Structure-activity correlations for beta-phenethylamines at human trace amine receptor 1

A cell line in which RD-HGA16 cells were stably transfected with the hTAAR 1 receptor was created and utilized to carry out a systematic evaluation of a series of beta-phenethylamines. Fair agreement was observed with data obtained for aryl and ethylene chain substituted analogs in an AV12-664 cell line in which hemagglutinin-tagged hTAAR 1 was stably co-expressed with rat G alpha(s). Analogs with multiple substituents as well as analogs with bulky groups were found to be partial agonists. Analogs in which the primary amino group was converted to a secondary or a tertiary amino group by N-methylation were also partial agonists. Comparative Molecular Field Analysis (CoMFA) using the potency data yielded a regression coefficient r(2) of 0.824. The steric field contribution to the model was 61% with the balance (39%) contributed by the electrostatic field. The collective results suggest that increasing steric bulk both at the amino nitrogen, particularly by N-dimethylation, and at the 4-position of the aromatic ring leads to low efficacy ligands.     

Timberol? Inhibits TAAR5-Mediated Responses to Trimethylamine and Influences the Olfactory Threshold in Humans

In mice, trace amine-associated receptors (TAARs) are interspersed in the olfactory epithelium and constitute a chemosensory subsystem that is highly specific for detecting volatile amines. Humans possess six putative functional TAAR genes. Human TAAR5 (hTAAR5) is highly expressed in the olfactory mucosa and was shown to be specifically activated by trimethylamine. In this study, we were challenged to uncover an effective blocker substance for trimethylamine-induced hTAAR5 activation. To monitor blocking effects, we recombinantly expressed hTAAR5 and employed a commonly used Cre-luciferase reporter gene assay. Among all tested potential blocker substances, Timberol®, an amber-woody fragrance, is able to inhibit the trimethylamine-induced hTAAR5 activation up to 96%. Moreover, human psychophysical data showed that the presence of Timberol® increases the olfactory detection threshold for the characteristic fishy odor of trimethylamine by almost one order of magnitude. In conclusion, our results show that among tested receptors Timberol® is a specific and potent antagonist for the hTAAR5-mediated response to trimethylamine in a heterologous system. Furthermore, our data concerning the observed shift of the olfactory detection threshold in vivo implicate that hTAAR5 or other receptors that may be inhibited by Timberol® could be involved in the high affinity olfactory perception of trimethylamine in humans.     

Tyramine and β-phenylethylamine,from fermented food products,as agonists for the human trace amine-associated receptor 1 (hTAAR1) in the stomach

The aromatic amines tyramine and β-phenylethylamine are abundant in fermented foods. Recently, a family of human trace amine-associated receptors (hTAARs) was discovered that responds to these compounds. This study examined the expression of hTAAR genes in five human organs. Among them, the stomach expressed hTAAR1 and hTAAR9. Interestingly, more hTAAR1 was expressed in the pylorus than in the other stomach regions. The CRE-SEAP reporter assay revealed that only hTAAR1 functioned as a G_s-coupled receptor in response to tyramine and β-phenylethylamine stimulation. The β-phenylethylamine-mediated hTAAR1 activity could be potentiated using 3-isobutyl-1-methylxanthine. These data suggest that tyramine and β-phenylethylamine in fermented foods act at hTAAR1 as agonists in the pylorus of stomach.     

BML-190 and AM251 act as inverse agonists at the human cannabinoid CB2 receptor: signalling via cAMP and inositol phosphates

The aminoalkylindole BML-190 and diarylpyrazole AM251 ligands have previously been shown to bind to cannabinoid CB(2) and CB(1) receptors, respectively. In HEK-293 cells stably expressing the human CB(2) receptor, BML-190 and AM251 potentiated the forskolin-stimulated accumulation of cAMP. Moreover, the CB(2) receptor can interact productively with 16z44, a promiscuous G alpha(16/z) chimera. BML-190 and AM251 reduce the basal levels of inositol phosphate production in cells expressing the CB(2) receptor and 16z44. These results demonstrate that BML-190 and AM251 act as inverse agonists at the human CB(2) receptor acting via G alpha(i/o) and G alpha(q) family-coupled pathways.     

