A novel adenylyl cyclase-activating serotonin receptor (5-HT7) implicated in the regulation of mammalian circadian rhythms.

We report the cloning and characterization of a novel serotonin receptor, designated as 5-HT7, which is coupled to the stimulation of adenylyl cyclase. 5-HT7 mRNA is expressed discretely throughout the CNS, predominantly in the thalamus and hypothalamus. 5-HT7 has a unique pharmacological profile that redefines agonist and antagonist classification of ligands previously thought to be "selective." The circadian phase of spontaneous neuronal activity of the rat suprachiasmatic nucleus of the hypothalamus advances in response to serotonin ligands with a pharmacological profile consistent exclusively with that of 5-HT7. These findings suggest a physiological role in the regulation of circadian rhythms for one subtype of serotonin receptor, 5-HT7, and provide a pharmacological test to evaluate its role in other neuronal systems.

Corticotrophin-releasing factor receptors: from molecular biology to drug design.

Corticotrophin-releasing factor (CRF) acts within both the brain and the periphery to coordinate the overall response of the body to stress. The involvement of the CRF systems in a variety of both CNS and peripheral disease states has stimulated great interest in this peptide as a potential site of therapeutic intervention. The recent cloning of multiple CRF receptor subtypes has precipitated a new era in CRF research that has allowed precise molecular, pharmacological and anatomical examination of mammalian CRF receptors. In this article, Derek Chalmers and colleagues highlight the major differences between the two classes of CRF receptors, CRF1 and CRF2, and a functionally related CRF-binding protein, and discuss the relevance of these sites to the ongoing development of CRF-based therapeutics.

Localization of ligand-binding domains of human corticotropin-releasing factor receptor: a chimeric receptor approach.

Two CRF receptors, CRFR1 and CRFR2, have recently been cloned and characterized. CRFR1 shares 70% sequence identity with CRFR2, yet has much higher affinity for rat/human CRF (r/hCRF) than CRFR2. As a first step toward understanding the interactions between rat/human CRF and its receptor, the regions that are involved in receptor-ligand binding and/or receptor activation were determined by using chimeric receptor constructs of the two human CRFR subtypes, CRFR1 and CRFR2, followed by generating point mutations of the receptor. The EC50 values in stimulation of intracellular cAMP of the chimeric and mutant receptors for the peptide ligand were determined using a cAMP-dependent reporter system. Three regions of the receptor were found to be important for optimal binding of r/hCRF and/or receptor activation. The first region was mapped to the junction of the third extracellular domain and the fifth transmembrane domain; substitution of three amino acids of CRFR1 in this region (Val266, Tyr267, and Thr268) by the corresponding CRFR2 amino acids (Asp266, Leu267, and Val268) increased the EC50 value by approximately 10-fold. The other two regions were localized to the second extracellular domain of the CRFR1 involving amino acids 175-178 and His189 residue. Substitutions in these two regions each increased the EC50 value for r/hCRF by approximately 7- to 8-fold only in the presence of the amino acid 266-268 mutation involving the first region, suggesting that their roles in peptide ligand binding might be secondary.

Molecular approach to hypothalamic rhythms: isolation of novel indoleamine receptor genes.

We have utilized polymerase chain reaction with primers corresponding to conserved amino acid sequences within membrane-spanning regions of known serotonin receptors to identify clones of four putative new indoleamine receptors. We have determined complete amino acid sequences of these four receptors, which fall into three subfamilies; two of these subfamilies are novel. The sites of expression within the brain have been determined for each of the genes. Expression in mammalian cells demonstrates that each new protein is a receptor for serotonin and that each has a distinct pharmacology when compared to known receptors. Two of the new receptors are coupled to cyclic adenosine monophosphate, one negatively (Gi) and one positively (Gs). The latter is a candidate for the serotonin receptor that mediates phase advances in circadian rhythms of the suprachiasmatic nucleus.

CRF2 alpha and CRF2 beta receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues.

We have recently described the cloning and characterization of a novel corticotropin-releasing factor receptor subtype (CRF2) from rat brain that exists in two alternatively spliced forms, CRF2 alpha and CRF2 beta. These forms differ in their N-terminal coding sequence which results in the production of two distinct receptors of 411 and 431 amino acids, respectively. To assess whether these two forms might represent distinct targets for CRF action, RNase protection and in situ hybridization studies were performed using specific N-terminal cRNA probes. The results showed a differential distribution of the mRNAs for these two receptor forms in the rat. The mRNA for CRF2 alpha is found almost exclusively in the brain, particularly in the hypothalamus, lateral septum, and olfactory bulb, whereas the mRNA for CRF2 beta appears to be both in the brain and in the periphery, with the greatest abundance in the heart and skeletal muscle. Thus, the data suggest that these alternatively spliced forms of the CRF2 receptor may represent functionally distinct CRF receptors. In addition, it highlights the importance of probe specificity for in situ hybridization studies.

