There are many examples of a single receptor coupling directly to more than one cellular signal transduction pathway. Although traditional receptor theory allows for activation of multiple cellular effectors by agonists, it predicts that the relative degree of activation of each effector pathway by an agonist (relative efficacy) must be the same. In the current experiments, we demonstrate that agonists at the human serotonin2A (5-HT2A) and 5-HT2C receptors activate differentially two signal transduction pathways independently coupled to the receptors [phospholipase C (PLC)-mediated inositol phosphate (IP) accumulation and phospholipase A2 (PLA2)-mediated arachidonic acid (AA) release]. The relative efficacies of agonists differed depending on which signal transduction pathway was measured. Moreover, relative to 5-HT, some 5-HT2C agonists (e.g., 3-trifluoromethylphenyl-piperazine) preferentially activated the PLC-IP pathway, whereas others (e.g., lysergic acid diethylamide) favored the PLA2-AA pathway. In contrast, when two dependent responses were measured (IP accumulation and calcium mobilization), agonist relative efficacies were not different. These data strongly support the hypothesis termed "agonist-directed trafficking of receptor stimulus" recently proposed by Kenakin [Trends Pharmacol Sci 16:232-238 (1995)]. Concentration-response curves to 5-HT2C agonists were fit well by a three-state model of receptor activation, suggesting that two active receptor states may be sufficient to explain pathway-dependent agonist efficacy. Rational drug design that optimizes preferential effector activity within a group of receptor-selective drugs holds the promise of increased selectivity in clinically useful agents.

Numerous research has pointed out that serotonin2c (5-HT 2C ) receptor, a subtype of 5-HT receptors belonging to the G-protein-coupled receptor superfamily, modulates the activity of mesencephalic dopamine (DA) neurons, the dysfunction of which is involved in devastating diseases such as schizophrenia, Parkinson's disease, and drug addiction. In the present study, using in vivo intracerebral microdialysis and Chinese hamster ovary (CHO) cells expressing 5-HT 2C receptors to identify appropriate 5-HT 2C receptor ligands, we sought to determine whether the property of 5-HT 2C receptors to spontaneously activate intracellular signaling pathways in vitro (constitutive activity) participates in the tonic inhibitory control that they exert on DA release in the rat striatum and nucleus accumbens in vivo. In

Peripheral opioid analgesia is increased substantially after inflammation. We evaluated the hypothesis that an inflammatory mediator, bradykinin (BK), evokes functional competence of the ␦-opioid receptor (DOR) for inhibiting trigeminal ganglia (TG) sensory neurons. We also evaluated whether BK evokes trafficking of the DOR to the plasma membrane. Rat TG cultures were pretreated with BK (

2,5-Dimethoxy-4-substituted phenylisopropylamines and phenethylamines are 5-hydroxytryptamine (serotonin) (5-HT) 2A/2C agonists. The former are partial to full agonists, whereas the latter are partial to weak agonists. However, most data come from studies analyzing phospholipase C (PLC)-mediated responses, although additional effectors [e.g., phospholipase A 2 (PLA 2 )] are associated with these receptors. We compared two homologous series of phenylisopropylamines and phenethylamines measuring both PLA 2 and PLC responses in Chinese hamster ovary-K1 cells expressing human 5-HT 2A or 5-HT 2C receptors. In addition, we assayed both groups of compounds as head shake inducers in rats. At the 5-HT 2C receptor, most compounds were partial agonists for both pathways. Relative efficacy of some phenylisopropylamines was higher for both responses compared with their phenethylamine counterparts, whereas for others, no differences were found. At the 5-HT 2A receptor, most compounds behaved as partial agonists, but unlike findings at 5-HT 2C receptors, all phenylisopropylamines were more efficacious than their phenethylamine counterparts. 2,5-Dimethoxyphenylisopropylamine activated only the PLC pathway at both receptor subtypes, 2,5-dimethoxyphenethylamine was selective for PLC at the 5-HT 2C receptor, and 2,5-dimethoxy-4-nitrophenethylamine was PLA 2 -specific at the 5-HT 2A receptor. For both receptors, the rank order of efficacy of compounds differed depending upon which response was measured. The phenylisopropylamines were strong head shake inducers, whereas their phenethylamine congeners were not, in agreement with in vitro results and the involvement of 5-HT 2A receptors in the head shake response. Our results support the concept of functional selectivity and indicate that subtle changes in ligand structure can result in significant differences in the cellular signaling profile.

