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Abstract:The apelin peptide was recently discovered and demonstrated to be the endogenous ligand for the G protein-coupled receptor, APJ. A search of the GenBank databases retrieved a rat expressed sequence tag partially encoding the preproapelin sequence. The GenBank search also revealed a human sequence on chromosome Xq25-26.1, containing the gene encoding preproapelin. We have used the rat sequence to screen a rat brain cDNA library to obtain a cDNA encoding the full-length open reading frame of rat preproapelin. This cDNA encoded a protein of 77 amino acids, sharing an identity of 82% with human preproapelin. Northern and in situ hybridization analyses revealed both human and rat apelin and APJ to be expressed in the brain and periphery. Both sequence and mRNA expression distribution analyses revealed similarities between apelin and angiotensin II, suggesting they that share related physiological roles. A synthetic apelin peptide was injected intravenously into male Wistar rats, resulting in immediate lowering of both systolic and diastolic blood pressure, which persisted for several minutes. Intraperitoneal apelin injections induced an increase in drinking behavior within the first 30 min after injection, with a return to baseline within 1 h.

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G-protein-coupled receptors (GPCRs) represent by far the largest class of targets for modern drugs. Virtually all therapeutics that are directed towards GPCRs have been designed using assays that presume that these receptors are monomeric. The recent realization that these receptors form homo-oligomeric and hetero-oligomeric complexes has added a new dimension to rational drug design. However, this important aspect of GPCR biology remains largely unincorporated into schemes to search for new therapeutics. This review provides a synopsis of the current thinking surrounding GPCR homo-oligomerization and hetero-oligomerization and shows how new models point towards unexplored avenues in the development of new therapies.

We demonstrate a heteromeric D1-D2 dopamine receptor signaling complex in brain that is coupled to Gq/11 and requires agonist binding to both receptors for G protein activation and intracellular calcium release. The D1 agonist SKF83959 was identified as a specific agonist for the heteromer that activated Gq/11 by functioning as a full agonist for the D1 receptor and a high-affinity partial agonist for a pertussis toxin-resistant D2 receptor within the complex. We provide evidence that the D1-D2 signaling complex can be more readily detected in mice that are 8 months in age compared with animals that are 3 months old, suggesting that calcium signaling through the D1-D2 dopamine receptor complex is relevant for function in the postadolescent brain. Activation of Gq/11 through the heteromer increases levels of calcium/calmodulin-dependent protein kinase II␣ in the nucleus accumbens, unlike activation of Gs/olf-coupled D1 receptors, indicating a mechanism by which D1-D2 dopamine receptor complexes may contribute to synaptic plasticity.heterooligomerization ͉ SKF83959 ͉ calcium signaling ͉ calcium/calmodulin-dependent protein kinase II␣ D iverse roles for each of the five dopamine receptors (D1-D5) have been shown to be initiated primarily through stimulation or inhibition of adenylyl cyclase (AC) via G s /olf or G i /o signaling proteins, respectively (1). There have been reports, however, of a D1-like receptor in brain that is coupled to G q /11, stimulating phospholipase C (PLC) and intracellular calcium release (2-5). Activation of this G q /11-coupled D1-like receptor by specific receptor agonists does not correlate with the ability of these same agonists to activate AC (4), suggesting that the G q /11-coupled D1-like receptor is a molecular entity distinct from the G s /olfcoupled D1 receptor.Molecular identification of the G q /11-coupled D1-like receptor has proven elusive because D1 receptor coupling to PLC has not been demonstrated in a variety of cell types in which the D1 receptor was expressed. We had postulated that G q /11 activation by D1 receptor agonists in brain could occur by concurrent activation of the D1 receptor and the D2 receptor (6). We have shown that heterologously coexpressed D1 and D2 dopamine receptors formed heterooligomers (7) and that coactivation of these receptors resulted in a PLC-dependent rise in intracellular calcium (6). We also demonstrated that D1 and D2 receptors could be coimmunoprecipitated from striatal membranes (6). These results suggested the possibility of a unique signaling complex in brain composed of PLC-coupled D1-D2 receptor heterooligomers.In this work we report the presence of such a D1-D2 dopamine receptor signaling complex in striatum that is coupled to rapid G q /11 signaling on activation of both receptors and which can be defined by a unique pharmacology. The complex was more readily detected in older mice and could modulate levels of calcium/calmodulin-dependent protein kinase II␣ (CaMKII␣) in the nucleus accumbens, indicating a potential role for the D...

