Oligomers of ERBB3 have two distinct interfaces that differ in their sensitivity to disruption by heregulin

ErbB receptors associate in a ligand-dependent or -independent manner, and overexpression of epidermal growth factor receptor (ErbB1) or ErbB2 results in ligand-independent activation. Ligand-independent activation is poorly understood, and dimerization alone is not sufficient for activation. ErbB receptors also form higher order oligomers, but the mechanism of oligomer formation and their contribution to signaling are not known. The kinase-deficient ErbB3 as well as its extracellular domains are particularly prone to ligand-independent oligomerization, and oligomers are destabilized by binding of the ligand heregulin. In contrast, ligand binding facilitates heterodimerization with ErbB2 and is expected to stabilize an extended conformation of the ErbB3 extracellular domain (ECD) in which the dimerization interface is exposed. In the absence of ligand, ErbB3 can adopt a closed conformation that is held together by an intramolecular tether. We used a constitutively extended form of the ErbB3-ECD to analyze the conformation of the ECD in oligomers and the mechanism of oligomer disruption by heregulin. The extended conformation of the ECD forms oligomers more readily, suggesting the crystallographically defined dimer interface is one of the interfaces involved in oligomerization. Heregulin destabilizes oligomeric complexes but not dimers, which are neither stabilized nor disrupted by ligand binding, indicating a distinct second interface in oligomers of ErbB3. Cross-linking and activation studies on membrane-embedded ErbB3/ErbB2 chimeras confirm this dual effect of heregulin. Most of the ErbB3-ECD on the cell surface is apparently kept in an open conformation through oligomerization, and the resulting oligomers adopt a conformation representing a state of reduced activity.     

D1 dopamine receptor stimulation increases GluR1 surface expression in nucleus accumbens neurons

The goal of this study was to understand how dopamine receptors, which are activated during psychostimulant administration, might influence glutamate-dependent forms of synaptic plasticity that are increasingly recognized as important to drug addiction. Regulation of the surface expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR1 plays a critical role in long-term potentiation, a well-characterized form of synaptic plasticity. Primary cultures of rat nucleus accumbens neurons were used to examine whether dopamine receptor stimulation influences cell surface expression of GluR1, detected using antibody to the extracellular portion of GluR1 and fluorescence microscopy. Surface GluR1 labeling on processes of medium spiny neurons and interneurons was increased by brief (5-15 min) incubation with a D1 agonist (1 microm SKF 81297). This effect was attenuated by the D1 receptor antagonist SCH 23390 (10 microm) and reproduced by the adenylyl cyclase activator forskolin (10 microm). Labeling was decreased by glutamate (10-50 microm, 15 min). These results are the first to demonstrate modulation of AMPA receptor surface expression by a non-glutamatergic G protein-coupled receptor. Normally, this may enable ongoing regulation of AMPA receptor transmission in response to changes in the activity of dopamine projections to the nucleus accumbens. When dopamine receptors are over-stimulated during chronic drug administration, this regulation may be disrupted, leading to inappropriate plasticity in neuronal circuits governing motivation and reward.     

Mechanisms of inverse agonism of antipsychotic drugs at the D(2) dopamine receptor: use of a mutant D(2) dopamine receptor that adopts the activated conformation

The antipsychotic drugs have been shown to be inverse agonists at the D(2) dopamine receptor. We have examined the mechanism of this inverse agonism by making mutations in residue T343 in the base of the sixth transmembrane spanning region of the receptor. T343R, T343S and T343K mutant D(2) dopamine receptors were made and the T343R mutant characterized in detail. The T343R mutant D(2) dopamine receptor exhibits properties of a receptor that resides more in the activated state, namely increased agonist binding affinity (independent of G-protein coupling and dependent on agonist efficacy), increased agonist potency in functional tests (adenylyl cyclase inhibition) and increased inverse agonist effects. The binding of agonists to the mutant receptor also shows sensitivity to sodium ions, unlike the native receptor, so that isomerization of the receptor to its inactive state may be driven by sodium ions. The binding of inverse agonists to the receptor is, however, unaffected by the mutation. We conclude that inverse agonism at this receptor is not achieved by the inverse agonist binding preferentially to the non-activated state of the receptor over the activated state. Rather the inverse agonist appears to bind to all forms of the receptor but then renders the receptor inactive.     

