Orphan G-protein-coupled receptors (GPCRs) are cloned proteins with structural characteristics common to the GPCRs but that bind unidentified ligands. Orphan GPCRs have been used as targets to identify novel transmitter molecules. Here we describe the isolation from brain extracts and the characterization of the natural ligand of a particular orphan GPCR (SLC-1) that is sequentially homologous to the somatostatin receptors. We show that the natural ligand of this receptor is the neuropeptide melanin-concentrating hormone (MCH). MCH is a cyclic peptide that regulates a variety of functions in the mammalian brain, in particular feeding behaviour. We demonstrate that nanomolar concentrations of MCH strongly activate SLC-1-related pathways through G(alpha)i and/or G(alpha)q proteins. We have analysed the tissue localization of the MCH receptor and find that it is expressed in several brain regions, in particular those involved in olfactory learning and reinforcement mechanisms, indicating that therapies targeting the MCH receptor should act on the neuronal regulation of food consumption.

The melanin-concentrating hormone (MCH) system is thought to be an important regulator of food intake. Recently the orphan G protein-coupled receptor SLC-1 was identified as the MCH receptor (MCHR). Preliminary analyses of MCHR mRNA distribution have supported a role for the MCH system in nutritional homeostasis. We report here a complete anatomical distribution of the MCHR mRNA. We have found high levels of expression of MCHR mRNA in most anatomical areas implicated in control of olfaction, with the exception of the main olfactory bulb. Dense labeling was also detected in the hippocampal formation, subiculum, and basolateral amygdala, all of which are important in learning and memory, and in the shell of the nucleus accumbens, a substrate for motivated behavior and feeding. Within the hypothalamus, MCHR mRNA was moderately expressed in the ventromedial nucleus, arcuate nucleus, and zona incerta, all of which serve key roles in the neuronal circuitry of feeding. In the brainstem, strong expression was observed in the locus coeruleus, which is implicated in arousal, as well as in nuclei that contribute to orofacial function and mastication, including the facial, hypoglossal, motor trigeminal, and dorsal motor vagus nuclei. In most regions there was a good correspondence between MCHR mRNA distribution and that of MCH-immunoreactive fibers. Taken together, these data suggest that MCH may act at various levels of the brain to integrate various aspects of feeding behavior. However, the extensive MCHR distribution throughout the brain suggests that this receptor may play a role in other functions, most notably reinforcement, arousal, sensorimotor integration, and autonomic control.

To explore membrane-permeable synthetic inhibitors that discriminate between endogenous calpain and proteasome in cells, we examined the inhibition of profiles against calpain and proteasome in vitro and in vivo of peptidyl aldehydes possessing di-leucine and tri-leucine. The tripeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLLal) strongly inhibited calpain and proteasome activities in vitro. The concentration required for 50% inhibition (IC50) of the casein-degrading activity of calpain was 1.25 microM, and the IC50s for the succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide (Suc-LLVY-MCA)- and benzyloxycarbonyl-leucyl-leucyl-leucine-4-methylcoumaryl -7-amide (ZLLL-MCA)-degrading activities of proteasome were 850 and 100 nM, respectively. On the other hand, the synthetic dipeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLal) strongly inhibited the casein degrading activity of calpain (IC50 1.20 microM), but the inhibition of proteasome was weak (IC50S for SucLLVY-MCA- and ZLLL-MCA-degrading activities were 120 and 110 microM, respectively). Thus, while calpain was inhibited by similar concentrations of ZLLal and ZLLLal, the inhibitory potencies of ZLLLal against the ZLLL-MCA- and Suc-LLVY-MCA-degrading activities in proteasome were 1,100 and 140 times stronger than those of ZLLal, respectively. To evaluate the effectiveness of these inhibitors on intracellular proteasome, the induction of neurite outgrowth in PC12 cells caused by proteasome inhibition was examined. ZLLLal and ZLLal initiated neurite outgrowth with optimal concentrations of 20 nM and 10 microM, respectively, again showing a big difference in the effective concentrations for the proteasome inhibition as in vitro. As for the effect on intracellular calpain, the concentration of ZLLLal and ZLLal required for the inhibition of the autolytic activation of calpain in rabbit erythrocytes were 100 and 100 microM or more, respectively. The almost equal inhibitory potencies of ZLLLal and ZLLal were in agreement with the inhibition of calpain in vitro. These differential effects of inhibitors against calpain and proteasome are potentially useful for identifying the functions of calpain and proteasome in cell physiology and pathology.

