Susan E. Senogles

Dopamine receptor subtypes: beyond the D1/D2 classification

The D1/D2 dopamine receptor classification is widely accepted. However, intense investigative efforts over the last several years using pharmacological, biochemical and behavioral approaches have produced results that are increasingly difficult to reconcile with the existence of only two dopamine receptor subtypes. Recent developments, including cloning of the cDNAs and/or genes for several members of the large family of G-protein-coupled receptors, have revealed that heterogeneity in the pharmacological or biochemical characteristics of individual receptors often indicates the presence of previously unsuspected molecular subtypes. In this article, Marc Caron and colleagues have assembled the main lines of evidence that suggest the presence of several novel subtypes for both D1 and D2 dopamine receptors and predict that molecular cloning will, in the near future, confirm their existence.

A Region of the Third Intracellular Loop of the Short Form of the D2 Dopamine Receptor Dictates Gi Coupling Specificity

The D2 dopamine receptor has two isoforms, the short form (D2s receptor) and the long form (D2l receptor), which differ by the presence of a 29-amino acid insert in the third cytoplasmic loop. Both the D2s and D2l receptors have been shown to couple to members of the Gαi family of G proteins, but whether each isoform couples to specific Gαi protein(s) remains controversial. In previous studies using Gαi mutants resistant to modification by pertussis toxin (GαiPT), we demonstrated that the D2s receptor couples selectively to Gαi2PT and that the D2l receptor couples selectively to Gαi3PT (Senogles, S. E. (1994) J. Biol. Chem. 269, 23120–23127). In this study, two point mutations of the D2s receptor were created by random mutagenesis (R233G and A234T). The two mutant D2s receptors demonstrated pharmacological characteristics comparable with those of the wild-type D2s receptor, with similar agonist and antagonist binding affinities. We used human embryonic kidney 293 cells stably transfected with Gαi1PT, Gαi2PT, or Gαi3PT to measure agonist-mediated inhibition of forskolin-stimulated cAMP accumulation before and after pertussis toxin treatment. The two mutant D2s receptors demonstrated a change in Gi coupling specificity compared with the wild-type D2s receptor. Whereas the wild-type D2s receptor coupled predominantly to Gαi2PT, mutant R233G coupled preferentially to Gαi3PT, and mutant A234T coupled preferentially to Gαi1PT. These results suggest that this region of the third cytoplasmic loop is crucial for determining Gi protein coupling specificity.

Dopaminergic Inhibition of Catecholamine Secretion from Chromaffin Cells: Evidence that Inhibition Is Mediated by D4 and D5 Dopamine Receptors

Abstract: Previous studies have suggested that activation of D2‐like dopamine receptors inhibits catecholamine secretion from adrenal chromaffin cells. The purpose of this study was to determine whether the activation of D1‐like receptors on chromaffin cells affects either catecholamine release from the cells or the inhibition of secretion by D2‐like dopamine receptors. Both D1‐ and D2‐selective agonists inhibited secretion elicited by dimethylphenylpiperazinium (DMPP), veratridine, and high K+ levels. The D1‐selective agonists 6‐chloro‐7,8‐dihydroxy‐3‐allyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine (Cl‐APB) and SKF‐38393 inhibited DMPP‐stimulated catecholamine secretion in a concentration‐dependent manner; 50% inhibition was obtained with ∼10 µM Cl‐APB and ∼100 µM SKF‐38393. Of the D2‐selective agonists, bromocriptine was a more potent inhibitor of DMPP‐stimulated catecholamine release than was quinpirole. The inhibition of secretion caused by Cl‐APB or SKF‐38393 was additive with the inhibition caused by bromocriptine. Pertussis toxin treatment (50 ng/ml, 18 h) attenuated the inhibitory effect of D2‐selective, but not D1‐selective, dopamine agonists. In addition, forskolin‐stimulated adenylyl cyclase activity was inhibited by D2‐selective, but not D1‐selective, agonists. Neither D1‐ nor D2‐selective agonists stimulated adenylyl cyclase activity in the cells, although cyclase activity was stimulated by forskolin, carbachol, and vasoactive intestinal peptide. DMPP‐stimulated Ca2+ uptake was inhibited by both D1‐ and D2‐selective dopamine agonists. PCR analysis was used to determine which of the dopamine receptor subtypes within the D1‐like and D2‐like subfamilies was responsible for the observed inhibition. PCR analysis indicated that mRNA for only D4 and D5 dopamine receptor subtypes was present in chromaffin cells. These combined data suggest that D1‐ and D2‐selective agonists inhibit Ca2+ uptake and catecholamine secretion by activating D4 and D5 dopamine receptors on chromaffin cells.