Dopamine receptors belong to a superfamily of receptors that exert their biological effects through guanine nucleotide-binding (G) proteins. Two main dopamine receptor subtypes have been identified, D1 and D2, which differ in their pharmacological and biochemical characteristics. D1 stimulates adenylyl cyclase activity, whereas D2 inhibits it. Both receptors are primary targets for drugs used to treat many psychomotor diseases, including Parkinson's disease and schizophrenia. Whereas the dopamine D1 receptor has been cloned, biochemical and behavioural data indicate that dopamine D1-like receptors exist which either are not linked to adenylyl cyclase or display different pharmacological activities. We report here the cloning of a gene encoding a 477-amino-acid protein with strong homology to the cloned D1 receptor. The receptor, called D5, binds drugs with a pharmacological profile similar to that of the cloned D1 receptor, but displays a 10-fold higher affinity for the endogenous agonist, dopamine. As with D1, the dopamine D5 receptor stimulates adenylyl cyclase activity. Northern blot and in situ hybridization analyses reveal that the receptor is neuron-specific, localized primarily within limbic regions of the brain; no messenger RNA was detected in kidney, liver, heart or parathyroid gland. The existence of a dopamine D1-like receptor with these characteristics had not been predicted and may represent an alternative pathway for dopamine-mediated events and regulation of D2 receptor activity.

We report the isolation of a cDNA clone named GPR54, which encodes a novel G protein-coupled receptor (GPCR). A PCR search of rat brain cDNA retrieved a clone partially encoding a GPCR. In a library screening this clone was used to isolate a cDNA with an open reading frame (ORF) encoding a receptor of 396 amino acids long which shared significant identities in the transmembrane regions with rat galanin receptors GalR1 (45%), GalR3 (45%) and GalR2 (44%). Northern blot and in situ hybridization analyses revealed that GPR54 is expressed in brain regions (pons, midbrain, thalamus, hypothalamus, hippocampus, amygdala, cortex, frontal cortex, and striatum) as well as peripheral regions (liver and intestine). In COS cell expression of GPR54 no specific binding was observed for 125 I-galanin. A recent BLAST search with the rat GPR54 ORF nucleotide sequence recovered the human orthologue of GPR54 in a 3.5 Mb contig localized to chromosome 19p13.3.z 1999 Federation of European Biochemical Societies.

Although dopamine D1 and D2 receptors belong to distinct subfamilies of dopamine receptors, several lines of evidence indicate that they are functionally linked. However, a mechanism for this linkage has not been elucidated. In this study, we demonstrate that agonist stimulation of co-expressed D1 and D2 receptors resulted in an increase of intracellular calcium levels via a signaling pathway not activated by either receptor alone or when only one of the co-expressed receptors was activated by a selective agonist. Calcium signaling by D1-D2 receptor co-activation was abolished following treatment with a phospholipase C inhibitor but not with pertussis toxin or inhibitors of protein kinase A or protein kinase C, indicating coupling to the G q pathway. We also show, by co-immunoprecipitation from rat brain and from cells co-expressing the receptors, that D1 and D2 receptors are part of the same heteromeric protein complex and, by immunohistochemistry, that these receptors are co-expressed and co-localized within neurons of human and rat brain. This demonstration that D1 and D2 receptors have a novel cellular function when co-activated in the same cell represents a significant step toward elucidating the mechanism of the functional link observed between these two receptors in brain.The dopamine D1 receptor has been shown to signal via G s and G olf proteins to stimulate adenylyl cyclase (1), and there have been reports of calcium signaling by D1 receptor-mediated phosphatidylinositol hydrolysis by phospholipase C (PLC), 1 presumably through G q coupling. Although PLC activity was not detected in studies on the D1 receptor expressed in COS7 cells (2) or in Chinese hamster ovary and baby hamster kidney cells (3), it has been shown that activation of the D1 receptor in brain tissue (4, 5) or in Xenopus oocytes injected with mRNA taken from striatum (6) resulted in phosphatidylinositol turnover. This discrepancy suggests the involvement of a factor in D1 receptor-mediated PLC activity that is present in native tissues but absent in heterologous expression systems. An examination of the above studies where PLC activation was observed indicates that, although D1 receptor-selective antagonists blocked phosphatidylinositol turnover demonstrating the involvement of the D1 receptor, the amount of agonist used to elicit the response was greater than 100 M, a concentration at which even selective ligands may bind other receptors. For example, SKF 81297, a highly D1-selective agonist, has a K i for the D1 receptor of ϳ1 nM but has a K i for the D2 receptor of ϳ900 nM (7). This analysis suggests the possibility that the D1 agonistmediated PLC stimulation by high concentrations of drugs observed in physiological systems may have resulted from the coincident activation of another receptor.There are considerable data indicating that the dopamine D1 and D2 receptors are functionally linked. For example, behavioral studies have shown that co-stimulation of the D1 receptor is essential for D2 agonists to produce maximal locomotor stim...