omology-based cloning approaches and genome-sequencing efforts have revealed the existence of a large number of human genes encoding 'orphan' G-protein-coupled receptors (GPCRs), receptors that bind unidentified natural ligands. Discovery of these natural ligands is the first necessary step in understanding the biological significance of the orphan GPCRs. We 1 and others 2 have developed an approach by which to successfully isolate endogenous ligands from complex tissue libraries. This approach, referred to as the orphan-receptor strategy 3 , uses orphan receptors as baits to isolate their native ligand from tissue extracts and has been successfully applied to identify new neuropeptides 4-6 . Here we apply this strategy to the orphan receptor GPR14 and show that it binds the bioactive peptide known as urotensin II.The complementary DNA encoding the orphan receptor GPR14, or SENR, was cloned using degenerate oligonucleotides directed at conserved regions of known GPCRs 7,8 . Phylogenetic analysis positioned the GPR14 sequence closely to somatostatin, opioid and galanin receptors 8 , indicating that the endogenous ligand of GPR14 may also be peptidergic. We set out to identify the natural ligand of GPR14 from peptide extracts prepared from a variety of different mammalian tissues. Extracts were fractionated by preparative reverse-phase high-performance liquid chromatography (rpHPLC) into 72 individual fractions, and aliquots were tested for induction of changes in intracellular Ca 2+ ([Ca 2+ ] i ) in Chinese hamster ovary (CHO) cells transiently transfected with GPR14 cDNA. Intracellular Ca 2+ changes were monitored using a fluorescence imaging plate reader (FLIPR) system 9 . A reproducible and robust change in Ca 2+ concentration was observed in two adjacent fractions (Fig. 1a). This change could not be detected either in nontransfected cell lines or in cells transfected with other orphan receptors. Highest levels of activity were detected in bovine hypothalamic tissue, which was consequently used for large-scale purification. The active component was purified over a seven-step purification strategy combining reverse-phase and cationic-exchange HPLCs. One single activity peak was detected, indicating that the activity can be attributed to a unique molecular entity. The bioactive compound was extremely scarce, preventing us from carrying out a total structural analysis. However, the Ca 2+ response to the active material showed a distinctive time course (Fig. 1a, inset). Furthermore, the active material was sensitive to trypsin (Fig. 1a, inset) and alkylating agents, leading us to conclude that the biological activity could be attributed to a peptide containing basic amino acid(s) and alkylation-sensitive amino acids.As GPR14 is similar to the somatostatin receptors we decided to screen somatostatin-like, cysteine-bridge-containing peptides on GPR14 and to compare their biological activities and physicochemical properties with that of the endogenous compound identified in our purification scheme. Peptides tested wer...

A novel real-time PCR assay system was developed to quantify the cytomegalovirus (CMV) genome load. The real-time PCR assay could detect from 6 to over 10(6) copies of CMV-DNA with a wide linear range. The virus load of immunocompromised patients with symptomatic CMV infections was quantified and compared to that of asymptomatic ones. In symptomatic patients, all 17 peripheral blood leukocytes were positive for CMV DNA, and its mean value was 10(3.3) copies/10(6) cells. On the other hand, only 9 of 38 samples (24%) were positive in the asymptomatic patients, and its mean titer was lower (10(2.0) copies/10(6) cells) than that of the symptomatic group (P = 0.002). In plasma, the virus genome was detected in 13 out of 17 samples from symptomatic patients (76%), and its mean value was 10(4.0) copies/ml. In contrast, for the asymptomatic group, only one out of 36 samples were positive (3%). Finally, this system was used to monitor two patients with CMV infections serially. The CMV DNA copy number changed with their clinical symptoms and anti-CMV therapy, and virtually paralleled the result of the pp65 antigenemia assay in both cases. In one patient with the cord blood transplantation, however, the CMV DNA became positive faster than the antigenemia assay. These results indicate that this assay is sensitive and useful for estimating the CMV genome load not only in peripheral blood leukocytes but also in plasma. It can be very helpful for diagnosing CMV-related diseases and monitoring the virus load in patients with CMV infections.