Molecular dynamics of dopamine at the D2 receptor.

A three-dimensional model of the dopamine D2 receptor, assumed to be a target of antipsychotic drug action, was constructed from its amino acid sequence. The model was based on structural similarities within the super-family of guanine nucleotide-binding regulatory (G) protein-coupled neuroreceptors and has seven alpha-helical transmembrane segments that form a central core with a putative ligand-binding site. The space between two residues postulated to be involved in agonist binding, Asp-80 and Asn-390, perfectly accommodated an anti-dopamine molecule. Molecular electrostatic potentials were mainly negative on the synaptic side of the receptor model and around aspartate residues lining the central core and positive in the cytoplasmic domains. The docking of dopamine into a postulated binding site was examined by molecular dynamics simulation. The protonated amino group became oriented toward negatively charged aspartate residues in helix 2 and helix 3, whereas the dopamine molecule fluctuated rapidly between different anti and gauche conformations during the simulation. The receptor model suggests that protonated ligands are attracted to the binding site by electrostatic forces and that protonated agonists may induce conformational changes in the receptor, leading to G-protein activation, by increasing the electrostatic potentials near Asp-80.     

Conformation of acetylcholine bound to the nicotinic acetylcholine receptor.

We report here the biologically active conformation of acetylcholine when bound to the high-affinity state of the receptor from Torpedo californica. The acetylcholine conformation was determined in the free and bound states by proton NMR two-dimensional nuclear Overhauser effects. In agreement with x-ray crystallographic data, acetylcholine in solution has an extended conformation with an average distance between the acetyl methyl and choline methyl protons of approximately equal to 5 A. When bound to the acetylcholine receptor, acetylcholine adopts a conformation where the acetyl methyl group is close (3.3 A) to the methyl groups of the choline moiety. This bent conformation places the oxygens adjacent to one another and allows the methyl groups to form an uninterrupted hydrophobic surface over the rest of the acetylcholine molecule. The significant difference between the free- and bound-state conformations implies that structure-activity studies based solely on molecular modeling strategies must be approached with caution.     

Conformation of the beta subunit of deglycosylated human chorionic gonadotropin in the interaction at receptor sites

The present study was performed to evaluate the relationship between the antagonist effect and the conformation of deglycosylated human chorionic gonadotropin (DGhCG) on the Leydig cell lutropin receptors. The maximum binding of [125I]DGhCG to anti-hCG beta antibody was decreased by 50%, while its binding profile to anti-hCG, anti-DGhCG and anti-hCG alpha antibodies remained unchanged, suggesting conformational changes in the beta subunit of DGhCG. However, the association of [125I]DGhCG to the binding sites of the receptors was much faster than that of [125I]hCG, and the ligand reached the binding equilibrium at 4 and 37 degrees C for 3 h and 15-30 min, respectively. Thus, the conformational changes in the beta subunit were not accompanied by loss of its receptor binding ability. The agonist properties of DGhCG which was bound to the receptors was fully restored by the addition of anti-hCG beta subunit antibody, while anti-hCG or anti-DGhCG restored only about 30% of the full agonist activity. This was probably due to a change of the conformation of the beta subunit to make it similar to that of the intact hormone. This restoration caused by anti-hCG beta was partially prevented by anti-hCG alpha. These facts indicate that some conformational change only in the beta subunit, not in the alpha subunit, of the deglycosylated hormone bound to the receptors is essential for the restoration of agonist properties.     

Opiates, prolactin, and the dopamine receptor.

The administration of a dopamine antagonist, chlorpromazine, and two opiates, morphine and methadone, resulted in a significant rise in serum PRL within 90-150 min. Prior administration of dopamine receptor agonists (apomorphine, levodopa, aand bromocriptine) blocked this effect. In contrast, cyproheptadine, a serotonin antagonist, did not. We suggest that the opiates induce hyperprolactinemia in man via dopamine receptor blockade.     