Distribution of D(5) dopamine receptor mRNA in rat ventromedial hypothalamic nucleus

Estrogen induces lordosis through, in part, estrogen receptor (ER)-mediated synthesis of progesterone receptors (PR) in the ventromedial nucleus (VMN). In vitro, PR is activated by the neurotransmitter dopamine through D1-like receptors (1). In vivo, lordosis is induced by dopamine, an effect mediated in part by PR and D(5) dopamine receptors. The purpose of the present study was to determine mRNA distribution of D1-like receptors in the female rat brain using RT-PCR combined with punchout microdissection techniques. Employing specific primers to D(5) and D(1) dopamine receptors, we found detectable expression levels of D(5) dopamine receptor mRNA in VMN as well as the arcuate nucleus/median eminence (ArcN/ME). In contrast, D(1) dopamine receptor mRNA was detected only in VMN. By using this highly sensitive and specific RT-PCR methodology, we have confirmed the presence of D(5) dopamine receptor mRNA in an area of the brain that regulates reproductive behavior through PR. The data support the previous observation that D(5) dopamine receptors in VMN contribute to facilitation of female reproductive behavior by D1-like agonists.     

Serine mutations in transmembrane V of the dopamine D1 receptor affect ligand interactions and receptor activation.

Several serines present in transmembrane domain V are conserved among members of the G-protein-coupled receptor family that bind catecholamines. Two of these serines that are present in the beta-adrenergic receptor were previously shown by site-directed mutagenesis to affect agonist binding and receptor activation (Strader, C. D., Candelore, M. R., Hill, W. S., Sigal, I. S., and Dixon, R. A. F. (1989) J. Biol. Chem. 264, 13572-13578). We investigated the role of the serines present in transmembrane V of another catecholamine receptor, the dopamine D1 receptor, by site-directed mutagenesis, and the results show that mutations at serines 198, 199, and 202 affect dopamine binding. The substitution of serine 198 or serine 199 by an alanine also affects the binding of several other agonist and antagonist dopaminergic compounds while an alanine substitution at serine 202 has no effect on the binding of these compounds. Moreover, each single serine mutation decreased the maximal cAMP accumulation elicited by a dopamine D1 partial agonist. These results suggest that serines present in transmembrane V of the D1 receptor affect ligand interactions and receptor signal transduction, but not entirely in the manner that would be predicted from the model proposed for the beta-adrenergic receptor.     

Hydrophobic Residues of the D2 Dopamine Receptor Are Important for Binding and Signal Transduction

Abstract: Dopamine receptors belong to the seven transmembrane helix-containing, G protein-coupled receptor superfamily. Mutagenesis studies suggest that dopamine and its analogues interact with aspartate-114 in helix 3 and two helix 5 serines (194 and 197) of the D₂ receptor. In addition to these amino acids, hydrophobic residues within the receptor core may be important not only for binding but also for receptor activation. Described is a site-directed mutagenesis investigation into the roles of these hydrophobic residues in the long isoform of the human D₂ receptor. Replacement of helix 6 phenylalanines (389 or 390) with alanines resulted in disrupted binding to several agonists and antagonists and impaired inhibition of adenylyl cyclase activity. Replacement of the helix 5 phenylalanine-198 with an alanine selectively disrupted [³H]N-0437 binding, whereas the affinities for other agonists and antagonists remained unchanged. This mutant remained functionally intact when stimulated with dopamine or bromocriptine. Replacement of the helix 7 phenylalanine-411 or the helix 6 leucine-387 with alanines produced receptors that bound agonists well but were unable to inhibit adenylyl cyclase. Based on these data, two conserved helix 6 phenylalanines (389 and 390) appear to be crucial for ligand binding, and phenylalanine-411 in helix 7 and leucine-387 in helix 6 may be important for propagating conformational changes from the agonist binding site(s) to G protein coupling domain(s) of the D₂ receptor.     

