The PDZ-binding domain is essential for the dendritic targeting of 5-HT2A serotonin receptors in cortical pyramidal neurons in vitro

The 5-HT(2A) serotonin receptor represents an important molecular target for atypical antipsychotic drugs and for most hallucinogens. In the mammalian cerebral cortex, 5-HT(2A) receptors are enriched in pyramidal neurons, within which 5-HT(2A) receptors are preferentially sorted to the apical dendrites. In primary cortical cultures, 5-HT(2A) receptors are sorted to dendrites and not found in the axons of pyramidal neurons. We identified a sorting motif that mediates the preferential targeting of 5-HT(2A) receptors to the dendrites of cortical pyramidal neurons in vitro. We constructed green fluorescent protein-tagged 5-HT(2A) receptors wherein potential sorting motifs were disrupted, and subsequently employed either the Semliki Forest virus or calcium phosphate for the transient expression of recombinant 5-HT(2A) receptors in cultured cortical pyramidal neurons. Using dual-labeling immunofluorescent confocal microscopy, we quantified the axonal and dendritic sorting patterns of endogenous and recombinant 5-HT(2A) receptors. We discovered that disruption of the PDZ-binding domain of the 5-HT(2A) receptor greatly attenuates the dendritic targeting of 5-HT(2A) receptors without inappropriately sorting 5-HT(2A) receptors to axons. The PDZ-binding domain is therefore a necessary signal for the preferential targeting of the 5-HT(2A) receptor to the dendritic compartment of cultured cortical pyramidal neurons, the first such role ascribed to this protein-protein interaction motif of any G protein-coupled receptor

Interactive effects of mGlu5 and 5-HT2A receptors on locomotor activity in mice

RationaleMetabotropic glutamate (mGlu) receptors have been suggested to play a role in neuropsychiatric disorders including schizophrenia, drug abuse, and depression. Because serotonergic hallucinogens increase glutamate release and mGlu receptors modulate the response to serotonin (5-HT)(2A) activation, the interactions between serotonin 5-HT(2A) receptors and mGlu receptors may prove to be important for our understanding of these diseases.ObjectiveWe tested the effects of the serotonergic hallucinogen and 5-HT(2A) agonist, 2,5-dimethoxy-4-methylamphetamine (DOM), and the selective 5-HT(2A) antagonist, M100907, on locomotor activity in the mouse behavioral pattern monitor (BPM) in mGlu5 wild-type (WT) and knockout (KO) mice on a C57 background.ResultsBoth male and female mGlu5 KO mice showed locomotor hyperactivity and diminished locomotor habituation compared with their WT counterparts. Similarly, the mGlu5-negative allosteric modulator 2-methyl-6-(phenylethynyl)pyridine (MPEP) also increased locomotor hyperactivity, which was absent in mGlu5 KO mice. The locomotor hyperactivity in mGlu5 receptor KO mice was potentiated by DOM (0.5 mg/kg, subcutaneously (SC)) and attenuated by M100907 (1.0 mg/kg, SC). M100907 (0.1 mg/kg, SC) also blocked the hyperactivity induced by MPEP.ConclusionsThese studies demonstrated that loss of mGlu5 receptor activity either pharmacologically or through gene deletion leads to locomotor hyperactivity in mice. Additionally, the gene deletion of mGlu5 receptors increased the behavioral response to the 5-HT(2A) agonist DOM, suggesting that mGlu5 receptors either mitigate the behavioral effects of 5-HT(2A) hallucinogens or that mGlu5 KO mice show an increased sensitivity to 5-HT(2A) agonists. Taken together, these studies indicate a functional interaction between mGlu5 and 5-HT(2A) receptors

Expression and Function of Serotonin 2A and 2B Receptors in the Mammalian Respiratory Network

Neurons of the respiratory network in the lower brainstem express a variety of serotonin receptors (5-HTRs) that act primarily through adenylyl cyclase. However, there is one receptor family including 5-HT2A, 5-HT2B, and 5-HT2C receptors that are directed towards protein kinase C (PKC). In contrast to 5-HT2ARs, expression and function of 5-HT2BRs within the respiratory network are still unclear. 5-HT2BR utilizes a Gq-mediated signaling cascade involving calcium and leading to activation of phospholipase C and IP3/DAG pathways. Based on previous studies, this signal pathway appears to mediate excitatory actions on respiration. In the present study, we analyzed receptor expression in pontine and medullary regions of the respiratory network both at the transcriptional and translational level using quantitative RT-PCR and self-made as well as commercially available antibodies, respectively. In addition we measured effects of selective agonists and antagonists for 5-HT2ARs and 5-HT2BRs given intra-arterially on phrenic nerve discharges in juvenile rats using the perfused brainstem preparation. The drugs caused significant changes in discharge activity. Co-administration of both agonists revealed a dominance of the 5-HT2BR. Given the nature of the signaling pathways, we investigated whether intracellular calcium may explain effects observed in the respiratory network. Taken together, the results of this study suggest a significant role of both receptors in respiratory network modulation

Oligomerization of the alpha 1a- and alpha 1b-adrenergic receptor subtypes. Potential implications in receptor internalization.

We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the alpha 1a- and alpha 1b-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the alpha 1a- and alpha 1b-AR can form homo-oligomers with similar transfer efficiency of approximately 0.10. Hetero-oligomers could also be observed between the alpha 1b- and the alpha 1a-AR subtypes but not between the alpha 1b-AR and the beta2-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the alpha 1b-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the alpha 1b-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type alpha 1b-AR. Confocal imaging revealed that oligomerization of the alpha1-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The alpha 1a-selective agonist oxymetazoline induced the co-internalization of the alpha 1a- and alpha 1b-AR, whereas the alpha 1b-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the alpha 1a- and alpha 1b-AR subtypes