A dopamine receptor in esophageal smooth muscle of the opossum.

We examined the possibility that dopamine may play a role in nerve-mediated "off" responses of esophageal body (EB) and relaxations of lower esophageal spincter (LES) smooth muscle. The effects of dopamine, epinine, and dopamine antagonists on EB and LES smooth muscle were studied on these responses. Dopamine and epinine caused a dose-related fall in basal LES muscle tension and in amplitude of EB muscle "off" responses. Threshold dose for both was about 10(-7) M, and maximal dose was about 10(-4) M. At high concentrations, they also caused repetitive transient contractions of both LES and EB muscle after the period of inhibition. These effects were antagonized by haloperidol, 10(-5) M, and bulbocapnine, 10(-5) M, but were not influenced by propranolol, 10(-5) M, nor by phenoxybenzamine, 10(-5) M. Neither haloperidol nor bulbocapnine influenced responses to electrical field stimulation. Tetrodotoxin 10(-7) M abolished the responses to electrical field stimulation but did not antagonize the effects of dopamine and epinine. EB and LES smooth muscle contain a dopamine receptor. It is unlikely that dopamine is involved in responses to electrical field stimulation.     

Distribution of D2 dopamine receptor mRNA in rat brain.

The distribution of mRNA coding for the D2 dopamine receptor was studied in the rat brain by in situ hybridization. A cDNA probe corresponding to the putative third cytosolic loop and sixth and seventh transmembrane domains of the rat D2 receptor was used to generate an 35S-labeled riboprobe to hybridize to D2 receptor mRNA. D2 mRNA was found both in dopamine projection fields and in regions associated with dopamine-containing cell bodies, suggesting both postsynaptic and presynaptic autoreceptor localization. Highest concentrations of D2 mRNA were found in neostriatum, olfactory tubercle, substantia nigra, ventral tegmental area, and the nucleus accumbens. This distribution is consistent with those reported with D2 receptor autoradiography.     

From the Cover: Evidence of positive selection acting at the human dopamine receptor D4 gene locus

Associations have been reported of the seven-repeat (7R) allele of the human dopamine receptor D4 (DRD4) gene with both attention-deficit/hyperactivity disorder and the personality trait of novelty seeking. This polymorphism occurs in a 48-bp tandem repeat in the coding region of DRD4, with the most common allele containing four repeats (4R) and rarer variants containing 2-11. Here we show by DNA resequencing/haplotyping of 600 DRD4 alleles, representing a worldwide population sample, that the origin of 2R-6R alleles can be explained by simple one-step recombination/mutation events. In contrast, the 7R allele is not simply related to the other common alleles, differing by greater than six recombinations/mutations. Strong linkage disequilibrium was found between the 7R allele and surrounding DRD4 polymorphisms, suggesting that this allele is at least 5-10-fold "younger" than the common 4R allele. Based on an observed bias toward nonsynonymous amino acid changes, the unusual DNA sequence organization, and the strong linkage disequilibrium surrounding the DRD4 7R allele, we propose that this allele originated as a rare mutational event that nevertheless increased to high frequency in human populations by positive selection.     

Phenotypical characterization of the rat striatal neurons expressing the D1 dopamine receptor gene.

In situ hybridization experiments were performed in rat brain sections from normal and 6-hydroxydopamine-treated rats in order to map and identify the neurons expressing the D1 receptor gene in the striatum and the substantia nigra. Procedures of combined in situ hybridization, allowing the simultaneous detection of two mRNAs in the same section or in adjacent sections, were used to characterize the phenotypes of the neurons expressing the D1 receptor gene. D1 receptor mRNA was found in neurons all over the caudate-putamen, the accumbens nucleus, and the olfactory tubercle but not in the substantia nigra. In the caudate-putamen and accumbens nucleus, most of the neurons containing D1 receptor mRNA were characterized as medium-sized substance P neurons and distinct from those containing D2 receptor mRNA. Nevertheless, 15-20% of the substance P neurons did not contain D1 receptor mRNA. The neurons containing preproenkephalin A mRNA did not contain D1 receptor mRNA but contained D2 receptor mRNA. A small number of cholinergic and somatostatinergic neurons exhibited a weak reaction for D1 receptor mRNA. These results demonstrate that dopamine acts on efferent striatal neurons through expression of distinct receptors--namely, D1 and D2 in separate cell populations (substance P and preproenkephalin A neurons, respectively)--and can also act on nonprojecting neurons through D1 receptor expression.     