Molecular basis of inverse agonism in a G protein-coupled receptor

G protein-coupled receptors (GPCRs) recognize a wide variety of extracellular ligands to control diverse physiological processes. Compounds that bind to such receptors can either stimulate, fully or partially (full or partial agonists), or reduce (inverse agonists) the receptors' basal activity and receptor-mediated signaling. Various studies have shown that the activation of receptors through binding of agonists proceeds by conformational changes as the receptor switches from a resting to an active state leading to G protein signaling. Yet the molecular basis for differences between agonists and inverse agonists is unclear. These different classes of compounds are assumed to switch the receptors' conformation in distinct ways. It is not known, however, whether such switching occurs along a linear 'on-off' scale or whether agonists and inverse agonists induce different switch mechanisms. Using a fluorescence-based approach to study the alpha2A-adrenergic receptor (alpha(2A)AR), we show that inverse agonists are differentiated from agonists in that they trigger a very distinct mode of a receptor's switch. This switch couples inverse agonist binding to the suppression of activity in the receptor.     

Structural Factors that Distinguish Dopamine D1 and D2 Agonists

To determine the structural features responsible for their selectivity as dopamine D1 agonists, a conformational analysis has been performed on an analog of nomifensine, dihydrexidine, a benzergoline, and an isochroman using the MM2-87 program. The preferred three dimensional structure of the hydroxylated phenyl ring of the nomifensine analog was found to differ from the other compounds with a substantial energy barrier to achieving the planar conformation of the other compounds which may explain its weak potency for D1 receptors. The preferred three dimensional structures of dihydrexidine and the benzergoline were found to differ significantly despite their molecular similarity. These conformational differences were also evident in crystal structures of the compounds or their analogs. The hypothesis that an equatorial ammonium hydrogen (or amine lone pair) is required for D1 agonist selectivity was tested by performing calculations on N-methyl equatorial and N-methyl axial analogs of the compounds. Calculations were also performed on nonselective dopamine agonists (apomorphine and 5,6-diOH- and 6,7-diOH aminotetralin) and dopamine D2-selective agonists ((+)-PHNO and an analog of quinpirole). The energy difference for the N-methyl axial conformations (or their equivalent) were found to be relatively small for the nonselective agonists and more substantial for the D2-selective agonists. This suggests that D2-selectivity may be associated with the relative unfavorability of the N-methyl axial conformation and provides an explanation for the decreased potency of tertiary amine analogs of the D1-selective agonists. In the benzergoline, where the energy difference is computed to be smaller, the addition of the N-methyl group appears to have a smaller deleterious effect on D1 activity. An N-methyl axial conformation has also been observed for the benzergoline in the crystal state suggesting that this conformation is energetically accessible.     

Synthesis and Pharmacology of the enantiomers of the potential atypical antipsychotic agents 5-OMe-BPAT and 5-OMe-(2,6-di-OMe)-BPAT.

The optically pure enantiomers of the potential atypical antipsychotic agents 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 5) and 5-methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}t etralin [5-OMe-(2,6-di-OMe)-BPAT, 6] were synthesized and evaluated for their in vitro binding affinities at alpha1-, alpha2-, and beta-adrenergic, muscarinic, dopamine D1, D2A, and D3, and serotonin 5-HT1A and 5-HT2 receptors. In addition, their intrinsic efficacies at serotonin 5-HT1A receptors were established in vitro. (S)- and (R)-5 had high affinities for dopamine D2A, D3, and serotonin 5-HT1A receptors, moderate affinities for alpha1-adrenergic and serotonin 5-HT2 receptors, and no affinity (Ki > 1000 nM) for the other receptor subtypes. (S)- and (R)-6 had lower affinities for the dopamine D2A and the serotonin 5-HT1A receptor, compared to (S)- and (R)-5, and hence showed some selectivity for the dopamine D3 receptor. The interactions with the receptors were stereospecific, since the serotonin 5-HT1A receptor preferred the (S)-enantiomers, while the dopamine D2A and D3 receptors preferred the (R)-enantiomers of 5 and 6. The intrinsic efficacies at the serotonin 5-HT1A receptor were established by measuring their ability to inhibit VIP-induced cAMP production in GH4ZD10 cells expressing serotonin 5-HT1A receptors. Both enantiomers of 5 behaved as full serotonin 5-HT1A receptor agonists in this assay, while both enantiomers of 6 behaved as weak partial agonists. The potential antipsychotic properties of (S)- and (R)-5 were evaluated by establishing their ability to inhibit d-amphetamine-induced locomotor activity in rats, while their propensity to induce extrapyramidal side-effects (EPS) in man was evaluated by determining their ability to induce catalepsy in rats. Whereas (R)-5 was capable of blocking d-amphetamine-induced locomotor activity, indicative of dopamine D2 receptor antagonism, (S)-5 even enhanced the effect of d-amphetamine, suggesting that this compound has dopamine D2 receptor-stimulating properties. Since both enantiomers also were devoid of cataleptogenic activity, they are interesting candidates for further exploring the dopamine D2/serotonin 5-HT1A hypothesis of atypical antipsychotic drug action.     

Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization

Adenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS-7 cells, long-term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS-15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long-term desensitization time-course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre-treatment of cells with R(-)-PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors.     

D2 dopamine receptor homodimerization is mediated by multiple sites of interaction,including an intermolecular interaction involving transmembrane domain 4

In this study, we examined the mechanisms of intermolecular interaction involved in D2 dopamine receptor dimer formation to develop an understanding of the quaternary structure of G protein-coupled receptors. The potential role of two mechanisms was investigated: disulfide bridges and hydrophobic interactions between transmembrane domains. D2 dopamine receptor oligomers were unaffected by treatment with a reducing agent; however, oligomers of the D1 dopamine receptor dissociated following a similar treatment. This observation suggested that other forces such as hydrophobic interactions were more robust in the D2 receptor than in the D1 receptor in maintaining oligomerization. To elucidate which transmembrane domains were involved in the intermolecular hydrophobic interactions, truncation mutants were generated by successive deletion of transmembrane domains from amino and/or carboxyl portions of the D2 dopamine receptor. Immunoblot analyses revealed that all the fragments were well expressed but only fragments containing transmembrane domain 4 were able to self-associate, suggesting that critical areas for receptor dimerization resided within this transmembrane domain. Disruption of the helical structure of transmembrane domain 4 in a truncated receptor capable of forming dimers interfered with its ability to self-associate; however, a similar disruption of the transmembrane domain 4 helix structure in the full-length receptor did not significantly affect dimerization. These results indicated that there are other sites of interaction involved in D2 receptor oligomer assembly in addition to transmembrane domain 4.     

D(1) dopamine receptor stimulation increases GluR1 phosphorylation in postnatal nucleus accumbens cultures

Postsynaptic interactions between dopamine and glutamate receptors in the nucleus accumbens are critical for acute responses to drugs of abuse and for neuroadaptations resulting from their chronic administration. We tested the hypothesis that D(1) dopamine receptor stimulation increases phosphorylation of the AMPA receptor subunit GluR1 at the protein kinase A phosphorylation site (Ser845). Nucleus accumbens cell cultures were prepared from postnatal day 1 rats. After 14 days in culture, GluR1 phosphorylation was measured by western blotting using phosphorylation site-specific antibodies. The D(1) receptor agonist SKF 81297 increased Ser845 phosphorylation in a concentration- dependent manner, with marked increases occurring within 5 min. This was prevented by the D(1) receptor antagonist SCH 23390 and the protein kinase A inhibitor H89, and reproduced by forskolin. The D(2) receptor agonist quinpirole attenuated the response to D(1) receptor stimulation. Neither D(1) nor D(2) receptor agonists altered GluR1 phosphorylation at Ser831, the site phosphorylated by protein kinase C and calcium/calmodulin-dependent protein kinase II. In other systems, phosphorylation of GluR1 at Ser845 is associated with enhancement of AMPA receptor currents. Thus, the present results suggest that AMPA receptor transmission in the nucleus accumbens may be augmented by concurrent D(1) receptor stimulation.     