Accelerated pseudogenization of trace amine‐associated receptor genes in primates

Trace amines (TAs) in the mammalian brain have been investigated for four decades. Trace amine‐associated receptors (TAARs) were discovered during the search for receptors activated by TAs. TAARs are considered a second class of vertebrate olfactory receptors and successfully proliferated in conjunction with adaptation to living on the ground to detect carnivore odors. Thus, therian mammals have a high number of TAAR genes due to rapid species‐specific gene duplications. In primate lineages, however, their genomes have significantly smaller numbers of TAAR genes than do other mammals. To elucidate the evolutionary force driving these patterns, exhaustive data mining of TAAR genes was performed for 13 primate genomes (covering all four infraorders) and two nonprimate euarchontan genomes. This study identified a large number of pseudogenes in many of these primate genomes and thus investigated the pseudogenization event process for the TAAR repertoires. The degeneration of TAARs is likely associated with arboreal inhabitants reducing their exposure to carnivores, and this was accelerated by the change in the nose shape of haplorhines after their divergence from strepsirrhines. Arboreal life may have decreased the reliance on the chemosensing of predators, suggestive of leading to the depauperation of TAAR subfamilies. The evolutionary deterioration of TAARs in primates has been reestablished in recently derived primates due to high selection pressure and probably functional diversity.     

Evolution of Brain-Expressed Biogenic Amine Receptors into Olfactory Trace Amine-Associated Receptors

The family of trace amine-associated receptors (TAARs) is distantly related to G protein-coupled biogenic aminergic receptors. TAARs are found in the brain as well as in the olfactory epithelium where they detect biogenic amines. However, the functional relationship of receptors from distinct TAAR subfamilies and in different species is still uncertain. Here, we perform a thorough phylogenetic analysis of 702 TAAR-like (TARL) and TAAR sequences from 48 species. We show that a clade of Tarl genes has greatly expanded in lampreys, whereas the other Tarl clade consists of only one or two orthologs in jawed vertebrates and is lost in amniotes. We also identify two small clades of Taar genes in sharks related to the remaining Taar genes in bony vertebrates, which are divided into four major clades. We further identify ligands for 61 orphan TARLs and TAARs from sea lamprey, shark, ray-finned fishes, and mammals, as well as novel ligands for two 5-hydroxytryptamine receptor 4 orthologs, a serotonin receptor subtype closely related to TAARs. Our results reveal a pattern of functional convergence and segregation: TARLs from sea lamprey and bony vertebrate olfactory TAARs underwent independent expansions to function as chemosensory receptors, whereas TARLs from jawed vertebrates retain ancestral response profiles and may have similar functions to TAAR1 in the brain. Overall, our data provide a comprehensive understanding of the evolution and ligand recognition profiles of TAARs and TARLs.     

Agonists for 13 trace amine-associated receptors provide insight into the molecular basis of odor selectivity

Trace amine-associated receptors (TAARs) are vertebrate olfactory receptors. However, ligand recognition properties of TAARs remain poorly understood, as most are "orphan receptors" without known agonists. Here, we identify the first ligands for many rodent TAARs and classify these receptors into two subfamilies based on the phylogeny and binding preference for primary or tertiary amines. Some mouse and rat orthologs have similar response profiles, although independent Taar7 gene expansions led to highly related receptors with altered ligand specificities. Using chimeric TAAR7 receptors, we identified an odor contact site in transmembrane helix III that functions as a selectivity filter. Homology models based on the β(2) adrenergic receptor structure indicate spatial proximity of this site to the ligand. Gain-of-function mutations at this site created olfactory receptors with radically altered odor recognition properties. These studies provide new TAAR ligands, valuable tools for studying receptor function, and general insights into the molecular pharmacology of G protein-coupled receptors.     

