dopamine D 5 receptors (Tiberi and Caron, 1994), serotonin 5-HT 2C recep-tors

ABSTRACT Single amino acid mutations in the third intracellular loop, as well as other domains of G protein-coupled receptors, have been shown to confer drastic changes in receptor properties and have been postulated to be responsible for various disease states. To determine whether an amino acid mutation can confer dramatic alterations in the 5-hydroxytryptamine 2A (5-HT 2A ) receptor, we mutated amino acid 322 to lysine (C322K), glutamate (C322E) or arginine (C322R). Transient expression of the mutant receptors revealed properties associated with constitutive activity. Radioligand binding studies revealed an increase in 5-HT affinity from 293 nM (native) to 86 nM (C322E), 25 nM (C322K) and 11 nM (C322R). 5-HT potency for stimulation of inositol phosphate production increased from 152 nM (native) to 61 nM (C322E) and 25 nM (C322K). Basal inositol phosphate levels in COS-7 cells expressing C322K and C322E mutant receptors were 8-fold and 4-fold higher, respectively, than cells expressing native 5-HT 2A receptors. Basal levels of inositol phosphate stimulated by C322K receptors represented 48% of total inositol phosphate production stimulated by native receptors in the presence of 10 M 5-HT. Antipsychotic drugs (chlorpromazine, clozapine, haloperidol, loxapine and risperidone) displayed inverse agonist activity by inhibiting C322K constitutive activation of phosphatidylinositol hydrolysis. These data indicate that amino acid 322 in the 5-HT 2A receptor plays an important role in maintaining the inactive conformation and provide further evidence that amino acid mutations can produce profound alterations in G protein-coupled receptor activity. The third intracellular loop of GPCR has been identified as a region that is crucial for receptor/G protein interactions (Strader et al., 1987; Recently, several GPCR have exhibited constitutive activity in vivo. Naturally occurring amino acid mutations in the luteinizing hormone recepto

Risperidone-Induced Inactivation and Clozapine-Induced Reactivation of Rat Cortical Astrocyte 5-Hydroxytryptamine7 Receptors: Evidence for In Situ G Protein-Coupled Receptor Homodimer Protomer Cross-Talk

We have reported previously novel drug-induced inactivation and reactivation of human 5-hydroxytryptamine7 (5-HT7) 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-HT7 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-HT7 receptors. As in the recombinant cell line, exposure of risperidone-inactivated astrocyte r5-HT7 receptors to competitive antagonists resulted in the reactivation of r5-HT7 receptors. The potencies of the reactivating drugs closely correlated with their affinities for h5-HT7 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-HT7 receptors but is an intrinsic property of 5-HT7 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 cross-talk offer new avenues of research into receptor dysfunction in disease states and the development of novel drugs

Human 5-HT7 Receptor-Induced Inactivation of Forskolin-Stimulated Adenylate Cyclase by Risperidone, 9-OH-Risperidone and Other “Inactivating Antagonists”

We have previously reported on the unusual human 5-hydroxytryptamine7 (h5-HT7) receptor-inactivating properties of risperidone, 9-OH-risperidone, bromocriptine, methiothepin, metergoline, and lisuride. Inactivation was defined as the inability of 10 μM 5-HT to stimulate cAMP accumulation after brief exposure and thorough removal of the drugs from HEK293 cells expressing h5-HT7 receptors. Herein we report that brief exposure of the h5-HT7 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 μM forskolin-stimulated adenylate cyclase activity with potencies similar to the drugs' affinities for the h5-HT7 receptor. The actions of the inactivating drugs were pertussis toxin-insensitive, indicating the lack of Gi in their mechanism(s) of action. Methiothepin and bromocriptine maximally inhibited 10 μM forskolin-stimulated adenylate cyclase, whereas the other drugs produced partial inhibition, indicating the drugs are inducing slightly different inactive conformations of the h5-HT7 receptor. Maximal effects of these inactivating drugs occurred within 15 to 30 min of exposure of the cells to the drugs. A Gs-mediated inhibition of forskolin-stimulated activity has never been reported. The inactivating antagonists seem to induce a stable conformation of the h5-HT7 receptor, which induces an altered state of Gs, 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

Pharmacological Analysis of the Novel, Rapid, and Potent Inactivation of the Human 5-Hydroxytryptamine7 Receptor by Risperidone, 9-OH-Risperidone, and Other Inactivating Antagonists

In a previous publication, using human 5-hydroxytryptamine7 (h5-HT7) receptor-expressing human embryonic kidney (HEK) 293 cells, we reported the rapid, potent inactivation of the h5-HT7 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-HT7 receptor: lisuride, bromocryptine, and metergoline. As inactivators, these drugs displayed potencies of 1, 80, and 321 nM, respectively. Pretreatment of 5-HT7-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-HT7 receptor, with IC50 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-HT7 receptors. These results indicate that risperidone and 9-OH risperidone may be producing 5-HT7 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-HT7 receptor and a G-protein