The genetic design of signaling cascades to record receptor activation.

We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.

Synthesis, protein levels, activity, and phosphorylation state of tyrosine hydroxylase in mesoaccumbens and nigrostriatal dopamine pathways of chronically food-restricted rats.

Chronic food restriction (FR) enhances the rewarding and motor-activating effects of abused drugs, and is accompanied by changes in dopamine (DA) dynamics and increased D-1 DA receptor-mediated cell signaling and transcriptional responses in nucleus accumbens (NAc). However, little is known about effects of FR on DA synthetic activity in the mesoaccumbens and nigrostriatal pathways. In Experiment 1 of the present study, tyrosine hydroxylase (TH) gene expression was measured in ventral tegmental area and substantia nigra, using real-time RT-PCR and in situ hybridization; no differences were observed between FR and ad libitum fed (AL) rats. In Experiment 2, TH protein levels, determined by Western blot, were found to be elevated in NAc and caudate-putamen (CPu) of FR relative to AL rats. In the absence of increased transcription, this may reflect a slowing of TH degradation. In Experiments 3 and 4, DA synthetic activity was assessed by Western blot measurement of TH phosphorylation at Ser40, and HPLC measurement of in vivo tyrosine hydroxylation rate, as reflected by DOPA accumulation following administration of a decarboxylase inhibitor (NSD-1015; 100 mg/kg, i.p.). Basal phospho-(Ser40)-TH levels did not differ between groups but DOPA accumulation was decreased by FR. Decreased DOPA synthesis, despite increased levels of TH protein, may reflect the inhibitory effect of increased DA binding to TH protein or decreased concentrations of cofactor tetrahydrobiopterin. Finally, in response to D-amphetamine (0.5 and 5.0 mg/kg, i.p.), phospho-(Ser40)-TH was selectively decreased in NAc of FR rats. This suggests increased feedback inhibition of DA synthesis-a possible consequence of postsynaptic receptor hypersensitivity, or increased extracellular DA concentration. These results indicate that FR increases TH protein levels, but may decrease the capacity for DA synthesis by decreasing TH activity. According to this scheme, the previously observed upregulation of striatal cell signaling and transcriptional responses to DA receptor agonist administration may include compensatory neuroadaptations.

Effects of the group I metabotropic glutamate receptor agonist, DHPG, and injection stress on striatal cell signaling in food-restricted and ad libitum fed rats.

BACKGROUND: Chronic food restriction augments the rewarding effect of centrally administered psychostimulant drugs and this effect may involve a previously documented upregulation of D-1 dopamine receptor-mediated MAP kinase signaling in nucleus accumbens (NAc) and caudate-putamen (CPu). Psychostimulants are known to induce striatal glutamate release, and group I metabotropic glutamate receptors (mGluR) have been implicated in the cellular and behavioral responses to amphetamine. The purpose of the present study was to evaluate whether chronic food restriction increases striatal MAP kinase signaling in response to the group I mGluR agonist, DHPG. RESULTS: Western immunoblotting was used to demonstrate that intracerebroventricular (i.c.v.) injection of DHPG (500 nmol) produces greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted as compared to ad libitum fed rats. Fos-immunostaining induced by DHPG was also stronger in CPu and NAc core of food-restricted relative to ad libitum fed rats. However, i.c.v. injection of saline-vehicle produced greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted relative to ad libitum fed rats, and this difference was not seen when subjects received no i.c.v. injection prior to sacrifice. In addition, although DHPG activated Akt, there was no difference in Akt activation between feeding groups. To probe whether the augmented ERK1/2 and CREB activation in vehicle-injected food-restricted rats are mediated by one or more GluR types, effects of an NMDA antagonist (MK-801, 100 nmol), AMPA antagonist (DNQX, 10 nmol), and group I mGluR antagonist (AIDA, 100 nmol) were compared to saline-vehicle. Antagonist injections did not diminish activation of ERK1/2 or CREB. CONCLUSIONS: These results indicate that a group I mGluR agonist induces phosphorylation of Akt, ERK1/2 and CREB in both CPu and NAc. However, group I mGluR-mediated signaling may not be upregulated in food-restricted rats. Rather, a physiological response to "i.c.v. injection stress" is augmented by food restriction and appears to summate with effects of the group I mGluR agonist in activating ERK1/2 and CREB. While the augmented cellular response of food-restricted rats to i.c.v. injection treatment represents additional evidence of enhanced CNS responsiveness in these subjects, the functional significance and underlying mechanism(s) of this effect remain to be elucidated.

The endogenous ligand Stunted of the GPCR Methuselah extends lifespan in Drosophila.

Many extracellular signals are transmitted to the interior of the cell by receptors with seven membrane-spanning helices that trigger their effects by means of heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins). These G-protein-coupled receptors (GPCRs) control various physiological functions in evolution from pheromone-induced mating in yeast to cognition in humans. The potential role of the G-protein signalling system in the control of animal ageing has been highlighted by the genetic revelation that mutation of a GPCR encoded by methuselah extends the lifespan of adult Drosophila flies. How methuselah functions in controlling ageing is not clear. A first essential step towards the understanding of methuselah function is to determine the ligands of Methuselah. Here we report the identification and characterization of two endogenous peptide ligands of Methuselah, designated Stunted A and B. Flies with mutations in the gene encoding these ligands show an increase in lifespan and resistance to oxidative stress. We conclude that the Stunted-Methuselah system is involved in the control of animal ageing.

Developmental changes in opioid peptides and their receptors in Cpe(fat)/Cpe(fat) mice lacking peptide processing enzyme carboxypeptidase E.

Carboxypeptidase E (CPE) is involved in the biosynthesis of a number of neuropeptides including opioid peptides. A point mutation in this gene results in a loss of enzyme activity, decrease in mature neuroendocrine peptides, and development of late onset obesity as seen in Cpe(fat)/Cpe(fat) mice. In this study, we examined the processing of peptides derived from prodynorphin and proenkephalin in various brain regions of these mice during development. At 6 to 8 weeks, an age prior to the onset of obesity, levels of dynorphin peptides are decreased in all brain regions, whereas levels of ir-Met-enkephalin are differentially altered. There is an accumulation of C-terminally extended forms of all three opioid peptides in Cpe(fat)/Cpe(fat) mice, consistent with a lack of CPE activity. Thus, it appears that there is no direct correlation between the level of mature opioid peptides and the development of obesity in these mice. Since altered levels of peptides can influence the opioid receptor system, we examined the functional activity of mu and kappa opioid receptors using [35S]guanosine-5'-O-(gamma-thio)-triphosphate binding assays. We find no differences in kappa receptor activity in Cpe(fat)/Cpe(fat) compared with control littermate mice. In contrast, the mu receptor activity is differentially altered in select regions of Cpe(fat)/Cpe(fat) mice in response to a mu-specific ligand. Taken together, these results suggest that the lack of CPE activity leads to alterations in the level of opioid peptides during development and that changes in peptide levels differentially affect opioid receptor activity in vivo.