Functional genomics: the search for novel neurotransmitters and neuropeptides

AbstractFunctional genomics can be defined as the search for the physiological role of a gene for which only its primary sequence is known. Most of the genes encoding proteins containing seven hydrophobic stretches code for G protein-coupled receptors (GPCRs). Although many of these have been shown to interact with known natural ligands, several bind ligands which have not been thus far isolated. These are the so-called orphan GPCRs. As an example of functional genomics, an `orphan receptor strategy' has been developed to identify the natural ligands of orphan GPCRs. The application of this strategy is bound to revolutionize our understanding of the diversity of the primary messengers which modulate synaptic transmission. This review discusses the basic concepts and some of the particular problems associated with the orphan receptor strategy. The strategy's potential is exemplified by its successes which culminated in the discovery of the neuropeptides `orphanin FQ/nociceptin' and `orexins/hypocretins'. The steps that led to the characterization of these neuropeptides are discussed as are some of the further studies that have addressed the roles of these neuropeptides. To conclude, some of the implications of the application of the orphan receptor strategy are discussed

Neuropeptide S receptor in GtoPdb v.2023.1

The neuropeptide S receptor (NPS receptor) responds to the 20 amino-acid peptide neuropeptide S derived from a precursor (NPS, P0C0P6). NPS activates its receptor at low nanomolar concentrations elevating intracellular cAMP and calcium levels [71]. Currently, some peptidic and small molecule NPS receptor antagonists are available as research tools [27, 79, 8, 59]. No NPS receptor ligands are currently used clinically

Neuropeptide S receptor in GtoPdb v.2021.3

The neuropeptide S receptor (NPS, provisional nomenclature [23]) responds to the 20 amino-acid peptide neuropeptide S derived from a precursor (NPS, P0C0P6)

Neuropeptide S receptor (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The neuropeptide S receptor (NPS, provisional nomenclature [18]) responds to the 20 amino-acid peptide neuropeptide S derived from a precursor (NPS, P0C0P6)

Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells.

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed

Urotensin II Modulates Rapid Eye Movement Sleep Through Activation of Brainstem Cholinergic Neurons

Urotensin II (UII) is a cyclic neuropeptide with strong vasoconstrictive activity in the peripheral vasculature. UII receptor mRNA is also expressed in the CNS, in particular in cholinergic neurons located in the mesopontine tegmental area, including the pedunculopontine tegmental (PPT) and lateral dorsal tegmental nuclei. This distribution suggests that the UII system is involved in functions regulated by acetylcholine, such as the sleep-wake cycle. Here, we tested the hypothesis that UII influences cholinergic PPT neuron activity and alters rapid eye movement (REM) sleep patterns in rats. Local administration of UII into the PPT nucleus increases REM sleep without inducing changes in the cortical blood flow. Intracerebroventricular injection of UII enhances both REM sleep and wakefulness and reduces slow-wave sleep 2. Intracerebroventricular, but not local, administration of UII increases cortical blood flow. Moreover, whole-cell recordings from rat-brain slices show that UII selectively excites cholinergic PPT neurons via an inward current and membrane depolarization that were accompanied by membrane conductance decreases. This effect does not depend on action potential generation or fast synaptic transmission because it persisted in the presence of TTX and antagonists of ionotropic glutamate, GABA, and glycine receptors. Collectively, these results suggest that UII plays a role in the regulation of REM sleep independently of its cerebrovascular actions by directly activating cholinergic brainstem neurons

Distribution of D2 dopamine receptor mRNA in the primate brain

1. The distribution of the messenger RNA (mRNA) encoding the D2dopamine receptor has been mapped in the monkey brain by hybridization.2. Using [35s]-labelled riboprobes corresponding to the region of the D2 dopamine receptor spanning the third cytosolic loop and the sixth and seventh transmembrane domains, specific hybridization was observed in a number of neural structures.3. High levels of mRNA expression were observed in the caudate, putamen, and claustrum. Significant amounts were also identified in the hippocampus, lateral geniculate nucleus, much of the cortex, amygdala, pons, and thalamus. High levels of this mRNA were also visualized in the substantia nigra, likely reflecting autoreceptor synthesis.4. While the distribution of D2 dopamine receptor mRNA was similar between the monkey and previously published maps in the rat, several differences were noted.5. These results demonstrate the feasibility of visualizing this mRNA in the primate brain, and suggest that a similar analysis of human postmortem brain material may be possible.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29649/1/0000738.pd

Site-directed mutagenesis of the human dopamine D2 receptor

Based on amino acid sequence and computer modeling, two conflicting three-dimensional models of the dopamine D2 receptor have been proposed. One model (Dahl et al., 1991, Proc. Natl. Acad. Sci. USA 88, 8111) suggests that dopamine interacts with aspartate 80 of transmembrane (TM) 2 and asparagine 390 of TM6 with the transmembranes arranged in a clockwise manner, while a second model (Hibert et al., 1991, Mol. Pharmacol. 40, 8) suggests that dopamine interacts with aspartate 114 of TM3 and the serines of TM5 (194 and 197) with the transmembranes arranged in a counterclockwise manner when viewed from the extracellular space. The present study tests the latter model by selectively mutating aspartate 114 and serines 194 and 197 of the human dopamine D2 receptor by site-directed mutagenesis. In addition, two methionines (116 and 117) were mutated to evaluate whether residues near aspartate (114) of the dopamine D2 receptor are critical in differentiating dopamine receptor agonists from adrenoceptor agonists. Removal of the negative charge with the mutation of aspartate (114) to either asparagine or glycine led to a total loss of both agonist and antagonist binding. Individual or dual methionine mutations in positions 116 and 117, to make the dopamine D2 binding pocket more closely resemble the [beta]2-adrenoceptor, did not result in a change in selectivity toward noradrenergic agonists or antagonists. The serine mutations revealed interesting differences between the dopamine D2 receptor and the adrenoceptors. In particular serine 197 appeared more important than serine 194 for agonist binding. In addition, the binding of one agonist (N-0437) was unaffected by individual serine mutations, while the binding of some antagonists, such as raclopride and spiperone, was significantly altered. These findings are discussed in relation to ligand structure and their interactions with the putative binding pocket.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29803/1/0000149.pd