Risperidone displays a novel mechanism of antagonism of the h5-HT 7 receptor. Pretreatment of the cells with 5 or 20 nM risperidone, followed by removal of the drug from the media, renders the 5-HT 7 receptors unresponsive to 10 M 5-HT for at least 24 h. Thus, risperidone seems to be producing a rapid, long-lasting inactivation of the h5-HT 7 receptor. Whole-cell radioligand binding studies indicate that risperidone interacts in an irreversible or pseudo-irreversible manner with the h5-HT 7 receptor, thus producing the inactivation. Internalization of the h5-HT 7 receptor was not detected by monitoring green fluorescent protein-labeled fluorescent forms of the h5-HT 7 receptor exposed to 20 nM risperidone. Ten other antagonists were tested for h5-HT 7 -inactivating properties, and only 9-OH-risperidone and methiothepin were found to demonstrate the same anomalous properties as risperidone. These results indicate that the h5-HT 7 receptor may possess unique structural features that allow certain drugs to induce a conformation resulting in an irreversible interaction in the intact membrane environment. This may indicate that the h5-HT 7 receptor is part of a subfamily of G-protein-coupled receptors (GPCRs) possessing this property or that many GPCRs have the potential to be irreversibly blocked, but only select drugs can induce this effect. At the very least, the possibility that highly prescribed drugs, such as risperidone, are irreversibly antagonizing GPCR function in vivo is noteworthy.

In a previous publication, using human 5-hydroxytryptamine 7 (h5-HT 7 ) receptor-expressing human embryonic kidney (HEK) 293 cells, we reported the rapid, potent inactivation of the h5-HT 7 receptor stimulation of cAMP production by three antagonists: risperidone, 9-OH-risperidone, and methiothepin (Smith et al., 2006). To better understand the drug-receptor interaction producing the inactivation, we 1) expanded the list of inactivating drugs, 2) determined the inactivating potencies and efficacies by performing concentration-response experiments, and 3) determined the potencies and efficacies of the inactivators as irreversible binding site inhibitors. Three new drugs were found to fully inactivate the h5-HT 7 receptor: lisuride, bromocryptine, and metergoline. As inactivators, these drugs displayed potencies of 1, 80, and 321 nM, respectively. Pretreatment of 5-HT 7 -expressing HEK cells with increasing concentrations of the inactivating drugs risperidone, 9-OH-risperidone, methiothepin, lisuride, bromocriptine, and metergoline potently inhibited radiolabeling of the h5-HT 7 receptor, with IC 50 values of 9, 5.5, 152, 3, 73, and 10 nM, respectively. We were surprised to find that maximal concentrations of risperidone and 9-OH-risperidone inhibited only 50% of the radiolabeling of h5-HT 7 receptors. These results indicate that risperidone and 9-OH risperidone may be producing 5-HT 7 receptor inactivation by different mechanisms than lisuride, bromocryptine, metergoline, and methiothepin. These results are not interpretable using the conventional model of G-protein-coupled receptor function. The complex seems capable of assuming a stable inactive conformation as a result of the interaction of certain antagonists. The rapid, potent inactivation of the receptor-G-protein complex by antagonists implies a constitutive, pre-existing complex between the h5-HT 7 receptor and a G-protein.

Rationale-The h5-HT 7 receptor is subject to inactivation by risperidone and 9-OH-risperidone, apparently through a pseudo-irreversible complex formed between these drugs and the receptor. Although risperidone and 9-OH-risperidone ("inactivating antagonists") completely inactivate the receptor, only 50% of the receptors form a pseudo-irreversible complex with these drugs.Objectives-This study aims to more fully determine the mechanism(s) responsible for the novel effects of risperidone and 9-OH-risperidone and to determine if the inactivation can be reversed (reactivation).Methods-The ability of non-inactivating drugs (competitive antagonists) to dissociate washresistant [ 3 H]risperidone binding from h5-HT 7 receptors was investigated. Also, the ability of noninactivating drugs to reactivate inactivated h5-HT 7 receptors was investigated, using cAMP accumulation as a functional endpoint.Results-The competitive (non-inactivating) antagonists clozapine and mesulergine released the wash-resistant [ 3 H]risperidone binding to the h5-HT 7 receptor. The competitive antagonists clozapine, SB269970, mianserin, cyproheptadine, mesulergine, and ICI169369 reactivated the risperidone-inactivated h5-HT 7 receptors in a concentration-dependent manner. The potencies for reactivation closely match the affinities of these drugs for the h5-HT 7 receptor (r 2 =0.95), indicating that the reactivating antagonists are binding to and producing their effects through the orthosteric binding site of the h5-HT 7 receptor. Bioluminescence resonance energy transfer analyses indicate that the h5-HT 7 receptor forms homodimers.Conclusions-The ability of the non-inactivating drugs to bind h5-HT 7 orthosteric sites and reverse the wash-resistant effects of risperidone or 9-OH-risperidone, also bound to h5-HT 7 orthosteric sites, is evidence for protomer-protomer interactions between h5-HT 7 homodimers. This is the first demonstration of a non-mutated G-protein-coupled receptor homodimer engaging in protomer-protomer interactions in an intact cell preparation.