1 The serotonin 2C (5-HT 2C ) receptor couples to both phospholipase C (PLC)-inositol phosphate (IP) and phospholipase A 2 (PLA 2 )-arachidonic acid (AA) signalling cascades. Agonists can dierentially activate these eectors (i.e. agonist-directed tracking of receptor stimulus) perhaps due to agonist-speci®c receptor conformations which dierentially couple to/activate transducer molecules (e.g. G proteins). Since editing of RNA transcripts of the human 5-HT 2C receptor leads to substitution of amino acids at positions 156, 158 and 160 of the putative second intracellular loop, a region important for G protein coupling, we examined the capacity of agonists to activate both the PLC-IP and PLA 2 -AA pathways in CHO cells stably expressing two major, fully RNA-edited isoforms (5-HT 2C-VSV , 5-HT 2C-VGV ) of the h5-HT 2C receptor. 2 5-HT increased AA release and IP accumulation in both 5-HT 2C-VSV and 5-HT 2C-VGV expressing cells. As expected, the potency of 5-HT for both RNA-edited isoforms for both responses was 10 fold lower relative to that of the non-edited receptor (5-HT 2C-INI ) when receptors were expressed at similar levels. 3 Consistent with our previous report, the ecacy order of two 5-HT receptor agonists (TFMPP and bufotenin) was reversed for AA release and IP accumulation at the non-edited receptor thus demonstrating agonist tracking of receptor stimulus. However, with the RNA-edited receptor isoforms there was no dierence in the relative ecacies of TFMPP or bufotenin for AA release and IP accumulation suggesting that the capacity for 5-HT 2C agonists to trac receptor stimulus is lost as a result of RNA editing. 4 These results suggest an important role for the second intracellular loop in transmitting agonistspeci®c information to signalling molecules.

The peripheral ␦ opioid receptor (DOR) is an attractive target for analgesic drug development. There is evidence that DOR can form heteromers with the -opioid receptor (KOR). As drug targets, heteromeric receptors offer an additional level of selectivity and, because of allosteric interactions between protomers, functionality. Here we report that selective KOR antagonists differentially altered the potency and/or efficacy of DOR agonists in primary cultures of adult rat peripheral sensory neurons and in a rat behavioral model of thermal allodynia. In vitro, the KOR antagonist nor-binaltorphimine (nor-BNI) enhanced the potency of [D-Pen enhanced the effect of DPDPE and decreased the effect of SNC80 to inhibit PGE 2 -stimulated thermal allodynia. In contrast to nor-BNI, the KOR antagonist 5Ј-guanidinonaltrindole (5Ј-GNTI) reduced the response of DPDPE both in cultured neurons and in vivo. Evidence for DOR-KOR heteromers in peripheral sensory neurons included coimmunoprecipitation of DOR with KOR, a DOR-KOR heteromer selective antibody augmented the antinociceptive effect of DPDPE in vivo, and the DOR-KOR heteromer agonist 6Ј-GNTI inhibited adenylyl cyclase activity in vitro as well as PGE 2 -stimulated thermal allodynia in vivo. Taken together, these data suggest that DOR-KOR heteromers exist in rat primary sensory neurons and that KOR antagonists can act as modulators of DOR agonist responses most likely through allosteric interactions between the protomers of the DOR-KOR heteromer.

The serotonin(2C) receptor (5-HT(2C)R) is a member of the serotonin(2) family of 7-transmembrane-spanning (7-TMS) receptors, which possesses unique molecular and pharmacological properties such as constitutive activity and RNA editing. The 5-HT(2C)R is widely expressed within the central nervous system, where is thought to play a major role in the regulation of neuronal network excitability. In keeping with its ability to modulate dopamine (DA) neuron function in the brain, the 5-HT(2C)R is currently considered as a major target for improved treatments of neuropsychiatric disorders related to DA neuron dysfunction, such as depression, schizophrenia, Parkinson's disease or drug addiction. The aim of this review is to provide an update of the functional status of the central 5-HT(2C)R, covering molecular, cellular, anatomical, biochemical and behavioral aspects to highlight its distinctive regulatory properties, the emerging functional significance of constitutive activity and RNA editing in vivo, and the therapeutic potential of inverse agonism.

Constitutive receptor activity/inverse agonism and functional selectivity/biased agonism are 2 concepts in contemporary pharmacology that have major implications for the use of drugs in medicine and research as well as for the processes of new drug development. Traditional receptor theory postulated that receptors in a population are quiescent unless activated by a ligand. Within this framework ligands could act as agonists with various degrees of intrinsic efficacy, or as antagonists with zero intrinsic efficacy. We now know that receptors can be active without an activating ligand and thus display “constitutive” activity. As a result, a new class of ligand was discovered that can reduce the constitutive activity of a receptor. These ligands produce the opposite effect of an agonist and are called inverse agonists. The second topic discussed is functional selectivity, also commonly referred to as biased agonism. Traditional receptor theory also posited that intrinsic efficacy is a single drug property independent of the system in which the drug acts. However, we now know that a drug, acting at a single receptor subtype, can have multiple intrinsic efficacies that differ depending on which of the multiple responses coupled to a receptor is measured. Thus, a drug can be simultaneously an agonist, an antagonist, and an inverse agonist acting at the same receptor. This means that drugs have an additional level of selectivity (signaling selectivity or “functional selectivity”) beyond the traditional receptor selectivity. Both inverse agonism and functional selectivity need to be considered when drugs are used as medicines or as research tools.