A region of the third intracellular loop of the short form of the D2 dopamine receptor dictates Gi coupling specificity

The D2 dopamine receptor has two isoforms, the short form (D2s receptor) and the long form (D2l receptor), which differ by the presence of a 29-amino acid insert in the third cytoplasmic loop. Both the D2s and D2l receptors have been shown to couple to members of the G alpha(i) family of G proteins, but whether each isoform couples to specific G alpha(i) protein(s) remains controversial. In previous studies using G alpha(i) mutants resistant to modification by pertussis toxin (G alpha(i)PT), we demonstrated that the D2s receptor couples selectively to G alpha(i2)PT and that the D2l receptor couples selectively to G alpha(i3)PT (Senogles, S. E. (1994) J. Biol. Chem. 269, 23120-23127). In this study, two point mutations of the D2s receptor were created by random mutagenesis (R233G and A234T). The two mutant D2s receptors demonstrated pharmacological characteristics comparable with those of the wild-type D2s receptor, with similar agonist and antagonist binding affinities. We used human embryonic kidney 293 cells stably transfected with G alpha(i1)PT, G alpha(i2)PT, or G alpha(i3)PT to measure agonist-mediated inhibition of forskolin-stimulated cAMP accumulation before and after pertussis toxin treatment. The two mutant D2s receptors demonstrated a change in G(i) coupling specificity compared with the wild-type D2s receptor. Whereas the wild-type D2s receptor coupled predominantly to G alpha(i2)PT, mutant R233G coupled preferentially to G alpha(i3)PT, and mutant A234T coupled preferentially to G alpha(i1)PT. These results suggest that this region of the third cytoplasmic loop is crucial for determining G(i) protein coupling specificity.     

Trace amine-associated receptor 1 is a stereoselective binding site for compounds in the amphetamine class

The demonstrated ability of amphetamine to functionally activate the rat trace amine associated receptor 1 (rTAAR1) and the subsequent reports of amphetamine activation of TAAR1 in rhesus monkey mouse, human, and human-rat chimeric TAAR1-expressing cell lines has led to speculation as to the role of this receptor in the central nervous system (CNS) responses associated with amphetamine and its analogs. The agonist potencies of ten pairs of enantiomeric amphetamines, including several with known CNS activity, at primate TAAR1 stably expressed in RD-HGA16 cells, robustly indicate the S-configuration to be associated with higher potency. Moreover, the rank order of potency to activate TAAR1 parallels the stimulant action reported by humans for the specific amphetamines. Taken together, these data suggest that TAAR1 is a stereoselective binding site for amphetamine and that activation of TAAR1 is involved in the modulation of the stimulant properties of amphetamine and its congeners. In addition, the observed parallel between hTAAR1 and rhTAAR1 responses supports the rhesus monkey as a highly translational model for developing novel TAAR1-directed compounds as therapeutics for amphetamine-related addictions.     

Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the alpha subunits of Gq family.

High efficiency transient transfection of Cos-7 cells was previously used to establish the functional coupling between G alpha q/G alpha 11 and phospholipase C beta 1 (Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817). Here the same system was used to study the functional coupling between other guanine nucleotide-binding regulatory protein (G-protein) alpha subunits and phospholipases and to study which G alpha subunits mediate the activation of phospholipase C by the alpha 1-adrenergic receptor subtypes, alpha 1 A, alpha 1 B, and alpha 1 C. We found that G alpha 14 and G alpha 16 behaved like G alpha 11 or G alpha q, i.e. they could activate endogenous phospholipases in Cos-7 cells in the presence of AIFn. The synergistic increase in inositol phosphate release in Cos-7 cells after they were cotransfected with cDNAs encoding G alpha subunits and phospholipase C beta 1 indicates that both G alpha 16 and G alpha 14 can activate phospholipase C beta 1. The activation of phospholipase C beta 1 was restricted to members of the Gq subfamily of alpha subunits. They activated phospholipase C beta 1 but not phospholipase C gamma 1, gamma 2, or phospholipase C delta 3. The cotransfection of Cos-7 cells with cDNAs encoding three different alpha 1-adrenergic receptors and G alpha q or G alpha 11 leads to an increase in norepinephrine-dependent inositol phosphate release. This indicates that G alpha q or G alpha 11 can mediate the activation of phospholipase C by all three subtypes of alpha 1-adrenergic receptors. With the same assay system, G alpha 16 and G alpha 14 appear to be differentially involved in the activation of phospholipase C by the alpha 1-adrenergic receptors. The alpha 1 B subtype receptor gave a ligand-mediated synergistic response in the cells cotransfected with either G alpha 14 or G alpha 16. However, the alpha 1 C receptor responded in cells cotransfected with G alpha 14 but not G alpha 16, and the alpha 1 A receptor showed little synergistic response in cells transfected with either G alpha 14 or G alpha 16. The ability of the alpha 1 A and alpha 1 C receptors to activate phospholipase C through G alpha q and G alpha 11 was also demonstrated in a cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evolutionary Patterns and Selective Pressures of Odorant/Pheromone Receptor Gene Families in Teleost Fishes

