Frederick J. Monsma

Molecular biology of dopamine receptors

The application of modern molecular biological methods has had an increasing and dramatic impact upon the discipline of molecular neuropharmacology. This is particularly true for the study of neurotransmitter receptors, where the use of recombinant DNA techniques has resulted in the cloning of multiple and sometimes unexpected receptor subtypes for a given neurotransmitter and, in some cases, the cloning of receptors for which no neurotransmitter is known. Within the past couple of years, it has become readily apparent that dopamine receptors will be no exception to this trend. Five different dopamine receptors have now been cloned and identified using molecular biological techniques, while only a few years ago only two receptor subtypes were thought to exist. David Sibley and Frederick Monsma review the molecular characteristics of the recently cloned dopamine receptors and discuss prospects for the cloning and identification of additional subtypes in this receptor family.

Molecular Cloning of a Novel G Protein‐Coupled Receptor Related to the Opiate Receptor Family

Abstract: A cDNA clone encoding a novel G protein‐linked receptor was isolated from a rat cerebral cortex cDNA library using a polymerase chain reaction‐amplified cDNA fragment as a probe. This 2.4‐kb clone encodes a 367 amino acid protein with seven putative transmembrane spanning domains. The protein is highly homologous to the cloned μ, δ, and κ opioid receptors and shares with them structural features such as three glycosylation sites in the amino terminus, a cyclic AMP‐dependent kinase phosphorylation site in the third cytoplasmic loop, an aspartic acid residue in the second transmembrane domain, and a palmitoylation site on the intracellular carboxy terminus. The receptor is also homologous with members of the somatostatin receptor family, yet it binds neither opiate nor somatostatin ligands. Northern blot analysis reveals two transcripts of 3.2 and 7.6 kb that are predominantly expressed in the cerebral cortex and hypothalamus. In situ hybridization analysis also shows a high abundance of mRNA in the cerebral cortex, hippocampus, amygdala, hypothalamus, thalamus, and dorsal raphe nuclei. It is suggested that the endogenous ligand for this receptor may represent a novel neuropeptide that may be closely related to the opiate peptide family.

Characterization of Novel Fluorescent Ligands with High Affinity for D1 and D2 Dopaminergic Receptors

Abstract: We have synthesized and characterized a series of novel fluorescently labeled ligands with high affinity and specificity for D1 and D2 dopamine receptors. D1‐selective probes were synthesized using (R,S)‐5‐(4′‐aminophenyl)‐8‐chloro‐2,3,4,5‐tetrahydro‐3‐methyl‐[1H]‐3‐benzazepin‐7‐ol, the 4′‐amino derivative of the high‐affinity, D1‐selective antagonist SCH‐23390, whereas D2‐selective probes were synthesized using the high‐affinity, D2‐selective antagonist N‐(p‐aminophenethyl)spiperone (NAPS). These ligands were coupled via spacer arms of various lengths to the fluorophores fluorescein and bodipy, which fluoresce in the yellow–green region, and to tetramethylrhodamine, which is a red fluorophore. The interaction of these fluorescent ligands with dopamine receptors was evaluated by examining their ability to compete for the binding of the radiolabeled antagonists [3H]SCH‐23390 or [3H]methylspiperone to rat striatal D1 or D2 dopamine receptors, respectively. We report here that these novel fluorescent ligands exhibit very high affinity and specificity for either D1 or D2 dopamine receptors. The availability of various fluorescent ligands with different emission maxima and with high affinity and specificity for D1 and D2 dopamine receptors will now permit investigations involving the visualization and localization of these receptor subtypes at the single cell and intracellular levels in the CNS and on intact cells in culture.

