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.

Although the perturbation of either the dopaminergic system or brain-derived neurotrophic factor (BDNF) levels has been linked to important neurological and neuropsychiatric disorders, there is no known signaling pathway linking these two major players. We found that the exclusive stimulation of the dopamine D1-D2 receptor heteromer, which we identified in striatal neurons and adult rat brain by using confocal FRET, led to the activation of a signaling cascade that links dopamine signaling to BDNF production and neuronal growth through a cascade of four steps: (i) mobilization of intracellular calcium through Gq, phospholipase C, and inositol trisphosphate, (ii) rapid activation of cytosolic and nuclear calcium/calmodulin-dependent kinase II␣, (iii) increased BDNF expression, and (iv) accelerated morphological maturation and differentiation of striatal neurons, marked by increased microtubule-associated protein 2 production. These effects, although robust in striatal neurons from D5 ؊/؊ mice, were absent in neurons from D1 ؊/؊ mice. We also demonstrated that this signaling cascade was activated in adult rat brain, although with regional specificity, being largely limited to the nucleus accumbens. This dopaminergic pathway regulating neuronal growth and maturation through BDNF may have considerable significance in disorders such as drug addiction, schizophrenia, and depression.brain-derived neurotrophic factor activation ͉ calcium signaling pathway ͉ calcium/calmodulin-dependent kinase II ͉ neuronal maturation ͉ GPCR oligomerization D opamine promotes neuronal differentiation, maintenance, and survival (1-4) by modulating the transcription of different genes. Little however, is known regarding the molecular events that govern these dopamine-mediated effects. Evidence has emerged indicating a positive relationship between functions mediated by dopamine and brain-derived neurotrophic factor (BDNF) and its receptor TrkB (2-7). However, a direct mechanism bridging dopamine signaling to BDNF has not yet been described. Classically, dopamine exerts its actions through D1-like (D1, D5) and D2-like (D2, D3, D4) receptors, which regulate activation or inhibition of cAMP accumulation, through Gs/olf or Gi/o proteins, respectively (8). Other signaling cascades have also been reported (9, 10), including phosphatidylinositol turnover in brain through D1-like receptor activation (11,12), but no such activation was observed when the cloned D1 receptor was expressed (13-15). These observations led us to the discovery of the dopamine D1-D2 receptor heterooligomer, which is able to mobilize intracellular calcium (15-18). However, the signaling cascade and the physiological functions of the dopamine D1-D2 receptor heteromer in brain are unknown.Because calcium is involved in the activation of BDNF signaling (19), we hypothesized that this pathway may be central to dopamine activation of BDNF and subsequent neuronal maturation and differentiation.In this context, we describe a signaling pathway that links dopamine action through the...