Background

Teleost fishes do not have a vomeronasal organ (VNO), and their vomeronasal receptors (V1Rs, V2Rs) are expressed in the main olfactory epithelium (MOE), as are odorant receptors (ORs) and trace amine-associated receptors (TAARs). In this study, to obtain insights into the functional distinction among the four chemosensory receptor families in teleost fishes, their evolutionary patterns were examined in zebrafish, medaka, stickleback, fugu, and spotted green pufferfish.

Methodology/Principal Findings

Phylogenetic analysis revealed that many lineage-specific gene gains and losses occurred in the teleost fish TAARs, whereas only a few gene gains and losses have taken place in the teleost fish vomeronasal receptors. In addition, synonymous and nonsynonymous nucleotide substitution rate ratios (K_A/K_S) in TAARs tended to be higher than those in ORs and V2Rs.

Conclusions/Significance

Frequent gene gains/losses and high K_A/K_S in teleost TAARs suggest that receptors in this family are used for detecting some species-specific chemicals such as pheromones. Conversely, conserved repertoires of V1R and V2R families in teleost fishes may imply that receptors in these families perceive common odorants for teleosts, such as amino acids. Teleost ORs showed intermediate evolutionary pattern between TAARs and vomeronasal receptors. Many teleost ORs seem to be used for common odorants, but some ORs may have evolved to recognize lineage-specific odors.     

Biogenic Trace Amine-Associated Receptors (TAARS) are encoded in avian genomes: evidence and possible implications

Recent studies of mammals and fish indicate that most trace amine-associated receptors (TAARs) may be involved in the detection of volatile biogenic compounds. It has therefore been suggested that this new class of "olfactory" receptors could be highly relevant for social communication and individual recognition. To determine if TAAR orthologues are encoded in avian genomes, we initiated BLAST searches of the Gallus gallus genome and public avian expressed sequence tags databases and performed associated phylogenetic analyses of the TAAR homologues identified. Our results suggest that a minimum of 3 TAAR paralogues are encoded in the G. gallus genome and that these are putative orthologues of the human/mouse genes TAAR1, TAAR2, and TAAR5. It is noteworthy that TAAR5 is activated by compounds that have been found in avian feces. We tentatively suggest that avian TAARs may compensate for the lack of an avian equivalent of the mammalian vomeronasal system and therefore may be important mediators of socially important avian chemical cues.     

Activation and regulation of platelet-activating factor receptor: role of G(i) and G(q) in receptor-mediated chemotactic, cytotoxic, and cross-regulatory signals

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role(s) of G(i) and G(q) in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing G alpha(i3) (PAFR-G alpha(i3)) or G alpha(q) (PAFR-G alpha(q)) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-G alpha(i3), or PAFR-G alpha(q) was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca2+ mobilization, phosphoinositide (PI) hydrolysis, and secretion of beta-hexosaminidase. PAFR-G alpha(i3) and PAFR-G alpha(q) mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-G alpha(i3), but not PAFR-G alpha(q), mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated beta arrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca2+ mobilization to PAF by all the receptors but only PAFR-G alpha(i3) activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that G(i) exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both G(i) and G(q) activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by G(i), the data suggest that differential activation of both G(i) and G(q) by PAFR likely mediate specific as well as redundant signaling pathways.     

