1) receptor sequence was found to facilitate antagonist binding up to 15-fold higher. In agreement with the binding data, astressin was a more potent functional antagonist at receptors expressing the Glu(70)Tyr(71) motif. These data show that the agonist- and antagonist-binding sites of the hCRF(1) receptor partially overlap and that two amino acids within the N terminus of the hCRF(1) receptor negatively influence binding and functional antagonism of astressin.

Molecular cloning and functional expression of the mouse CRF2(a) receptor splice variant.

The mouse corticotropin-releasing factor (CRF) type 2a receptor (CRF2(a)) splice variant was cloned by a PCR-based approach. The corresponding cDNA was found to encode a 411-amino acid polypeptide with highest sequence homology to the rat CRF2(a) receptor. By semiquantitative reverse transcriptase PCR (RT-PCR) analysis, the CRF2(b) mRNA was mainly found in the heart and skeletal muscle with only low level expression in the brain. In contrast, CRF2(a) mRNA was restricted to the brain with major expression sites in the cortex, hippocampus, hypothalamus and telencephalon. Binding and cyclic AMP stimulation studies showed a similar ligand selective profile for both mCRF2 receptor splice variants. A notable exception however, was urotensin I which displayed a approximately 3-fold higher affinity for the CRF2(a) receptor and also stimulated cyclic AMP production in mCRF2(a)-transfected cells with a approximately 3-fold higher potency than in mCRF2(b)-transfected cells. These data show that the mouse like other mammalian species expresses two ligand-selective CRF2 receptor splice variants and that the mCRF2(a) receptor is the predominant central CRF2 receptor in the mouse.

Desensitization of human CRF2(a) receptor signaling governed by agonist potency and ßarrestin2 recruitment.

The primary goal was to determine agonist-specific regulation of CRF2(a) receptor function. Exposure of human retinoblastoma Y79 cells to selective (UCN2, UCN3 or stresscopins) and non-selective (UCN1 or sauvagine) agonists prominently desensitized CRF2(a) receptors in a rapid, concentration-dependent manner. A considerably slower rate and smaller magnitude of desensitization developed in response to the weak agonist CRF. CRF1 receptor desensitization stimulated by CRF, cortagine or stressin1-A had no effect on CRF2(a) receptor cyclic AMP signaling. Conversely, desensitization of CRF2(a) receptors by UCN2 or UCN3 did not cross-desensitize Gs-coupled CRF1 receptor signaling. In transfected HEK293 cells, activation of CRF2(a) receptors by UCN2, UCN3 or CRF resulted in receptor phosphorylation and internalization proportional to agonist potency. Neither protein kinase A nor casein kinases mediated CRF2(a) receptor phosphorylation or desensitization. Exposure of HEK293 or U2OS cells to UCN2 or UCN3 (100nM) produced strong ßarrestin2 translocation and colocalization with membrane CRF2(a) receptors while CRF (1µM) generated only weak ßarrestin2 recruitment. ßarrestin2 did not internalize with the receptor, however, indicating that transient CRF2(a) receptor-arrestin complexes dissociate at or near the cell membrane. Since deletion of the ßarrestin2 gene upregulated Gs-coupled CRF2(a) receptor signaling in MEF cells, a ßarrestin2 mechanism restrains Gs-coupled CRF2(a) receptor signaling activated by urocortins. We further conclude that the rate and extent of homologous CRF2(a) receptor desensitization are governed by agonist-specific mechanisms affecting GRK phosphorylation, ßarrestin2 recruitment, and internalization thereby producing unique signal transduction profiles that differentially affect the stress response.

Molecular and cell signaling targets for PTSD pathophysiology and pharmacotherapy.

The reasons for differences in vulnerability or resilience to the development of posttraumatic stress disorder (PTSD) are unclear. Here we review key genetic diatheses and molecular targets especially signaling pathways that mediate responses to trauma and severe stress and their potential contribution to the etiology of PTSD. Sensitization of glucocorticoid receptor (GR) signaling and dysregulation of GR modulators FKBP5, STAT5B, Bcl-2, and Bax have been implicated in PTSD pathophysiology. Furthermore, Akt, NFκB, MKP-1, and p11, which are G protein-coupled receptor (GPCR) pathway molecules, can promote or prevent sustained high anxiety- and depressive-like behavior following severe stress. Agonist-induced activation of the corticotropin releasing factor CRF(1) receptor is crucial for survival in the context of serious danger or trauma, but persistent CRF(1) receptor hypersignaling when a threatening or traumatic situation is no longer present is maladaptive. CRF(1) receptor single nucleotide polymorphisms (SNPs) can confer susceptibility or resilience to childhood trauma while a SNP for the PAC1 receptor, another class B1 GPCR, has been linked genetically to PTSD. GRK3 phosphorylation of the CRF(1) receptor protein and subsequent binding of ßarrestin2 rapidly terminate Gs-coupled CRF(1) receptor signaling by homologous desensitization. A deficient GRK-ßarrestin2 mechanism would result in excessive CRF(1) receptor signaling thereby contributing to PTSD and co-morbid posttraumatic depression. Clinical trials are needed to assess if small molecule CRF(1) receptor antagonists are effective prophylactic agents when administered immediately after trauma. ßarrestin2-biased agonists for CRF receptors and possibly other GPCRs implicated in PTSD, however, may prove to be novel pharmacotherapy with greater selectivity and therapeutic efficacy. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.