We have previously reported on the unusual human 5-hydroxytryptamine 7 (h5-HT 7 ) receptor-inactivating properties of risperidone, 9-OH-risperidone, bromocriptine, methiothepin, metergoline, and lisuride. Inactivation was defined as the inability of 10 M 5-HT to stimulate cAMP accumulation after brief exposure and thorough removal of the drugs from HEK293 cells expressing h5-HT 7 receptors. Herein we report that brief exposure of the h5-HT 7 receptor-expressing cells to inactivating drugs, followed by removal of the drugs, results in potent and efficacious irreversible inhibition of forskolin-stimulated adenylate cyclase activity. Pretreatment, followed by removal of the inactivating drugs inhibited 10 M forskolin-stimulated adenylate cyclase activity with potencies similar to the drugs' affinities for the h5-HT 7 receptor. The actions of the inactivating drugs were pertussis toxin-insensitive, indicating the lack of G i in their mechanism(s) of action. Methiothepin and bromocriptine maximally inhibited 10 M forskolin-stimulated adenylate cyclase, whereas the other drugs produced partial inhibition, indicating the drugs are inducing slightly different inactive conformations of the h5-HT 7 receptor. Maximal effects of these inactivating drugs occurred within 15 to 30 min of exposure of the cells to the drugs. A G s -mediated inhibition of forskolin-stimulated activity has never been reported. The inactivating antagonists seem to induce a stable conformation of the h5-HT 7 receptor, which induces an altered state of G s , which, in turn, inhibits forskolin-mediated stimulation of adenylate cyclase. These and previous observations indicate that the inactivating antagonists represent a unique class of drugs and may reveal GPCR regulatory mechanisms previously unknown. These drugs may produce innovative approaches to the development of therapeutic drugs.

We have reported previously novel drug-induced inactivation and reactivation of human 5-hydroxytryptamine 7 (5-HT 7 ) receptors in a recombinant cell line. To explain these novel observations, a homodimer structure displaying protomer-protomer cross-talk was proposed. To determine whether these novel observations and interpretations are due to an artifactual G protein-coupled receptor (GPCR) mechanism unique to the recombinant cell line, we explored the properties of r5-HT 7 receptors expressed by cortical astrocytes in primary culture. As in the recombinant cell line, risperidone, 9-OH-risperidone, methiothepin, and bromocriptine were found to potently inactivate r5-HT 7 receptors. As in the recombinant cell line, exposure of risperidone-inactivated astrocyte r5-HT 7 receptors to competitive antagonists resulted in the reactivation of r5-HT 7 receptors. The potencies of the reactivating drugs closely correlated with their affinities for h5-HT 7 receptors. These results indicate the novel inactivating and reactivating property of drugs is not due to an artifact of the recombinant cell line expressing h5-HT 7 receptors but is an intrinsic property of 5-HT 7 receptors in vitro and ex vivo. This evidence suggests that a native (nonmutated) GPCR, in its native membrane environment (cortical astrocyte primary culture), can function as a homodimer with protomer-protomer cross-talk. Homodimers may be a common GPCR structure. The experimental design used in our studies can be used to explore the properties of other GPCRs in their native forms in recombinant cells, primary cultures expressing the endogenous GPCRs, and possibly in vivo. The homodimer structure and protomer-protomer crosstalk offer new avenues of research into receptor dysfunction in disease states and the development of novel drugs.

Recent studies have indicated that the 5‐HT1ER, discovered in human brain tissue (Leonhardt et al, 1989), is not expressed in mouse or rat brain, but is expressed in guinea pig brain (Bai et al, 2004). Thus there have been few reports on 5‐HT1ER drug development. In order to establish an animal model that will allow 5‐HT1ER drug development we identified regions of the guinea pig brain that when homogenized exhibit 5‐CT, mesulergine, and sumatriptan ‐insensitive 3H5‐HT binding (characteristic of the 5‐HT1ER). The hippocampus was determined to have a receptor density sufficiently high for further analysis. In hippocampal homogenates, 100nM 5‐CT, 30nM ritanserin, and 100nM LY344864 were used to block high‐affinity 3H5‐HT binding at non‐1E binding sites. The Kd value of 3H5‐HT was found to be 5.7±0.7nM which is indistinguishable from the cloned receptor Kd of 5.7±0.6nM. Drug affinities for the hippocampal and cloned 5‐HT1ER binding were identical. These findings indicate the 3H‐5‐HT binding is specific to the guinea pig 5‐HT1ER and levels of receptors can be determined in various brain tissues. Autoradiographical analysis and signal transduction studies of 5‐HT1ER in guinea pig brain can thus be determined. This should provide important insights into possible functions of the 5‐HT1ER receptor and may predict the possible therapeutic potential of selective h5‐HT1ER agonists and antagonists.(Supported by MH56650)

The h5‐HT7R is one of 13 5‐HT receptors expressed in humans. It is involved in the control of cognition, emotion, and cardiovascular tone. Recently we reported that a 30 min exposure to nM levels of the antipsychotic drug risperidone results in an inactivation of the h5‐HT7R‐mediated stimulation of cAMP production (Smith et al, Mol. Pharmacol., 2006). To further investigate this unusual effect of this highly prescribed drug we have been testing other drugs for similar inactivating properties. 9‐OH‐risperidone, the active metabolite of risperidone, and methiothepin, a potent 5‐HT receptor antagonist, also potently inactivate the receptor. Preliminary studies indicate amoxapine, ritanserin, metergoline, lisuride, bromocryptine, SB‐269970 also produce this effect: clozapine, TFMPP and tenilapine did not produce the inactivating effects. It appears that a majority of h5‐HT7 receptor antagonists produce this unusual and dramatic effect. h5‐HT7‐mediated MAPK phosphorylation is also inactivated by risperidone, 9‐OH‐risperidone, and methiothepin, consistent with data indicating an irreversible or pseudo‐irreversible interaction is occurring between the drugs and the receptor. Further studies are underway to investigate the mechanism and in vivo relevance of these observations.Supported by MH56650 (M.T.)