Dopamine D1 and D2 Receptor Immunoreactivities in the Arcuate-Median Eminence Complex and their Link to the Tubero-Infundibular Dopamine Neurons

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and/or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region.Key words: Dopamine D1 and D2 receptors, tubero-infundibular dopamine neurons, dopamine receptor colocalization, arcuate-median eminence complex, volume transmission, luteinizing hormone releasing hormone     

Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells

Mammalian D1 and D2 dopamine receptors were stably expressed in Drosophila Schneider-2 (S2) cells and screened for their pharmacological properties. Saturable, dose-dependent, high affinity binding of the D1-selective antagonist [3H]SCH-23390 was detected only in membranes from S2 cells induced to express rat dopamine D1 receptors, while saturable, dose-dependent, high affinity binding of the D2-selective antagonist [3H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D2 receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D1 and D2 receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D1 receptor bound the (+)-enantiomer of the D1-selective agonist SKF38393 with higher affinity than the (-)-enantiomer, while the dopamine D2 receptor bound the (-)-enantiomer of the D2-selective agonist norpropylapomorphine with higher affinity than the (+)-enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPgammaS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D1 and D2 receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D1 and D2 dopamine receptors free of promiscuous G protein contaminants.     

In vivo CREB phosphorylation mediated by dopamine and NMDA receptor activation in mouse hippocampus and caudate nucleus

The pattern of CREB phosphorylation was investigated in the caudate nucleus and hippocampus 10 min or 3 h after i.p. injection of dopamine or NMDA receptor agonists alone, or in combination with antagonists. Ten minutes after C57BL/6 J mice were injected with either the dopamine D1 receptor agonist SKF-38393 hydrobromide or NMDA, immunoreactivity of phosphorylated CREB (pCREB) was significantly increased in all parts of the caudate nucleus but not in hippocampal regions. However, 3 h after the injection of SKF-38393, pCREB levels in the caudate nucleus did not differ significantly from the pCREB levels in control animals, whereas pCREB levels were still elevated 3 h after NMDA injection. Except for the D1 receptor antagonist SCH-23390, which induced CREB phosphorylation in the caudate nucleus, dopamine and NMDA receptor antagonists had little effect on pCREB levels by themselves. However, the NMDA receptor antagonist CGS-19755 injected i.p. blocked both the NMDA- and SKF-38393-induced rise of pCREB levels in the caudate nucleus. Similarly, the D1 receptor antagonist SCH-23390 inhibited the effects produced by SKF-38393 or NMDA. Interestingly, the D2 receptor antagonist sulpiride also blocked the SKF-38393-triggered rise of pCREB. The results demonstrated that NMDA and dopamine receptors modulate pCREB levels in the caudate nucleus and suggest mutual permissive roles for both receptors.     

Assessment of striatal D1 and D2 dopamine receptor-G protein coupling by agonist-induced [35S]GTP gamma S binding

The dopamine receptor-mediated modulation of guanosine 5'-O-(gamma-[35S]thio)triphosphate ([35S]GTP gamma S) binding has been characterized in rat striatal membranes. In optimized experimental conditions, the potency of dopamine was 4.47 microM [3.02-6.61 microM] and a maximal response representing 54.8 +/- 4.5% increase above basal level was observed. Data obtained with different agonists and antagonists clearly revealed that the most important fraction of this response was reflecting D2 receptor activation. Further analysis with specific antagonists also supported evidence for the involvement of D1 dopamine receptors. The potencies of compounds interacting with D1 and D2 receptors were deduced from [35S]GTP gamma S binding experiments and compared with their binding affinities for these receptors measured in similar experimental conditions. A good correlation between these parameters was observed, supporting the applicability of this technique for the study of dopamine receptors in the central nervous system.     

Ligand-independent oligomerization of natriuretic peptide receptors. Identification of heteromeric receptors and a dominant negative mutant.

Activation of many single-transmembrane receptors requires ligand-induced receptor oligomerization. We have examined the oligomerization of the atrial natriuretic peptide receptor, NPR-A, using epitope-tagged receptor in a co-immunoprecipitation assay. Unlike other single-transmembrane receptors, NPR-A oligomerized in a ligand-independent fashion. Extracellular receptor sequences were both necessary and sufficient for oligomer formation. NPR-A was also able to oligomerize with the related natriuretic peptide receptor, NPR-B. A truncated NPR-A lacking most of the cytoplasmic domain blocked activation of the full-length receptor, presumably through formation of an inactive heteromer. These results indicate that oligomerization of this single-transmembrane receptor is important for the transduction of a conformational change across the plasma membrane but are not consistent with models in which natriuretic peptide receptor oligomerization serves merely to bring intracellular domains together.     