Cloning and characterization of the human corticotropin-releasing factor-2 receptor complementary deoxyribonucleic acid.

Two CRF receptor subtypes (CRF1 and CRF2 receptors) with distinct brain localizations and pharmacological profiles have recently been cloned and characterized. For the CRF2 receptor subtype, at least 2 splice forms with different 5'-coding sequences (CRF2 alpha and CRF2 beta) have been identified in rat. In this article, we report the genomic structure and the corresponding complementary DNA (cDNA) sequence of the human CRF2 receptor. The gene coding for human CRF2 receptor consists of at least 12 exons and spans approximately 30 kilobases. The cDNA sequence in the protein-coding region is 94% identical to that of the reported rat CRF2 alpha receptor. At present, there is no evidence for the existence of a CRF2 beta receptor homolog in humans. The encoded receptor is 411 amino acids in length and is 70% identical to the human CRF1 receptor, with least sequence homology in the N-terminal extracellular domain (47% identical). Cells transfected with the full-length human CRF2 receptor cDNA responded to rat/human CRF and sauvagine by increasing the intracellular cAMP level, with EC50 values of approximately 20 and 1 nM, respectively. The CRF- and sauvagine-induced accumulation of intracellular cAMP could be competitively inhibited by the CRF receptor antagonist D-Phe-CRF. This pharmacological profile was comparable to that of the rat CRF2 alpha receptor. The relative abundance of the CRF2 receptor messenger RNA appears to be lower in humans than in rats for the tissues studied thus far.

Anatomical distribution of prolactin-releasing peptide and its receptor suggests additional functions in the central nervous system and periphery.

A recently identified neuropeptide with PRL-releasing capabilities binds to and activates a previously known orphan G protein-coupled receptor, GPR10. We initiated a study to define the pharmacology of the peptide/receptor interaction and to identify the distribution of the peptide and its receptor in the central nervous system to elucidate sites of action of the peptide. The PRL-releasing peptide (PrRP) is a C-terminally amidated, 31-amino acid peptide derived from a 98-amino acid precursor. Radioiodinated PrRP-(1-31) binds to its receptor with high affinity (1 nM) and stimulates calcium mobilization in CHOK1 cells stably transfected with the receptor. A series of N-terminal deletions reveals that the PrRP-(12-31) amino acid is equipotent to PrRP-(1-31). Further N-terminal deletions reduce the affinity of the ligand considerably, although PrRP-(25-31) is still able to compete for binding and behaves as an agonist. The arginine residues at position 26 and 30 are critical for binding, as substitution with either lysine or citrulline reduces the affinity substantially. In situ hybridization reveals a distinct tissue distribution for both the peptide and receptor messenger RNAs. The receptor is expressed abundantly in the reticular thalamic nucleus, periventricular hypothalamus, dorsomedial hypothalamus, nucleus of the solitary tract, area postrema, anterior pituitary, and adrenal medulla. The peptide messenger RNA is expressed in the dorsomedial hypothalamus, nucleus of the solitary tract, ventrolateral reticular nucleus, and intestine. This tissue distribution suggests an alternative function of PrRP than its purported hypophysiotropic function, such as a potential role for PrRP in the central feedback control of neuroendocrine and autonomic homeostasis. Further work using selective agonists and antagonists should help define additional physiological roles of this novel mammalian neuropeptide.

Ser(13)-phosphorylated PYY from porcine intestine with a potent biological activity.

We have isolated a posttranslationally modified form of peptide YY (PYY) from porcine intestine and shown by MALDI-TOF and electrospray tandem mass spectrometry that it is phosphorylated at Ser(13). Phospho-PYY exhibits high affinity for binding to neuropeptide Y (NPY) receptors Y1, Y2 and Y5. The IC(50) values with the Y1, Y2, and Y5 receptor subtypes were for NPY 2.4, 3.1, and 3.3 nM, for PYY 2.3, 0.94, and 3.2 nM, and for phospho-PYY 4.6, 2.2, and 5.5 nM, respectively. Phospho-PYY potently inhibits forskolin-stimulated cAMP accumulation in SK-N-MC cells with an IC(50) value of 0.5 nM compared to 0.15 nM for non-phosphorylated PYY. The finding of phosphorylation of PYY is unusual among hormonal peptides, and emphasizes the importance of direct protein analysis of gene products.

trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine: a highly potent selective dopamine D1 full agonist.

trans-10,11-Dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phenan thridine (4a, dihydrexidine) has been found to be a highly potent and selective agonist of the dopamine D1 receptor in rat brain. Dihydrexidine had an EC50 of approximately 70 nM in activating dopamine-sensitive rat striatal adenylate cyclase and a maximal stimulation equal to or slightly greater than that produced by dopamine. Dihydrexidine had an IC50 of 12 nM in competing for [3H]SCH23390 (1a) binding sites in rat striatal homogenate, and of 120 nM versus [3H]spiperone. These data demonstrate that dihydroxidine has about ten-fold selectivity for D1/D2 receptors. More importantly, however, is the fact that dihydrexidine is a full agonist. Previously available agents, such as SKF38393 (1b), while being somewhat more selective for the D1 receptor, are only partial agonists. The isomeric cis-dihydroxybenzo[a]-phenanthridine neither stimulated cAMP synthesis nor inhibited the cAMP synthesis induced by dopamine. The cis isomer also lacked appreciable affinity for [3H]-1a binding sites. N-Methylation of the title compound decreased affinity for D1 sites about 7-8-fold and markedly decreased ability to stimulate adenylate cyclase. Addition of an N-n-propyl group reduced affinity for D1 sites by about 50-fold and essentially abolished the ability to stimulate adenylate cyclase. However, this latter derivative had twice the affinity of the D2-selective agonist quinpirole for the D2 receptor. The results are discussed in the context of a conceptual model for the agonist state of the D1 receptor.

Cloning of a cDNA encoding a novel interleukin-1 receptor related protein (IL 1R-rp2).

We have identified and isolated both the rat and human cDNAs for a novel putative receptor related to the interleukin-1 type 1 receptor. We have named this protein interleukin 1 receptor related protein two (IL 1R-rp2). The rat cDNA for IL1R-rp2 was first identified using oligonucleotides of degenerate sequence in a polymerase chain reaction (PCR) paradigm with rat brain mRNA as the template. The protein encoded by both of these cDNAs are 561 amino acids long and exhibit 42% and 26% overall identity with the interleukin-1 type 1 and type 2 receptors, respectively. RNase protection assays from rat tissues revealed a predominant expression for IL 1R-rp2 in the lung and epididymis with lower levels detected in the testis and cerebral cortex. By in situ hybridization we were able to determine that the expression in rat brain appeared to be non-neuronal and associated with the cerebral vasculature. When expressed transiently in COS-7 cells the receptor was incapable of high affinity binding to either [125I]-recombinant human IL 1 alpha or [125I]-recombinant human IL 1 beta. Together, these data demonstrate the existence of a novel protein that is related to the interleukin-1 receptor but does not bind IL-1 by itself.

Acute wake-promoting actions of JNJ-5207852, a novel, diamine-based H3 antagonist.

1 1-[4-(3-piperidin-1-yl-propoxy)-benzyl]-piperidine (JNJ-5207852) is a novel, non-imidazole histamine H3 receptor antagonist, with high affinity at the rat (pKi=8.9) and human (pKi=9.24) H3 receptor. JNJ-5207852 is selective for the H3 receptor, with negligible binding to other receptors, transporters and ion channels at 1 microm. 2 JNJ-5207852 readily penetrates the brain tissue after subcutaneous (s.c.) administration, as determined by ex vivo autoradiography (ED50 of 0.13 mg kg(-1) in mice). In vitro autoradiography with 3H-JNJ-5207852 in mouse brain slices shows a binding pattern identical to that of 3H-R-alpha-methylhistamine, with high specific binding in the cortex, striatum and hypothalamus. No specific binding of 3H-JNJ-5207852 was observed in brains of H3 receptor knockout mice. 3 In mice and rats, JNJ-5207852 (1-10 mg kg(-1) s.c.) increases time spent awake and decreases REM sleep and slow-wave sleep, but fails to have an effect on wakefulness or sleep in H3 receptor knockout mice. No rebound hypersomnolence, as measured by slow-wave delta power, is observed. The wake-promoting effects of this H3 receptor antagonist are not associated with hypermotility. 4 A 4-week daily treatment of mice with JNJ-5207852 (10 mg kg(-1) i.p.) did not lead to a change in body weight, possibly due to the compound being a neutral antagonist at the H3 receptor. 5 JNJ-5207852 is extensively absorbed after oral administration and reaches high brain levels. 6 The data indicate that JNJ-5207852 is a novel, potent and selective H3 antagonist with good in vitro and in vivo efficacy, and confirm the wake-promoting effects of H3 receptor antagonists.