Cloning of the cDNA and gene for a human D2 dopamine receptor.

A clone encoding a human D2 dopamine receptor was isolated from a pituitary cDNA library and sequenced. The deduced protein sequence is 96% identical with that of the cloned rat receptor with one major difference: the human receptor contains an additional 29 amino acids in its putative third cytoplasmic loop. Southern blotting demonstrated the presence of only one human D2 receptor gene. Two overlapping phage containing the gene were isolated and characterized. DNA sequence analysis of these clones showed that the coding sequence is interrupted by six introns and that the additional amino acids present in the human pituitary receptor are encoded by a single exon of 87 base pairs. The involvement of this sequence in alternative splicing and its biological significance are discussed.     

Multiple human D5 dopamine receptor genes: a functional receptor and two pseudogenes.

Three genes closely related to the D1 dopamine receptor were identified in the human genome. One of the genes lacks introns and encodes a functional human dopamine receptor, D5, whose deduced amino acid sequence is 49% identical to that of the human D1 receptor. Compared with the human D1 dopamine receptor, the D5 receptor displayed a higher affinity for dopamine and was able to stimulate a biphasic rather than a monophasic intracellular accumulation of cAMP. Neither of the other two genes was able to direct the synthesis of a receptor. Nucleotide sequence analysis revealed that these two genes are 98% identical to each other and 95% identical to the D5 sequence. Relative to the D5 sequence, both contain insertions and deletions that result in several in-frame termination codons. Premature termination of translation is the most likely explanation for the failure of these genes to produce receptors in COS-7 and 293 cells even though their messages are transcribed. We conclude that the two are pseudogenes. Blot hybridization experiments performed on rat genomic DNA suggest that there is one D5 gene in this species and that the pseudogenes may be the result of a relatively recent evolutionary event.     

Novel insights in dopamine receptor physiology

The dopaminergic system has a pivotal role in the central nervous system but also plays important roles in the periphery, mainly in the endocrine system. Dopamine exerts its functions via five different receptors, named D(1)-D(5), belonging to the category of G protein coupled membrane receptors. Dopamine receptors are heterogeneously expressed in different cells, tissues and organs, where they stimulate or inhibit different functions, including neurotransmission and hormone synthesis and secretion. In particular, the dopamineric system has a pivotal role in the physiological regulation of the hypothalamus-pituitary-adrenal axis. Recent data have demonstrated the expression and function of dopamine receptors not only in endocrine organs but also in endocrine tumors, mainly those belonging to the hypothalamus-pituitary-adrenal axis, and also in the so-called 'neuroendocrine' tumors. These data confirm the important role of the dopaminergic system in this endocrine axis, as well as in the neuroendocrine system. This review summarizes the main structural and functional characteristics of dopamine receptors, emphasizing the most recent novelties, and focused on the physiological and pathological regulation of the hypothalamus-pituitary-adrenal axis by the dopaminergic system. In addition, the recent findings on the relationship between dopamine receptors and neuroendocrine tumors are summarized.     

Preparation and characterization of an antibody specific to the rat dopamine D2 receptor.

