Evidence from gene knockout studies implicates Asc-1 as the primary transporter mediating d-serine reuptake in the mouse CNS.

In the mammalian central nervous system, transporter-mediated reuptake may be critical for terminating the neurotransmitter action of D-serine at the strychnine insensitive glycine site of the NMDA receptor. The Na(+) independent amino acid transporter alanine-serine-cysteine transporter 1 (Asc-1) has been proposed to account for synaptosomal d-serine uptake by virtue of its high affinity for D-serine and widespread neuronal expression throughout the brain. Here, we sought to validate the contribution of Asc-1 to D-serine uptake in mouse brain synaptosomes using Asc-1 gene knockout (KO) mice. Total [(3)H]D-serine uptake in forebrain and cerebellar synaptosomes from Asc-1 knockout mice was reduced to 34 +/- 5% and 22 +/- 3% of that observed in wildtype (WT) mice, respectively. When the Na(+) dependent transport components were removed by omission of Na(+) ions in the assay buffer, D-serine uptake in knockout mice was reduced to 8 +/- 1% and 3 +/- 1% of that measured in wildtype mice in forebrain and cerebellum, respectively, suggesting Asc-1 plays a major role in the Na(+) independent transport of D-serine. Potency determination of D-serine uptake showed that Asc-1 mediated rapid high affinity Na(+) independent uptake with an IC(50) of 19 +/- 1 microm. The remaining uptake was mediated predominantly via a low affinity Na(+) dependent transporter with an IC(50) of 670 +/- 300 microm that we propose is the glial alanine-serine-cysteine transporter 2 (ASCT2) transporter. The results presented reveal that Asc-1 is the only high affinity D-serine transporter in the mouse CNS and is the predominant mechanism for D-serine reuptake.

Pharmacological and functional characterisation of dopamine D4 receptors in the rat retina.

In the retina, activation of dopamine receptors, particularly the D2-like family (D2, D3, D4 receptor subtypes), with quinpirole suppresses the light sensitive cAMP pool and inhibits melatonin synthesis in photoreceptor cells. We have characterised rat retinal D4 receptors using the D4 selective radioligand [(125)I] L-750667 which bound specifically and saturably to rat retinal membranes with high affinity (K(d) 0.06+/-0.02 nM) and exhibited a D4 receptor pharmacology. Comparison of the binding kinetics of [(125)I] L-750667 and [(3)H] spiperone revealed B(max) values of 134+/-27 fmol/mg and 219+/-47 fmol/mg respectively, indicating that the dopamine D4 receptor is a major component of D2-like dopamine receptors in the rat retina. Modulation of retinal cAMP levels by quinpirole was used to evaluate the functional relevance of rat retinal dopamine D4 receptors. Quinpirole (0.03-3 micro ) produced a dose-related decrease of the light sensitive cAMP pool which was reversed by haloperidol, clozapine and the D4 selective antagonist, L-745870 with a rank order of potency suggesting that the quinpirole effect is due to activation of the dopamine D4 receptors. The D2 selective ligand L-741626 had no effect on the quinpirole response confirming that the D4 receptor is the major receptor subtype mediating dopamine induced suppression of adenylate cyclase in the retina.

Identification of signal transduction pathways used by orphan g protein-coupled receptors.

The superfamily of GPCRs have diverse biological roles, transducing signals from a range of stimuli, from photon recognition by opsins to neurotransmitter regulation of neuronal function. Of the many identified genes encoding GPCRs, >130 are orphan receptors ( i.e., their endogenous ligands are unknown), and this subset represents putative novel therapeutic targets for pharmaceutical intervention in a variety of diseases. As an initial step toward drug discovery, determining a biological function for these newly identified receptors is of vital importance, and thus identification of a natural ligand(s) is a primary aim. There are several established methods for doing this, but many have drawbacks and usually require some in-depth knowledge about how the receptor functions. The technique described here utilizes a transcription-based reporter assay in live cells. This allows the determination of the signal transduction pathway any given oGPCR uses, without any prior knowledge of the endogenous ligand. This can therefore reduce the redundancy of effort involved in screening ligands at a given receptor in multiple formats (i.e., Galpha(s), Galpha(i/0), and Galpha(q) assays), as well as ensuring that the receptor targeted is capable of signaling if appropriately activated. Such knowledge is often laboriously obtained, and for almost all oGPCRs, this kind of information is not yet available. This technology can also be used to develop inverse agonist as well as agonist sensitive high throughput assays for oGPCRs. The veracity of this approach is demonstrated, using a number of known GPCRs. The likely signaling pathways of the GPR3, GPR12, GPR19, GPR21, and HG55 oGPCRs are shown, and a high throughput assay for GPR26 receptors developed. The methods outlined here for elucidation of the signal transduction pathways for oGPCRs and development of functional assays should speed up the process of identification of ligands for this potentially therapeutically useful group of receptors.