Characterization of a neuronal cell line expressing native human melanin-concentrating hormone receptor 1 (MCHR1)

Melanin-concentrating hormone (MCH), an orexigenic neuropeptide in mammals, activates a G-protein coupled receptor, MCHR1. It is expected that antagonists of MCHR1 function will prove therapeutically useful as anti-obesity agents. Intracellular signaling by MCHR1 has been investigated primarily using non-neural cell lines expressing the recombinant receptor, in which MCHR1 has been shown to couple to G alpha(i/o) and G alpha(q) G-proteins. While these cell lines have been widely utilized to discover and optimize small molecule antagonists, it is unknown whether the intracellular signaling pathways in these cells accurately reflect those in neurons. Thus, we sought to develop a neurally derived cell line endogenously expressing MCHR1. IMR32, a human neuroblastoma cell line, has been shown to express MCHR1 mRNA; however, we were unable to detect either MCH-binding or MCH-stimulated Ca++-mobilization in these cells. Following transfection of IMR32 cells with a plasmid encoding human G alpha(16) G-protein, we isolated a cell line, I3.4.2, which responded to MCH in Ca++-mobilization assays. We found that the expression level of MCHR1 mRNA in I3.4.2 cells was 2000-fold higher than in the parent cell line. Using [125I]MCH saturation-binding to I3.4.2 cell membranes, we estimated the Bmax as 0.72 pmol/mg protein and the Kd as 0.35 nM. We report that Ca++-mobilization in I3.4.2 cells was insensitive to pertussis toxin (Ptx) treatment, indicating that signaling was via G alpha(q) G-proteins. Furthermore, negative results in cAMP accumulation assays confirmed the lack of signaling via the G alpha(i/o) G-proteins. Our results suggest that the I3.4.2 cell line may be useful for characterization of MCHR1 activity in a neural-derived cell line.     

Chimeric G proteins extend the range of insect cell-based functional assays for human G protein-coupled receptors

We previously described a functional assay for G protein-coupled receptors (GPCRs) based on stably transformed insect cells and using the promiscuous G protein Galpha16. We now show that, compared with Galpha16, the use of chimeric Galphaq subunits with C-terminal modifications (qi5-HA, qo5-HA, or qz5-HA) significantly enhances the ability of insect cells to redirect Gi-coupled GPCRs into a Gq-type signal transduction pathway. We coexpressed human Gi-coupled GPCRs, G protein alpha subunits (either a chimeric Galphaq or Galpha16), and the calcium-sensitive reporter protein aequorin in Sf9 cells using a nonlytic protein expression system, and measured agonist-induced intracellular calcium flux using a luminometer. Three of the GPCRs (serotonin 1A, 1D, and dopamine D2) were functionally redirected into a Gq-type pathway when coexpressed with the chimeric G proteins, compared with only one (serotonin 1A) with Galpha16. We determined agonist concentration-response relationships for all three receptors, which yielded EC50 values comparable with those achieved in mammalian cell-based assay systems. However, three other Gi-coupled GPCRs (the opioid kappa1 and delta1 receptors, and serotonin 1E) were not coupled to calcium flux by either the G protein chimeras or Galpha16. Possible reasons and solutions for this result are discussed.     

A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines

Insect cells are an underexplored resource for functional G-protein-coupled receptor (GPCR) assays, despite a strong record in biochemical (binding) assays. Here we describe the use of vectors capable of creating stably transformed insect cell lines to generate a cell-based functional GPCR assay. This assay employs the luminescent photoprotein aequorin and the promiscuous G-protein subunit Galpha16 and is broadly applicable to human GPCRs. We demonstrate that the assay can quantitate ligand concentration-activity relationships for seven different human GPCRs, can differentiate between partial and full agonists, and can determine rank order potencies for both agonists and antagonists that match those seen with other assay systems. Human Galpha16 improves signal strength but is not required for activity with some receptors. The coexpression of human and bovine betagamma subunits and/or phospholipase Cbeta makes no difference to agonist efficacy or potency. Two different receptors expressed in the same cell line respond to their specific agonists, and two different cell lines (Sf9 and High 5) are able to functionally detect the same expressed GPCR. Sf9 cells have the capability to produce fully functional human receptors, allied to a low background of endogenous receptors, and so are a valuable system for investigating orphan GPCRs and receptor dimerization.     