Antibodies specific to the dopamine D2 receptor have been raised in rabbits using synthetic peptides. The resulting antiserum was sensitive to picogram quantities of peptide as measured by enzyme-linked immunoassay and was shown to have a 33% cross-reactivity with partially purified D2 receptor protein. No detectable cross-reactivity with similarly prepared fungal membranes was observed. D2 receptor preparations from normal rat pituitary cells were used in Western blot analysis. Bands of M(r) = 95,000 and 34,000 were detected in these preparations with a third faint band at 120,000. These correspond to the pituitary D2 receptor.     

The Conformation of Cholinergic Molecules at Nicotinic Nerve Receptors

A correlation of the crystal structure analyses of the potent nicotinic agonists acetylcholine, acetyl-alpha-methylcholine, lactoylcholine, 1,1-dimethyl-4-phenylpiperazine, and nicotine allows one to determine the conformation of cholinergic agonists relevant to nicotinic nerve receptors.     

D3 dopamine receptor directly interacts with D1 dopamine receptor in immortalized renal proximal tubule cells

D3 receptors act synergistically with D1 receptors to inhibit sodium transport in renal proximal tubules; however, the mechanism by which this occurs is not known. Because dopamine receptor subtypes can regulate and interact with each other, we studied the interaction of D3 and D1 receptors in rat renal proximal tubule (RPT) cells. The D3 agonist PD128907 increased the immunoreactive expression of D1 receptors in a concentration- and time-dependent manner; these effects were blocked by the D3 antagonist U99194A. PD128907 also transiently (15 minutes) increased the amount of cell surface membrane D1 receptors. Laser confocal immunofluorescence microscopy showed that D3 receptor and D1 receptor colocalized in RPT cells more distinctly in Wistar-Kyoto rats than in spontaneously hypertensive rats (SHRs). In addition, D3 and D1 receptors could be coimmunoprecipitated, and this interaction was increased after D3 receptor agonist stimulation for 24 hours in Wistar-Kyoto rats but not in SHRs. We propose that the synergistic effects of D3 and D1 receptors may be caused by a D3 receptor-mediated increase in total, as well as cell surface membrane D1 receptor expression, and direct D3 and D1 receptor interaction, both of which are impaired in SHRs.     

The dopamine D2 receptor is expressed in GH3 cells.

Some pituitary tumours respond to dopamine by decreasing the release of prolactin and/or GH and by inhibition of tumour growth. Certain tumours are unresponsive. Dopamine D2 receptor high-affinity binding is impaired in these tumours, and the rat GH3 cell line behaves in a similar way. The hypothesis that the dopamine-binding defect results from impaired D2 receptor gene expression has been tested in the present study. On Northern blots, D2 receptor mRNA was present in both normal rat pituitary cells and in GH3 cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis identified a putative D2 receptor protein in normal and GH3 cell membranes. The lack of effect of dopamine in GH3 cells does not reflect the absence of D2 receptor gene expression.     

Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia.

Within the basal ganglia, acetylcholine and dopamine play a central role in the extrapyramidal control of motor function. The physiologic effects of these neurotransmitters are mediated by a diversity of receptor subtypes, several of which have now been cloned. Muscarinic acetylcholine receptors are encoded by five genes (m1-m5), and of the two known dopamine receptor subtypes (D1 and D2) the D2 receptor gene has been characterized. To gain insight into the physiological roles of each of these receptor subtypes, we prepared oligodeoxynucleotide probes to localize receptor subtype mRNAs within the rat striatum and substantia nigra by in situ hybridization histochemistry. Within the striatum, three muscarinic (m1, m2, m4) receptor mRNAs and the D2 receptor mRNA were detected. The m1 mRNA was expressed in most neurons (greater than 80%); the m2 mRNA, in neurons which were both very large and rare; and the m4 and D2 mRNAs, in 40-50% of the neurons, one-third of which express both mRNAs. Within the substantia nigra, pars compacta, only the m5 and D2 mRNAs were detected, and most neurons expressed both mRNAs. These data provide anatomical evidence for the identity of the receptor subtypes which mediate the diverse effects of muscarinic and dopaminergic drugs on basal ganglia function.