D2L, D2S, and D3 dopamine receptors stably transfected into NG108-15 cells couple to a voltage-dependent potassium current via distinct G protein mechanisms

The D2‐like dopamine (DA) receptor family has continued to expand and now includes the D2‐short (D2S) and D2‐long (D2L) receptor isoforms and the D3 and D4 receptors. The D2 receptor isoforms differ in length by 29 amino acids within the third cytoplasmic loop, a region of the receptor believed to be important for G protein coupling. This observation has led to the hypothesis that the two isoforms of the D2 receptor may utilize different signal transduction pathways when present in the same cell. The D2 and D3 receptors, although mostly different, show some common amino acid sequences within the third cytoplasmic loop. Thus, it is possible that the D2 and D3 receptors may employ similar signal transduction pathways. To test these hypotheses directly, NG108‐15 neuroblastoma‐glioma hybrid cells were stably transfected to express either the D2S, D2L, or D3 DA receptors. All transfected but not untransfected NG108‐15 cells demonstrated a dose‐dependent reduction in the peak whole‐cell potassium (K+) current in response to receptor activation by DA or the DA receptor agonists quinpirole (QUIN) and apomorphine (APO). The modulation of K+ current by D2S receptor stimulation was prevented by pretreatment of the cells with cholera toxin (20 μg/ml for 18 h), whereas pertussis toxin pretreatment (500 ng/ml for 4 h) completely blocked the effects of D2L and D3 receptor activation. These observations suggest that the signal transduction mechanisms involved in coupling the two isoforms of the D2 receptor to the K+ current are different, whereas the D2L and D3 receptor coupling mechanisms may be similar. In direct support of this hypothesis, it was observed that the intracellular application of a polyclonal antibody that is specific for the G0α subunit completely blocked the ability of D2L and D3 receptors to modulate outward K+ currents. In contrast, the D2S‐mediated modulation of K+ currents was blocked by intracellular application of an antibody recognizing GSα but not G0α. These findings demonstrate that D2S and D2L receptors are able to couple to a common effector in a cell via two G protein pathways.

Expression of Functional D2 Dopamine Receptors Following Differentiation of Y‐79 Human Retinoblastoma Cells

Y‐79 human retinoblastoma cells grown in serumfree medium in monolayer culture have previously been shown to undergo differentiation in response to dibutyryl cyclic AMP (Bt2cAMP). We report here that Y‐79 cells treated in this manner also express very high levels of functional D2 dopamine receptors. In control Y‐79 cells, cultured in suspension, D2 dopamine receptors, quantified via saturation analysis with the D2 antagonist [3H]methylspiperone, are expressed at a level of ∼3 fmol/106 cells (∼1,800 receptor sites/cell). Differentiation is initiated by attachment of the cells to the culture dish with poly‐D‐lysine and fibronectin and continued culture in serum‐free medium. After 8 days in serumfree culture, differentiation is further induced with continuous Bt2cAMP treatment. Using this differentiation protocol, D2 receptor levels increase up to a maximum of 30 fmol/106 cells (18,000 receptors/cell) on day 20, the limit of culture viability. Cultures of 15–17 days (7–9 days of Bt2cAMP treatment) expressing receptor levels of 15–20 fmol/106 cells are used for pharmacological and functional characterization of D2 dopamine receptors. The pharmacology of competition for [3H]methylspiperone binding to differentiated Y‐79 (dY‐79) cell membranes by a series of dopaminergic antagonists verifies the D2 receptor nature of this site, exhibiting appropriate affinities and the following rank order of potency: YM‐09151‐2 ∼ spiperone > domperidone ∼ (+)‐butaclamol ∼ fluphenazine > chlorpromazine > (–)‐sulpiride > (+)‐sulpiride > promethazine > (+)‐SCH 23390 ≫ (–)‐butaclamol. Inhibition of [3H]methylspiperone binding by dopaminergic agonists in dY‐79 cell membranes indicates the presence of multiple agonist affinity states, which can be converted to a homogeneous low‐affinity state by addition of guanine nucleotides. In addition, incubation with dopamine or other D2‐selective agonists results in an ∼50% reduction in forskolin‐stimulated cyclic AMP production in lysed dY‐79 cells, which can be blocked by D2‐selective antagonists. These data indicate that the D2 receptors expressed in these cells are indeed functional. The ability to induce high levels of expression of functional D2 dopamine receptors in a cultured human cell line should provide an excellent model system with which to examine the molecular events involved in the expression and regulation of this receptor system.