Primary structure of mouse secretogranin III and its absence from deficient mice.

The rat brain- and pituitary-specific 1B1075 mRNA encodes a chromogranin/secretogranin-like protein, called secretogranin III (SgIII), that is a component of intracellular dense core vesicles. In order to study the function of this gene product in a mouse model system, we have isolated the murine homolog of the rat 1B1075 mRNA. This mRNA contains 2163 bp encoding a putative protein of 421 amino acids. Cleavage of the strong putative signal sequence would yield a mature protein of 51 kDa. The sequence of the encoded murine protein preserves the structural features that suggest SgIII is a member of the granin family, and allowed us to recognize and correct errors in our published rat sequence.

Alternative splicing of the histamine H(3) receptor mRNA at the third cytoplasmic loop is not detectable in humans.

Histamine regulates neurotransmitter release in the central and peripheral nervous systems through H(3) presynaptic receptors. cDNAs coding for human, guinea pig and rat histamine H(3) receptors have recently been cloned. Cloning of rat and guinea pig H(3) receptors demonstrated the existence of multiple isoforms displaying deletions in the third cytoplasmic loop coding region. We investigated whether a similar splicing pattern might also occur in the human H(3) gene. Using both RT-PCR and RNase protection assays, we detected H(3) receptor mRNA expression in human brain, testes and a cell line expressing recombinant human H(3) receptor (SK-N-MC/H(3)). In all samples tested by both detection methods, only the long mRNA form was detected. We could not find any evidence that humans express other forms equivalent to that seen in the rat or guinea pig. If the alternative splicing seen in rats and guinea pigs presents itself through pharmacological variation, our current findings then have implication for the use of rats or guinea pigs as model system for the development of therapeutic targeting the human H(3) receptor.

Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time, age and genetic background.

The hypocretins (1 and 2) have emerged as key regulators of sleep and wakefulness. We developed a high-throughput enzyme immunoassay (EIA) to measure total brain hypocretin levels from large numbers of mice. Hypocretin levels were not altered by circadian time or age. However, significant differences in one or both hypocretin peptides were observed between different mouse strains. We studied hypocretin levels in knockout and transgenic mouse models with obesity, circadian gene mutations or monoaminergic defects. Compared to controls, only histamine receptor knockouts had lower hypocretin levels. This was most pronounced in H1 receptor knockouts suggesting the existence of a positive feedback loop between hypocretin and histaminergic neurons.

Corticotropin-releasing factor CRF1, but not CRF2, receptors mediate anxiogenic-like behavior.

The recent identification and differential localization in brain of three binding sites for corticotropin-releasing factor (CRF)-like peptides (CRF1 and CRF2 receptors as well as CRF-binding protein) suggest the existence of functionally distinct neurobiological systems which mediate CRF activation. For instance, evidence from receptor knockdown and pharmacological studies suggest involvement of the CRF1 receptor in anxiogenic-like behavior and the CRF-binding protein in learning and memory processes. The present studies examined the potential functional significance of the CRF2 receptor in relation to the CRF1 receptor using two animal models of anxiety and endocrine reactivity to a stressor. CRF1 and CRF2 receptor knockdown was achieved and confirmed autoradiographically within brain regions relevant to behavioral reactivity to stressors by chronic, central administration of antisense oligonucleotides. CRF1 but not CRF2, know down produced a significant anxiolytic-like effect in the Defensive Withdrawal relative to vehicle-treated and two missense oligonucleotide negative control groups. In contrast, neither antisense treatment altered endocrine or behavioral reactivity to a swim stressor. Thus, the present data support the reported role of CRF1 receptors in the mediation of anxiogenic-like behavior and suggest a functionally distinct for role for CRF2 receptors in brain.

Guanine nucleotide binding proteins and the regulation of cyclic AMP synthesis in NS20Y neuroblastoma cells: role of D1 dopamine and muscarinic receptors.

