The suprachiasmatic nucleus (SCN) controls the circadian rhythm of physiological and behavioural processes in mammals. Here we show that prokineticin 2 (PK2), a cysteine-rich secreted protein, functions as an output molecule from the SCN circadian clock. PK2 messenger RNA is rhythmically expressed in the SCN, and the phase of PK2 rhythm is responsive to light entrainment. Molecular and genetic studies have revealed that PK2 is a gene that is controlled by a circadian clock (clock-controlled). Receptor for PK2 (PKR2) is abundantly expressed in major target nuclei of the SCN output pathway. Inhibition of nocturnal locomotor activity in rats by intracerebroventricular delivery of recombinant PK2 during subjective night, when the endogenous PK2 mRNA level is low, further supports the hypothesis that PK2 is an output molecule that transmits behavioural circadian rhythm. The high expression of PKR2 mRNA within the SCN and the positive feedback of PK2 on its own transcription through activation of PKR2 suggest that PK2 may also function locally within the SCN to synchronize output.

We have previously reported the selective amplification of several rat striatal cDNA sequences that encode guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. One of these sequences (R226) exhibited high sequence identity (58%) with the two previously cloned adenosine receptors. A full-length cDNA clone for R226 has been isolated from a rat brain cDNA library. The cDNA clone encodes a protein of 320 amino acids that can be organized into seven transmembrane stretches. R226 has been expressed in COS-7 and CHO cells and membranes from the transfected cells were screened with adenosine receptor radioligands. R226 could bind the nonselective adenosine agonist tritiated N-ethyladenosine 5'-uronic acid ([3H]NECA) and A1-selective agonist radioiodinated N6-2-(4-amino-3-iodophenyl)-ethyladenosine ([125I]APNEA) but not A1-selective antagonists tritiated 1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) and 8-(4-[([[(2-aminoethyl)amino]carbonyl]methyl)oxy]-phenyl)-1, 3-dipropylxanthine ([3H]XAC) or the A2-selective agonist ligands tritiated 2-[4-(2-carboxyethyl)phenyl]ethyl-amino 5'-N-ethylcarboxamidoadenosine ([3H]CGS21680) and radioiodinated 2-[4-([2-[(4-aminophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl)phenyl]ethylamino 5'-N-ethylcarboxamidoadenosine. Extensive characterization with [125I]APNEA showed that R226 binds [125I]APNEA with high affinity (Kd = 15.5 +/- 2.4 nM) and the specific [125I]APNEA binding could be inhibited by adenosine ligands with a potency order of (R)-N6-phenyl-2-propyladenosine (R-PIA) = NECA greater than S-PIA greater than adenosine greater than ATP = ADP but not by antagonists XAC, isobutylmethylxanthine, and DPCPX. In R226 stably transfected CHO cells, adenosine agonists R-PIA, NECA, and CGS21680 inhibited by 40-50% the forskolin-stimulated cAMP accumulation through a pertussis toxin-sensitive G protein with an EC50 of 18 +/- 5.6 nM, 23 +/- 3.5 nM, and 144 +/- 34 nM, respectively. Based on these observations we conclude that R226 encodes an adenosine receptor with non-A1 and non-A2 specificity, and we thus name it the A3 adenosine receptor. mRNA analyses revealed that the highest expression of R226 was in the testis and low-level mRNAs were also found in the lung, kidneys, heart, and some parts of the central nervous system such as cortex, striatum, and olfactory bulb. The high-expression level of the A3 receptor in the testis suggests a possible role for adenosine in reproduction.

We previously described two mammalian secreted proteins, prokineticin 1 and prokineticin 2, that potently contract gastrointestinal smooth muscle. Prokineticin 1 has also been shown to promote angiogenesis by stimulating proliferation, migration, and fenestration of endocrine organ-derived endothelial cells. Here we report the cloning and characterization of two closely related G protein-coupled receptors as receptors for prokineticins. Expression of prokineticin receptors in heterologous systems shows that these receptors bind to and are activated by nanomolar concentrations of recombinant prokineticins. Activation of prokineticin receptors leads to mobilization of calcium, stimulation of phosphoinositide turnover, and activation of p44/p42 MAPK signaling pathways that are consistent with the effects of prokineticins on smooth muscle contraction and angiogenesis. mRNA expression analysis reveals that prokineticin receptors are expressed in gastrointestinal organs, endocrine glands, and other tissues. Diseases involving altered gastrointestinal (GI)1 motility are among the most common human disorders (1, 2). Understanding the physiological functions and mechanisms of action of GI motility regulatory factors is a prerequisite for developing effective treatments for GI motility disorders (3, 4). We have previously discovered two mammalian proteins, prokineticin 1 and 2 (PK1 and PK2, respectively), that are potentially important regulators of GI motility (5). Prokineticins are ϳ10-kDa cysteine-rich secreted proteins expressed in the GI tract and several other tissues. Recently a second function for PK1 was reported. This protein, which LeCouter et al. (6) named endocrine gland vascular endothelial growth factor, stimulates cell proliferation, migration, and fenestrations in several endothelial cell lines derived from endocrine glands. In addition, expression of PK1 from recombinant adenovirus injected into the mouse ovary stimulates angiogenesis (6). Thus, prokineticins regulate diverse biological functions that include contraction of GI smooth muscle and angiogenesis. Based on pharmacological evidence, we have demonstrated that a receptor(s) for prokineticins belongs to the family of G protein-coupled receptors (GPCRs) (5). Here we report the cloning of two closely related G protein-coupled receptors as receptors for prokineticins. EXPERIMENTAL PROCEDURES Cloning of Prokineticin Receptor 1 (PKR1) and ProkineticinReceptor 2 (PKR2) cDNAs-Human PKR1 and PKR2 sequences were identified in human sequence genome searches as described previously (7). Fulllength cDNAs were cloned from Marathon RACE Ready cDNA (CLON-TECH) by PCR. The PKR1 gene was amplified from testis Marathon RACE Ready cDNA using PCR and the following oligonucleotide primers: 5Ј-ggtgacatcagccttgcagacattgccc and 5Ј-ATGTGCATCCAAGCACA-CTAGTCAGTGTCC. This was followed by nested PCR using the following oligonucleotide primers: 5Ј-CACCATGGAGACCACCATGGG-GTTCATG and 5Ј-ATGTGCATCCAAGCACACTAGTCAGTGTCC. PKR2 was amplified from pooled testis and fetal brain Marathon...

