David L. Willins

Serotonin 5-HT2A receptors are expressed on pyramidal cells and interneurons in the rat cortex.

The distribution of 5‐HT2A receptors in rat cortex was evaluated using newly developed antibodies. Each of three antibodies tested identified an identical pattern of 5‐HT2A‐like immunoreactivity (5‐HT2A‐li) in rat cortex with 5‐HT2A‐li showing a widespread distribution. The majority of 5‐HT2A‐li cells displayed a pyramidal morphology. While a minority, some cortical neurons with a bipolar morphology displayed 5‐HT2A‐li as well. Dual‐label fluorescence confocal microscopic studies with a 5‐HT2A antibody and a mouse monoclonal antibody to parvalbumin, a marker of a subset of gamma aminobutyric acid (GABA)ergic interneurons in the cortex, demonstrated that although some cells expressing 5‐HT2A‐li were interneurons, most were not. Synapse 27:79–82, 1997.

5-Hydroxytryptamine2-Family Receptors (5-Hydroxytryptamine2A, 5-Hydroxytryptamine2B, 5-Hydroxytryptamine2C): Where Structure Meets Function

5-Hydroxytryptamine2 (serotonin2, 5-HT2)-family receptors are important for mediating many physiological functions, including vascular and nonvascular smooth muscle contraction, platelet aggregation, modulation of perception, mood, anxiety, and feeding behavior. A large number of psychopharmaceuticals, including atypical antipsychotic drugs, antidepressants, anxiolytics, and hallucinogens, mediate their actions, at least in part, via interactions with various 5-HT2-family receptors. This review article summarizes information about structure-function aspects of 5-HT2-family receptors. Evidence is presented that implies that conserved aromatic and charged residues are essential for ligand binding to 5-HT2A receptors. Additionally, findings are reviewed that are consistent with the hypothesis that residues located in intracellular loops 2 and 3 (i2 and i3) mediate coupling to specific G(alpha)-subunits such as G(alpha q). Studies are reviewed that suggest that 5-HT2-family receptors may be down-regulated by both agonists and antagonists, and usually this down-regulation is due to post-transcriptional mechanisms. Finally, a model for regulation of 5-HT2-family receptors by receptor-mediated endocytosis is advanced, and the particular structural features responsible for the various endocytotic pathways are emphasized. Taken together, these results suggest that discrete domains of the receptor structure are important for ligand binding, G-protein coupling, and internalization.

The Dynamin-dependent, Arrestin-independent Internalization of 5-Hydroxytryptamine 2A (5-HT2A) Serotonin Receptors Reveals Differential Sorting of Arrestins and 5-HT2A Receptors during Endocytosis

5-Hydroxytryptamine 2A (5-HT2A) receptors, a major site of action of clozapine and other atypical antipsychotic medications, are, paradoxically, internalized in vitro and in vivo by antagonists and agonists. The mechanisms responsible for this paradoxical regulation of 5-HT2A receptors are unknown. In this study, the arrestin and dynamin dependences of agonist- and antagonist-mediated internalization were investigated in live cells using green fluorescent protein (GFP)-tagged 5-HT2A receptors (SR2-GFP). Preliminary experiments indicated that GFP tagging of 5-HT2A receptors had no effect on either the binding affinities of several ligands or agonist efficacy. Likewise, both the native receptor and SR2-GFP were internalized via endosomes in vitro. Experiments with a dynamin dominant-negative mutant (dynamin K44A) demonstrated that both agonist- and antagonist-induced internalization were dynamin-dependent. By contrast, both the agonist- and antagonist-induced internalization of SR2-GFP were insensitive to three different arrestin (Arr) dominant-negative mutants (Arr-2 V53D, Arr-2-(319–418), and Arr-3-(284–409)). Interestingly, 5-HT2A receptor activation by agonists, but not antagonists, induced greater Arr-3 than Arr-2 translocation to the plasma membrane. Importantly, the agonist-induced internalization of 5-HT2A receptors was accompanied by differential sorting of Arr-2, Arr-3, and 5-HT2A receptors into distinct plasma membrane and intracellular compartments. The agonist-induced redistribution of Arr-2 and Arr-3 into intracellular vesicles and plasma membrane compartments distinct from those involved in 5-HT2A receptor internalization implies novel roles for Arr-2 and Arr-3 independent of 5-HT2A receptor internalization and desensitization.

Clozapine and other 5-hydroxytryptamine-2A receptor antagonists alter the subcellular distribution of 5-hydroxytryptamine-2A receptors in vitro and in vivo

In this study, we demonstrate that clozapine and other atypical antipsychotic drugs induce a paradoxical internalization of 5-hydroxytryptamine-2A receptors in vitro and a redistribution of 5-hydroxytryptamine-2A receptors in vivo. We discovered that clozapine, olanzapine, risperidone and the putative atypical antipsychotic drug MDL 100,907 all induced 5-hydroxytryptamine-2A receptor internalization in fibroblasts stably expressing the 5-hydroxytryptamine-2A receptor in vitro. Two 5-hydroxytryptamine-2A antagonists (mianserin and ritanserin), which have been demonstrated to reduce negative symptoms in schizophrenia, also caused 5-hydroxytryptamine-2A receptor internalization. Four different drugs, each devoid of 5-hydroxytryptamine-2A antagonist activity, had no effect on the subcellular distribution of 5-hydroxytryptamine-2A receptors in vitro. Treatment of rats for seven days with clozapine induced an increase in intracellular 5-hydroxytryptamine-2A receptor-like immunoreactivity in pyramidal neurons, while causing a decrease in labeling of apical dendrites in the medial prefrontal cortex. This redistribution of 5-hydroxytryptamine-2A receptors in pyramidal neurons was also seen when rats were chronically treated with another atypical antipsychotic drug, olanzapine. The typical antipsychotic drug haloperidol, however, did not induce a redistribution of 5-hydroxytryptamine-2A receptors in pyramidal neurons in the medial prefrontal cortex. Taken together, these results demonstrate that several atypical antipsychotic drugs with high 5-hydroxytryptamine-2A receptor affinities induce a redistribution of 5-hydroxytryptamine-2A receptors both in vivo and in vitro. It is conceivable that the loss of 5-hydroxytryptamine-2A receptors from the apical dendrites of pyramidal neurons is important for the beneficial effects of atypical antipsychotic drugs and other 5-hydroxytryptamine-2A antagonists in schizophrenia.