D1 dopamine receptors on NS20Y neuroblastoma cells stimulate adenylate cyclase activity, whereas muscarinic receptors on the same cells negatively regulate adenylate cyclase. To determine the mechanisms which underlie these processes, cyclic AMP accumulation was measured in intact cells following either cholera or pertussis toxin treatment. Pretreatment with pertussis toxin (100 ng/ml), which ribosylated greater than 95% of inhibitory quinine nucleotide binding protein (Gi), caused the complete loss of muscarinic induced inhibition. Conversely, pertussis toxin did not affect the ability of dihydrexidine (1 microM, a full efficacy D1 agonist), PGE1 (100 nM), or forskolin (1 microM, a direct activator) to stimulate cAMP accumulation. Both the dihydrexidine-induced stimulation and the carbachol-induced inhibition of cyclic AMP accumulation were unaffected by either removal of extracellular calcium, or increased intracellular calcium caused by the addition of the calcium ionophore A23187. Cholera toxin dose- and time-dependently induced large accumulations of cAMP. At low cholera toxin concentrations, the effects of dihydrexidine (300 nM) were additive with those of cholera toxin. At cholera toxin concentrations greater than 100 ng/ml, dihydrexidine became ineffective in stimulating further cAMP synthesis. Conversely, forskolin (1 microM) still caused marked increases in cAMP accumulation after all cholera toxin treatments. Dihydrexidine-stimulated cAMP accumulation was additive with forskolin-stimulated cAMP accumulation at low forskolin concentrations (10 nM-3 microM), but synergistic at high concentrations (3-100 microM). Additionally, forskolin was much more potent after cholera toxin treatment, suggesting that an activated stimulatory guanine nucleotide binding protein (Gs) may be required for full activation of adenylate cyclase by forskolin in this cell type.(ABSTRACT TRUNCATED AT 250 WORDS)

The developmental profile of the corticotropin releasing factor receptor (CRF2) in rat brain predicts distinct age-specific functions.

Corticotropin releasing factor (CRF) activates two known receptor types, CRF1, and CRF2. In the adult rat brain, CRF2 has a distinct distribution pattern, suggesting that it may mediate functions exclusive of CRF1. The goal of this study was to determine the age-dependent distribution of CRF2-messenger RNA (CRF2-mRNA) in the rat brain. Brains from rats sacrificed under stress-free conditions on fetal days (F) 15, 16, 17 and 19, and postnatal days 1, 3, 5, 7, 9, 12, 15, 25, 49, and 90 (adult) were analyzed using semiquantitative in situ hybridization histochemistry. The onset and distribution of CRF2-mRNA in the developing rat brain revealed important differences from the adult expression pattern: earliest expression of CRF2-mRNA was observed in the ventromedial hypothalamus (VMH) on F16. High levels of CRF2-mRNA were present in the fronto-parietal cortex in the fetal and early postnatal brain but not later. Conversely, no CRF2-mRNA was detectable in the ventroposterior (lateral and medial) thalamic nuclei prior to postnatal day 7. Distinct developmental profiles of CRF2-mRNA were also observed in the lateral septum, medial, basal and cortical amygdala nuclei, and in several hippocampal fields. In conclusion, CRF2 is expressed in the hypothalamus on F16, prior to the detection of CRF itself in the paraventricular nucleus. The differential levels and distributions of CRF2-mRNA in hypothalamic and limbic brain regions indicate a precise regulation of this receptor's expression during development, as shown for CRF1. Regulation of the levels of CRF2 may modulate the effects of CRF (and related ligands) on target neurons, consistent with differential maturation of the functions mediated by this receptor.

Functional studies of bradykinin receptors in Chinese hamster ovary cells stably expressing the human B2 bradykinin receptor.

Bradykinin B1 and B2 receptors, members of the G-protein coupled receptor superfamily, are involved in inflammation and pain. Chinese hamster ovary (CHO) cells stably expressing the human B2 bradykinin receptor (CHO-B2) were used to characterize the signal transduction pathways associated with this receptor and its regulation. The selective B2 antagonist [3H]NPC17731 but not the selective B1 antagonist [3,4-prolyl-3,4-(3)H(N)]-[des-Arg10,Leu9]kallidin ([3H]DALKD) bound to CHO-B2 cell membranes with a Kd of 0.77 nM and a Bmax of 1087 fmol/mg protein. [3H]NPC17731 binding was inhibited by bradykinin ligands in the order: NPC17731 > bradykinin > kallidin >> DALKD > [des-Arg10] kallidin (DAKD), consistent with the pharmacological profile of B2 bradykinin receptors. The B2 agonist bradykinin and the B1/B2 agonist kallidin, but not the B1 agonist DAKD, increased [35S]GTP gamma S binding to the CHO-B2 cell membranes. The B2 bradykinin receptors were co-immunoprecipitated with G alpha q/11. In response to bradykinin stimulation, coupling of the B2 receptors to G alpha q/11 was increased by 10-fold. Bradykinin and kallidin, but not DAKD, induced intracellular calcium release in CHO-B2 cells, which was blocked by NPC17731 but not by DALKD. These results demonstrate that B2 bradykinin receptors directly coupled to G alpha q/11 to regulate intracellular calcium release. CHO-B2 cell is a useful system that can be applied to study the effect of potential agents that may influence the B2 receptor function.