The distribution and function of neurons coexpressing the dopamine D1 and D2 receptors in the basal ganglia and mesolimbic system are unknown. We found a subset of medium spiny neurons coexpressing D1 and D2 receptors in varying densities throughout the basal ganglia, with the highest incidence in nucleus accumbens and globus pallidus and the lowest incidence in caudate putamen. These receptors formed D1-D2 receptor heteromers that were localized to cell bodies and presynaptic terminals. In rats, selective activation of D1-D2 heteromers increased grooming behavior and attenuated AMPA receptor GluR1 phosphorylation by calcium/calmodulin kinase II␣ in nucleus accumbens, implying a role in reward pathways. D1-D2 heteromer sensitivity and functional activity was up-regulated in rat striatum by chronic amphetamine treatment and in globus pallidus from schizophrenia patients, indicating that the dopamine D1-D2 heteromer may contribute to psychopathologies of drug abuse, schizophrenia, or other disorders involving elevated dopamine transmission. Dopaminergic signaling within basal ganglia occurs within at least two populations of GABAergic medium spiny neurons (MSNs), 2 containing the neuropeptides dynorphin (DYN) and substance P, or containing enkephalin (ENK) (1, 2). There is controversy as to the colocalization of dopamine D1 and D2 receptors (D1R, D2R) within these two neuronal subtypes. It is generally agreed that whereas D1R is largely segregated to the DYN/substance P-expressing direct striatonigral pathway, D2R is predominantly localized to the ENK-expressing indirect striatopallidal pathway, which is supported by recent studies using fluorophore-tagged promoter elements of D1R and D2R in bacterial artificial chromosome transgenic mice to quantify the cells expressing the receptors within these pathways (3, 4). Although strict segregation of D1R and D2R has been suggested (5, 6) it was noted in these studies that although ϳ60% of MSNs in nucleus accumbens (NAc) expressed D1R, ϳ50% expressed D2R. This indicated a certain fraction of NAc MSNs expressed both receptors, a finding consistent with numerous studies showing colocalization of D1R and D2R in a proportion of striatal neurons (7-11).In keeping with these findings, we have shown that D1R and D2R interacted in striatum to form a D1-D2 heteromeric complex that could be immunoprecipitated (9, 12). The D1-D2 heteromer was distinct from its constituent receptors in that it coupled to Gq/11 to activate phospholipase C and generate intracellular calcium release, representing a novel signaling pathway directly linking dopamine action to calcium (9, 13). However, despite growing evidence that neurons coexpressing D1R and D2R embody a significant fraction of MSNs in NAc, their regional distribution, phenotypic characterization, and functional role within basal ganglia are entirely unknown. Given the apparent relationship between dopamine receptor expression and DYN or ENK content, we hypothesized that neurons expressing the D1-D2 heteromer would contain both DYN and ...

The existence of dimers and oligomers for many G protein-coupled receptors has been described by us and others. Since many G protein-coupled receptor subtypes are highly homologous to each other, we examined whether closely related receptors may interact with each other directly and thus have the potential to create novel signaling units. Using -and ␦-opioid receptors, we show that each receptor expressed individually was pharmacologically distinct and could be visualized following electrophoresis as monomers, homodimers, homotetramers, and higher molecular mass oligomers. When -and ␦-opioid receptors were coexpressed, the highly selective synthetic agonists for each had reduced potency and altered rank order, whereas endomorphin-1 and Leu-enkephalin had enhanced affinity, suggesting the formation of a novel binding pocket. No heterodimers were visualized in the membranes coexpressing -and ␦-receptors by the methods available. However, heterooligomers were identified by the ability to co-immunoprecipitate -receptors with ␦-receptors and vice versa using differentially epitope-tagged receptors. In contrast to the individually expressed -and ␦-receptors, the coexpressed receptors showed insensitivity to pertussis toxin and continued signal transduction, likely due to interaction with a different subtype of G protein.In this study, we provide, for the first time, evidence for the direct interaction of -and ␦-opioid receptors to form oligomers, with the generation of novel pharmacology and G protein coupling properties.Opioid receptors have distinct pharmacological profiles and discrete but overlapping distributions in brain. The relatively recent cloning of opioid receptors has established that the products of three genes form the known subtypes (the -, ␦-, and -opioid receptors) that interact with the complex family of endogenous opioid peptides (reviewed in Ref. 1). The endogenous opioid peptide-receptor systems mediate important physiological functions related to pain perception, locomotion, motivation, reward, autonomic function, immunomodulation, and hormone secretion.The analysis of the contribution of each receptor type to the various opioid functions documented has been limited by the selectivity and cross-reactivity of the available opioid ligands and the postulation that multiple receptor subtypes are present. Since the cloning of the opioid receptors, the individual pharmacological and biochemical profiles of the -, ␦-, and -opioid receptors have been better defined; however, there are many aspects of opioid receptor biology that still remain poorly understood. A major problem that still remains is that the pharmacology of opioid receptors in brain tissue predicts a greater number of receptor subsites than revealed by opioid receptor cloning (1, 2). One possible explanation may be that the precise receptor-effector interactions present in endogenous brain regions may have not been adequately replicated in the heterologous expression systems in which the cloned receptors have been studied or, alternativel...