Serotonin 5-HT2C agonists selectively inhibit morphine-induced dopamine efflux in the nucleus accumbens

In vivo microdialysis was used to compare the effects of serotonergic drugs on morphine- and cocaine-induced increases in extracellular dopamine (DA) concentrations in the rat nucleus accumbens (NAc). Systemic administration of the 5-HT2A/2C agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (2.5 mg/kg, s.c. ) prevented the increase in extracellular DA in the NAc produced by morphine (5 mg/kg, i.p.). In contrast, this dose of DOI had no effect on the ability of cocaine (10 mg/kg, i.p.) to increase extracellular DA concentrations in the NAc. A 5-HT2C selective agonist, 6-chloro-2-[1-piperazinyl]-pyrazine (MK-212, 5 mg/kg, s.c.) also inhibited morphine-induced increases in extracellular DA concentrations in the NAc. Pretreatment of rats with the selective 5-HT2A antagonist, amperozide, had no effect on morphine-induced elevation of NAc DA concentrations. In order to determine if inhibition of the firing of 5-HT neurons contributes to the serotonin agonist-mediated inhibition of morphine-induced accumbens DA release, rats were pretreated with the 5-HT1A agonist, 8-OHDPAT. At a dose of 100 microg/kg (sc), 8-OHDPAT did not interfere with morphines ability to increase DA concentrations in the NAc. These results suggest that the activation of 5-HT2C receptors selectively inhibits morphine-induced DA release in the NAc in a manner which is independent of the inhibition of 5-HT neurons.

Activation is Hallucinogenic and Antagonism is Therapeutic: Role of 5-HT2A Receptors in Atypical Antipsychotic Drug Actions

This review summarizes recent studies with 5-hydroxytryptamine2A (5-HT2A) receptors, which represent the major site of action of hallucinogens and a likely site for atypical antipsychotic drug actions. We present evidence demonstrating that atypical antipsychotic drugs, as a group, have a preferentially high affinity for 5-HT2A receptors, compared with their affinities for other neurotransmitter receptors. The 5-HT2A receptor blockade seen with atypical antipsychotic drugs is probably an essential factor in explaining many of the unique features of atypical antipsychotic drugs. Atypical antipsychotic drugs have high affinities for several other 5-HT receptors (5-HT2C, 5-HT6, and 5-HT7), and the potential role of these novel 5-HT receptors in atypical antipsychotic drug action is also summarized.

What's All the RAVE about Receptor Internalization?

physiologic role of the perineurial barrier. dhh null mice transduction and cell surface receptor expression. Recent studies have suggested that, in some cases, GPCR-may provide a useful model for investigations of the biological function of this barrier, both in physiologic mediated endocytosis may also be important for mediating other biological actions of receptor occupancy. and pathologic settings. They may have an ancillary benefit for investigations of other aspects of peripheral These other actions influenced by receptor endocytosis may include physiologic responses such as tolerance, nerve development, since the absence of a perineurial barrier renders the nerves in these mice much more dependence, and even the therapeutic actions of various drugs (see Roth et al., 1998, for a recent review). accessible to experimental manipulation. These studies also raise the question of whether Dhh plays a role in For GPCRs in general, activation is usually followed by a cascade of events that includes receptor phosphoryla-the development of tight junctions between endothelial cells within the endoneurium and, by analogy, in the tion, desensitization, and internalization. It has been clear for some time, however, that efficacy does not blood–brain barrier. Finally, as the initial recruitment of mesenchymal cells around axon–Schwann cell units in always directly correlate with receptor phosphorylation, desensitization, and internalization (Keith et al., 1996; Yu embryonic peripheral nerves was unaffected in the dhh null mice, other signals distinct from Dhh must be in-et al., 1997; see figure). In some cases, even antagonists, which cannot activate receptors, cause GPCR internal-volved and remain to be identified. By demonstrating a key role of Schwann cells and Dhh ization (Willins et al., 1999). With these findings as a backdrop, Whistler et al. in the formation of the perineurium and the nerve–tissue (1999) present intriguing data in this issue of Neuron diffusion barrier, the authors have opened a new and which suggest that the differential biological actions of promising window into these and other unresolved drugs may depend, in part, on their abilities to induce questions of peripheral nerve development. endocytosis. They use ␮ opioid receptors, which mediate many of the analgesic, rewarding, and addictive properties of opioids (Matthes et al., 1996), as a model James L. Salzer system in which to probe the consequences of GPCR differences in agonist efficacy. accomplish this, they construct a chimeric ␮/␦ opioid Press). receptor is replaced with the C terminus of the ␦ recep-725–730. sponse to morphine (see figure). A …