Adenosine and dopamine receptor antagonist binding in the rat ventral and dorsal striatum: lack of changes after a neonatal bilateral lesion of the ventral hippocampus.

There is experimental evidence from radioligand binding experiments for the existence of strong antagonistic interactions between different subtypes of adenosine and dopamine receptors in the striatum, mainly between adenosine A1 and dopamine D1 and between adenosine A2A and dopamine D2 receptors. These interactions seem to be more powerful in the ventral compared to the dorsal striatum, which might have some implications for the treatment of schizophrenia. The binding characteristics of different dopamine and adenosine receptor subtypes were analysed in the different striatal compartments (dorsolateral striatum and shell and core of the nucleus accumbens), by performing saturation experiments with the dopamine D1 receptor antagonist [125I]SCH-23982, the dopamine D2-3 receptor antagonist [3H]raclopride, the adenosine A1 receptor antagonist [3H]DPCPX and the adenosine A2A receptor antagonist [3H]SCH 58261. The experiments were also performed in rats with a neonatal bilateral lesion of the ventral hippocampus (VH), a possible animal model of schizophrenia. Both dopamine D2-3 and adenosine A2A receptors follow a similar pattern, with a lower density of receptors (40%) in the shell of the nucleus accumbens compared with the dorsolateral caudate-putamen. A lower density of adenosine A1 receptors (20%) was also found in the shell of the nucleus accumbens compared with the caudate-putamen. On the other hand, dopamine D1 receptors showed a similar density in the different striatal compartments. Therefore, differences in receptor densities cannot explain the stronger interactions between adenosine and dopamine receptors found in the ventral, compared to the dorsal striatum. No statistical differences in the binding characteristics of any of the different adenosine and dopamine receptor antagonists used were found between sham-operated and VH-lesioned rats.     

Pharmacological characterization of dopamine receptors in the corpus allatum of Manduca sexta larvae

Dopamine receptors previously identified in corpora allata (CA) of Manduca sexta last instars on the basis of dopamine effects on JH (juvenile hormone)/JH acid biosynthesis and cyclic AMP (cAMP) accumulation, were characterized pharmacologically. For this study, a broad spectrum of agonists or antagonists of D1, D2, D3 or D4 dopamine receptors, together with the dopamine metabolite N-acetyl-dopamine, other neurotransmitters and their agonists/antagonists, were tested for their effects on gland activity and cAMP production. The lack of effect of other neurotransmitters supports the specificity of the effect of dopamine and the dopamine specificity of the receptors. Only the D2 receptor antagonist spiperone had a potent effect on JH biosynthesis and cAMP formation by CA taken on day 0 of the last stadium, when dopamine stimulates both activities and thus appears to be acting via a D1-like receptor. Several other D2 receptor antagonists, and D1, D2/D1 and D4,3/D2 receptor antagonists were less effective. Thus, the D1-like receptor of the Manduca CA appears to be distinct pharmacologically from vertebrate D1 receptors. By contrast, a number of D2 agonists/antagonists had a significant effect on JH acid biosynthesis and cAMP production by the CA from day 6 of the last stadium, when dopamine inhibits both activities and thus appears to be acting via a D2-like receptor. Certain D1-specific agonists/antagonists were equally effective. The Manduca D2-like receptor therefore bears some pharmacological resemblance to vertebrate D2 receptors. N-acetyl dopamine acted as a dopamine agonist with day 6 CA, the first identified function for an N-acetylated biogenic amine in insects. Dopamine was found to have the same differential affect on the formation of cAMP in homogenates of day 0 and day 6 brains as it did with CA, and in the same concentration range. Dopamine receptor agonists/antagonists affecting cAMP formation by day 0 and day 6 CA homogenates had similar effects with brain homogenates. By contrast, dopamine only stimulated cAMP formation by homogenates of day 0 and day 6 abdominal or ventral nerve cord. These results suggest that D1- and D2-like dopamine receptors of Manduca are regionally as well as temporally localized.     

Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes

Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCR oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.