Evolution of trace amine associated receptor (TAAR) gene family in vertebrates: lineage-specific expansions and degradations of a second class of vertebrate chemosensory receptors expressed in the olfactory epithelium

The trace amine-associated receptors (TAARs) form a specific family of G protein-coupled receptors in vertebrates. TAARs were initially considered neurotransmitter receptors, but recent study showed that mouse TAARs function as chemosensory receptors in the olfactory epithelium. To clarify the evolutionary dynamics of the TAAR gene family in vertebrates, near-complete repertoires of TAAR genes and pseudogenes were identified from the genomic assemblies of 4 teleost fishes (zebrafish, fugu, stickleback, and medaka), western clawed frogs, chickens, 3 mammals (humans, mice, and opossum), and sea lampreys. Database searches revealed that fishes had many putatively functional TAAR genes (13-109 genes), whereas relatively small numbers of TAAR genes (3-22 genes) were identified in tetrapods. Phylogenetic analysis of these genes indicated that the TAAR gene family was subdivided into 5 subfamilies that diverged before the divergence of ray-finned fishes and tetrapods. In tetrapods, virtually all TAAR genes were located in 1 specific region of their genomes as a gene cluster; however, in fishes, TAAR genes were scattered throughout more than 2 genomic locations. This possibly reflects a whole-genome duplication that occurred in the common ancestor of ray-finned fishes. Expression analysis of zebrafish and stickleback TAAR genes revealed that many TAARs in these fishes were expressed in the olfactory organ, suggesting the relatively high importance of TAARs as chemosensory receptors in fishes. A possible evolutionary history of the vertebrate TAAR gene family was inferred from the phylogenetic and comparative genomic analyses.     

Receptor-mediated inhibition of G protein-coupled inwardly rectifying potassium channels involves G(alpha)q family subunits, phospholipase C, and a readily diffusible messenger

G protein-coupled inwardly rectifying K+ (GIRK) channels can be activated or inhibited by distinct classes of receptor (G(alpha)i/o- and G(alpha)q-coupled), providing dynamic regulation of cellular excitability. Receptor-mediated activation involves direct effects of G(beta)gamma subunits on GIRK channels, but mechanisms involved in GIRK channel inhibition have not been fully elucidated. An HEK293 cell line that stably expresses GIRK1/4 channels was used to test G protein mechanisms that mediate GIRK channel inhibition. In cells transiently or stably cotransfected with 5-HT1A (G(alpha)i/o-coupled) and TRH-R1 (G(alpha)q-coupled) receptors, 5-HT (5-hydroxytryptamine; serotonin) enhanced GIRK channel currents, whereas thyrotropin-releasing hormone (TRH) inhibited both basal and 5-HT-activated GIRK channel currents. Inhibition of GIRK channel currents by TRH primarily involved signaling by G(alpha)q family subunits, rather than G(beta)gamma dimers: GIRK channel current inhibition was diminished by Pasteurella multocida toxin, mimicked by constitutively active members of the G(alpha)q family, and reduced by minigene constructs that disrupt G(alpha)q signaling, but was completely preserved in cells expressing constructs that interfere with signaling by G(beta)gamma subunits. Inhibition of GIRK channel currents by TRH and constitutively active G(alpha)q was reduced by, an inhibitor of phospholipase C (PLC). Moreover, TRH- R1-mediated GIRK channel inhibition was diminished by minigene constructs that reduce membrane levels of the PLC substrate phosphatidylinositol bisphosphate, further implicating PLC. However, we found no evidence for involvement of protein kinase C, inositol trisphosphate, or intracellular calcium. Although these downstream signaling intermediaries did not contribute to receptor-mediated GIRK channel inhibition, bath application of TRH decreased GIRK channel activity in cell-attached patches. Together, these data indicate that receptor-mediated inhibition of GIRK channels involves PLC activation by G(alpha) subunits of the G(alpha)q family and suggest that inhibition may be communicated at a distance to GIRK channels via unbinding and diffusion of phosphatidylinositol bisphosphate away from the channel.