Neurogenesis persists in the olfactory bulb (OB) of the adult mammalian brain. New interneurons are continually added to the OB from the subventricular zone (SVZ) via the rostral migratory stream (RMS). Here we show that secreted prokineticin 2 (PK2) functions as a chemoattractant for SVZ-derived neuronal progenitors. Within the OB, PK2 may also act as a detachment signal for chain-migrating progenitors arriving from the RMS. PK2 deficiency in mice leads to a marked reduction in OB size, loss of normal OB architecture, and the accumulation of neuronal progenitors in the RMS. These findings define an essential role for G protein-coupled PK2 signaling in postnatal and adult OB neurogenesis.

Many structural determinants for G protein-coupled receptor (GPCR) functions have been defined, but little is known concerning the regulation of their transport from the endoplasmic reticulum (ER) to the cell surface. Here we show that a carboxy-terminal hydrophobic motif, FxxxFxxxF, which is highly conserved among GPCRs, functions independently as an ER-export signal for the dopamine D1 receptor. A newly identified ER-membrane-associated protein, DRiP78, binds to this motif. Overexpression or sequestration of DRiP78 leads to retention of D1 receptors in the ER, reduced ligand binding, and a slowdown in the kinetics of receptor glycosylation. Our results indicate that DRiP78 may regulate the transport of a GPCR by binding to a specific ER-export signal.

Gonadotropin-releasing hormone (GnRH) deficiency in the human I55fsX1]). Another asymptomatic brother was heterozygous for the deletion, whereas both parents (deceased) had normal reproductive histories. The identified deletion results in a truncated PROK2 protein of 27 amino acids (rather than 81 in its mature form) that lacks bioactivity. In addition, Prok2 ؊/؊ mice with olfactory bulb defects exhibited disrupted GnRH neuron migration, resulting in a dramatic decrease in GnRH neuron population in the hypothalamus as well as hypogonadotropic hypogonadism. Homozygous loss-of-function PROK2 mutations cause both KS and nIHH.gonadotropin-releasing hormone deficiency

Loss-of-function mutations in PROK2 and PROKR2 underlie both KS and nIHH.

The motility of gastrointestinal tract is regulated by classical neurotransmitters, neuropeptides, and humoral agents. Two novel human cDNAs have been cloned based on their sequence similarity to a frog skin secretion protein, Bv8, and a nontoxic protein of mamba snake venom. These human cDNAs encode two secreted proteins of 86 and 81 amino acids. Northern blot hybridization has revealed that these cDNAs are expressed in gastrointestinal tract, particularly the stomach. Recombinant proteins with authentic N-terminal sequences have been produced in Escherichia coli and refolded into functional proteins by careful control of protein aggregation. Mass spectrometry has confirmed the formation of five pairs of disulfide bonds. The refolded recombinant proteins potently contract gastrointestinal smooth muscle with EC(50) values in the subnanomolar range. The contractile effects of the recombinant proteins are specific for gastrointestinal smooth muscle, because they have no effect on vascular or respiratory smooth muscle. To reflect their potent and specific effects on gastrointestinal smooth muscle cells, we have named these recombinant proteins prokineticins. Ligand binding studies with iodinated prokineticin revealed the presence of a high-affinity site in ileal smooth muscle. The displacement of specific binding by GTP gamma S suggests that the prokineticin receptor may belong to the family of G protein-coupled receptors. Experiments with verapamil and nifedipine revealed that calcium influx is essential for the contractile activity of prokineticins on gastrointestinal smooth muscle. In summary, we have identified two novel endogenous regulators of gastrointestinal motility. The availability of recombinant prokineticins should provide novel therapeutic agents for disorders involving impaired gastrointestinal motility.