D2s dopamine receptor mediates phospholipase D and antiproliferation.

The D2 dopamine receptor, short form (D2s) has been shown to stimulate phospholipase D (PLD) activity independent of activation of phospholipase C (PLC) activity in GH4 derived cells stably transfected with the D2s receptor [Mol. Pharm. 58 (2000) 455]. Agonist activation of D2s has been shown to mediate the inhibition of growth in the same cell line [J. Biol. Chem. 276 (1992) 24169; Endocrinology 134 (1994) 783]. In the present study, D2s-HEK 293 cells were generated using Epstein-Barr virus (EBV) based vectors. The stimulation of PLD by D2s can be augmented by the transfection of Rho A, but not Cdc 42 or Rac and nullified by transfection of N19 Rho A, a dominant negative form of Rho A. Addition of ethanol, at 0.5% reduced the ability of dopamine agonists to inhibit growth in D2s-HEK 293 cells, suggesting that PLD is involved in the antiproliferative effects of D2s signaling. In addition, the expression of N19 Rho A ablated the ability of the D2s to inhibit [3H]thymidine incorporation, while the expression of N19 Cdc 42 or N17 Rac had no effect. These results suggest that the D2s stimulation of PLD is Rho A dependent and lies along the signaling pathway which leads to the antiproliferative effects of D2s receptor activation.

D2 dopamine receptor-mediated antiproliferation in a small cell lung cancer cell line, NCI-H69.

The D2 dopamine receptor agonist bromocriptine has been used clinically for reducing tumor mass of pituitary adenomas arising from lactotroph origins. As well, bromocriptine has been shown to have an antiproliferative effect on primary lactotrophs and lactotroph-derived cell lines. The presence of D2 dopamine-like receptors on NCI-H69 cells was previously established by the use of [125I]iodosulpride binding and has been confirmed in this study by use of reverse transcription PCR with receptor-specific primers. The reverse transcription PCR analysis of NCI-H69 cells demonstrates that both the D2s and D2l are expressed in NCI-H69 cells, with D2s having the higher relative expression. The activation of the D2R results in an inhibition of growth of NCI-H69 cells as assessed by the incorporation of [3H]thymidine; a process not sensitive to pertussis toxin. In NCI-H69 cells, the D2 dopamine-like receptor is coupled to the inhibition of forskolin-stimulated cAMP accumulation and to the stimulation of phospholipase D. The receptor-mediated inhibition of cAMP accumulation is ablated by overnight treatment with pertussis toxin but the stimulation of phospholipase D mediated by dopaminergic agonists is not. These data suggest that the phospholipase D pathway is responsible for the antiproliferative effects of D2 dopamine-like receptors agonists in small cell lung cancer cells. In support of this hypothesis, the inhibition of [3H]thymidine incorporation mediated by dopaminergic agonists was shown to be sensitive to the presence of ethanol. Taken together, these data suggest that the D2 dopamine-like receptor activates phospholipase D, which ultimately leads to an inhibition of growth of this small cell lung cancer cell line.

Postmortem stability of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase in rat striatum.

Abstract: The stability of dopamine‐sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase was measured in homogenates of rat striatal tissue frozen from 0 to 24 h postmortem. ATPase, GTPase, and Mg2+‐dependent guanylate cyclase activities showed no significant change over this period. Mn2+‐dependent guanylate cyclase activity was stable for 10 h postmortem. Basal and dopamine‐stimulated adenylate cyclase activity decreased markedly during the first 5 h. However, when measured in washed membrane preparations, these adenylate cyclase activities remained stable for at least 10 h. Therefore, the postmortem loss of a soluble activator, such as GTP, may decrease the adenylate cyclase activity in homogenates. These results are not consistent with an earlier suggestion that there is a postmortem degradation of the enzyme itself. Other kinetic parameters of dopamine‐sensitive adenylate cyclase can also be measured independently of postmortem changes. Thus, it is possible to investigate kinetic parameters of dopamine‐sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase in human brain obtained postmortem.

