Evidence that pimozide is not a partial agonist of dopamine receptors.

The dopamine receptor antagonist pimozide, at concentrations up to 10 nM, competitively antagonized the inhibitory action of a pomorphine on prolactin (PRL) secretion by cultured rat pituitary cells. At higher concentrations pimozide as well as the analogues clopimozide and penfluridol suppressed PRL secretion. The latter effect could not be reversed by dopamine antagonists devoid of intrinsic effects on PRL release. Suppression of PRL release was also observed with compounds which were devoid of dopamine receptor agonistic or antagonistic properties such as R 6694 and R 5052, structurally related to pimozide, and also with loperamide. The inhibitory action of pimozide on PRL release resembled that of the calcium antagonist flunarizine. Concentration effect curves showed parallel slopes and the effect of both compounds could be reversed by increasing the concentration of calcium ions (Ca²⁺). Both flunarizine and pimozide were also capable of inhibiting releasing factor-stimulated luteinizing hormone secretion, an effect not shared by apomorphine. Pimozide and the various structurally related compounds used, also antagonized Ca²⁺-induced smooth muscle contractions of the isolated caudal artery of the rat.The present findings indicate that pimozide is a competitive antagonist without partial agonistic activity on apomorphine-sensitive dopamine receptors in the pituitary and that its inhibitory effect on PRL release as well as on vascular smooth muscle contractions is due to interference with a Ca²⁺-dependent mechanism of the stimulus-effect coupling process.     

8-O-acetylharpagide is not an ecdysteroid agonist

We have reinvestigated the activity of 8-O-acetylharpagide, an iridoid glucoside, as an ecdysteroid agonist. Elbrecht et al. (Insect Biochem. Mol. Biol. 26 (1996) 519) isolated a preparation of this compound from Ajuga reptans L. and ascribed ecdysteroid agonist activity on the basis of the induction of an ecdysteroid-like response in Drosophila melanogaster KcO cells, the displacement of [3H]ponasterone A from the Drosophila receptor and the activation of an ecdysteroid-regulated gene in a transactivation assay. We provide evidence that the agonist activity derives from contaminating ecdysteroids; A. reptans is a species rich in ecdysteroids. Purified 8-O-acetylharpagide is not active in the D. melanogaster B(II) cell bioassay, neither as an agonist nor as an antagonist, nor does it displace [3H]ponasterone A from dipteran or lepidopteran ecdysteroid receptor complexes.     

Meclizine is an agonist ligand for mouse constitutive androstane receptor (CAR) and an inverse agonist for human CAR

The constitutive androstane receptor (CAR, NR1I3) is a key regulator of xenobiotic and endobiotic metabolism. The ligand-binding domains of murine (m) and human (h) CAR are divergent relative to other nuclear hormone receptors, resulting in species-specific differences in xenobiotic responses. Here we identify the widely used antiemetic meclizine (Antivert; Bonine) as both an agonist ligand for mCAR and an inverse agonist for hCAR. Meclizine increases mCAR transactivation in a dose-dependent manner. Like the mCAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, meclizine stimulates binding of steroid receptor coactivator 1 to the murine receptor in vitro. Meclizine administration to mice increases expression of CAR target genes in a CAR-dependent manner. In contrast, meclizine suppresses hCAR transactivation and inhibits the phenobarbital-induced expression of the CAR target genes, cytochrome p450 monooxygenase (CYP)2B10, CYP3A11, and CYP1A2, in primary hepatocytes derived from mice expressing hCAR, but not mCAR. The inhibitory effect of meclizine also suppresses acetaminophen-induced liver toxicity in humanized CAR mice. These results demonstrate that a single compound can induce opposite xenobiotic responses via orthologous receptors in rodents and humans.     

Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist

The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a "steric doorstop" holding the protein in an open and active conformation.     

Ivermectin is not an agonist at a GABA receptor in tick salivary glands

The avermectins, broad-spectrum anti-parasitic agents which are effective against ticks, act principally by stimulating -aminobutyric acid (GABA) receptors directly or indirectly. GABA is known to potentiate dopamine-induced fluid secretion in the salivary glands of female ixodid ticks. We thus tested wheter 10 M ivermectin would have an effect similar to GABA in the isolated salivary gland preparation ofDermacentor andersoni. Ivermectin was not an agonist at this GABA receptor.     

3-Ethoxy-beta-carboline: a high affinity benzodiazepine receptor ligand with partial inverse agonist properties

3-Ethoxy-beta-carboline binds with high affinity to benzodiazepine receptors in the central nervous system (Ki approximately equal to 10.1, 15.3, and 25.3 nM in rat cerebellum, cerebral cortex, and hippocampus, respectively). This compound has pharmacological actions reminiscent of benzodiazepine receptor partial inverse agonists such as FG 7142 and 3-carboethoxy-beta-carboline. Thus, while not a convulsant, 3-ethoxy-beta-carboline potentiated the convulsant actions of pentylenetetrazole in mice. Furthermore, this compound reduced both the time spent and the total entries in the open arms of an elevated plus maze and also inhibited stress-induced ulcer formation, effects that are also observed with benzodiazepine receptor inverse agonists. These findings suggest that 3-ethoxy-beta-carboline is a partial inverse agonist at benzodiazepine receptors which may prove useful for in vivo studies since it has a higher affinity for benzodiazepine receptors and better solubility than the commonly used partial inverse agonist FG 7142. Furthermore, 3-ethoxy-beta-carboline appears to be less vulnerable to metabolic degradation than ester analogs with a similar pharmacological profile such as 3-carboethoxy-beta-carboline.     