Structural and Functional Evidence for Testosterone Activation of GPRC6A in Peripheral Tissues.

G protein-coupled receptor (GPCR) family C group 6 member A (GPRC6A) is a multiligand GPCR that is activated by cations, L-amino acids, and osteocalcin. GPRC6A plays an important role in the regulation of testosterone (T) production and energy metabolism in mice. T has rapid, transcription-independent (nongenomic) effects that are mediated by a putative GPCR. We previously found that T can activate GPRC6A in vitro, but the possibility that T is a ligand for GPRC6A remains controversial. Here, we demonstrate direct T binding to GPRC6A and construct computational structural models of GPRC6A that are used to identify potential binding poses of T. Mutations of the predicted binding site residues were experimentally found to block T activation of GPRC6A, in agreement with the modeling. Using Gpr6ca(-/-) mice, we confirmed that loss of GPRC6A resulted in loss of T rapid signaling responses and elucidated several biological functions regulated by GPRC6A-dependent T rapid signaling, including T stimulation of insulin secretion in pancreatic islets and enzyme expression involved in the biosynthesis of T in Leydig cells. Finally, we identified a stereo-specific effect of an R-isomer of a selective androgen receptor modulator that is predicted to bind to and shown to activate GPRC6A but not androgen receptor. Together, our data show that GPRC6A directly mediates the rapid signaling response to T and uncovers previously unrecognized endocrine networks.

D3 dopamine receptor activates phospholipase D through a pertussis toxin-insensitive pathway

Within the dopamine receptor family, the D(3) dopamine receptors function remains inadequately described. The D(3) receptor has been shown to couple to inhibition of adenylyl cyclase, stimulation of mitogenesis, and regulation of K(+) and Ca(2+) currents, all in a pertussis toxin (PTX)-sensitive manner. Here we report D(3) receptor activation of the phospholipase D (PLD) enzyme in HEK 293 cells heterologously expressing the human D(3) receptor. Activation by agonist is dose dependent and displays the pharmacology expected of the D(3) receptor. The D(3) receptor specific antagonists AJ-76 and U99194A ablated the increase in activity by the preferring D(3) agonist (+) 7-OH DPAT. In addition, the D(3) receptor-mediated activation of PLD is not mediated by G-proteins of the G(i)/G(o) family, as pretreatment with PTX had no effect. PLD activation is a novel finding for the D(3) receptor, and is the first example of an effector system where D(3) signals without G(i)/G(o) protein intermediates.

D3 dopamine receptor signals to activation of phospholipase D through a complex with Rho

J. Neurochem. (2009) 112, 963–971.

Differential inhibition of secretagogue-stimulated sodium uptake in adrenal chromaffin cells by activation of D4 and D5 dopamine receptors

Abstract: Recent studies have demonstrated that D1‐selective and D2‐selective dopamine receptor agonists inhibit catecholamine secretion and Ca2+ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1‐like dopamine receptor subfamily, and D4, a member of the D2‐like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na+, which could explain inhibition of secretion and Ca2+ uptake by dopamine agonists. D1‐selective agonists preferentially inhibited both dimethylphenylpiperazinium‐ (DMPP) and veratridine‐stimulated 22Na+ influx into chromaffin cells. The D1‐selective agonists chloro‐APB hydrobromide (CI‐APB; 100 µM) and SKF‐38393 (100 µM) inhibited DMPP‐stimulated Na+ uptake by 87.5 ± 2.3 and 59.7 ± 4.5%, respectively, whereas the D2‐selective agonist bromocriptine (100 µM) inhibited Na+ uptake by only 22.9 ± 5.0%. Veratridine‐stimulated Na+ uptake was inhibited 95.1 ± 3.2 and 25.7 ± 4.7% by 100 µM CI‐APB or bromocriptine, respectively. The effect of CI‐APB was concentration dependent. A similar IC50 (∼18 µM) for inhibition of both DMPP‐ and veratridine‐stimulated Na+ uptake was obtained. The addition of 8‐bromo‐cyclic AMP (1 mM) had no effect on either DMPP‐ or veratridine‐stimulated Na+ uptake. These observations suggest that D1‐selective agonists are inhibiting secretagogue‐stimulated Na+ uptake in a cyclic AMP‐independent manner.