Sarmesin is a partial agonist of angiotensin-II receptors in rabbit, but not in rat, aortic rings

Sarmesin, [Sar1, Tyr(Me)4]angiotensinII], has been reported to be a competitive angiotensin II (AII) receptor antagonist in rat smooth muscle preparations (Scanlon et al., (1984), Life Science 34, 317-321). In the present study, sarmesin displaced AII from its binding sites in rat aortic smooth muscle cells and in a rabbit aorta membrane preparation (IC50 5 and 6 nM resp.; Ki 4.1 and 5.3 resp.) In rabbit aortic rings, sarmesin (0.003-3 microM) produced concentration-dependent contractions (ED50 89 nM) and this effect was inhibited by saralasin. No contraction was observed in the rat aorta up to 100 microM. In rabbit aortic rings, sarmesin, at the same concentrations that produced contraction, inhibited contractions induced by AII in a competitive manner (pA2 7, 26). These results indicate that, in rabbit aortic rings sarmesin is a partial agonist of AII receptors.     

Deactivation and proton transfer in light-induced metarhodopsin II/metarhodopsin III conversion: a time-resolved fourier transform infrared spectroscopic study

Vertebrate rhodopsin shares with other retinal proteins the 11-cis-retinal chromophore and the light-induced 11-cis/trans isomerization triggering its activation pathway. However, only in rhodopsin the retinylidene Schiff base bond to the apoprotein is eventually hydrolyzed, making a complex regeneration pathway necessary. Metabolic regeneration cannot be short-cut, and light absorption in the active metarhodopsin (Meta) II intermediate causes anti/syn isomerization around the retinylidene linkage rather than reversed trans/cis isomerization. A new deactivating pathway is thereby triggered, which ends in the Meta III "retinal storage" product. Using time-resolved Fourier transform infrared spectroscopy, we show that the identified steps of receptor activation, including Schiff base deprotonation, protein structural changes, and proton uptake by the apoprotein, are all reversed. However, Schiff base reprotonation is much faster than the activating deprotonation, whereas the protein structural changes are slower. The final proton release occurs with pK approximately 4.5, similar to the pK of a free Glu residue and to the pK at which the isolated opsin apoprotein becomes active. A forced deprotonation, equivalent to the forced protonation in the activating pathway, which occurs against the unfavorable pH of the medium, is not observed. This explains properties of the final Meta III product, which displays much higher residual activity and is less stable than rhodopsin arising from regeneration with 11-cis-retinal. We propose that the anti/syn conversion can only induce a fast reorientation and distance change of the Schiff base but fails to build up the full set of dark ground state constraints, presumably involving the Glu(134)/Arg(135) cluster.     

Design of a partial PPARdelta agonist

Structure based ligand design was used in order to design a partial agonist for the PPARdelta receptor. The maximum activation in the transactivation assay was reduced from 87% to 39%. The crystal structure of the ligand binding domain of the PPARdelta receptor in complex with compound 2 was determined in order to understand the structural changes which gave rise to the decrease in maximum activation.     

Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst.

The oligonucleotide [5'-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5'-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity. The radioactive products from [5'-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli 'endonuclease III' is, after all, not an endonuclease.     

The DNA polymerase III holoenzyme contains γ and is not a trimeric polymerase

There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX₃δδ’χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-C_tag) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-C_tag per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III₂τ₂ γδδ’χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F’ episome.     

The 650 and chromophore in Escherichia coli is an 'oxy-' or oxygenated compound, not the oxidized form of cytochrome oxidase d: an hypothesis

The form of cytochrome d in Escherichia coli and Azotobacter vinelandii that shows an absorption maximum at 648 to 652 nm ('cytochrome d650') is generally regarded as the oxidized form of this terminal oxidase. Membranes from E. coli grown under oxygen-limited conditions, when treated with ferricyanide, do not reveal cytochrome d650, whereas a sharp symmetrical band at 652 nm results from the reaction of the reduced enzyme with O2 at either room temperature or after flash photolysis of the CO-liganded form at -130 degrees C. Electron paramagnetic resonance spectroscopy of cytochrome d650 trapped at -130 degrees C shows that its spectrum is indistinguishable from the CO-liganded form and does not reveal resonances of high spin ferric haem previously attributed to cytochrome d. An hypothesis is proposed in which cytochrome d650 is an early intermediate in the reaction of reduced cytochrome d650 and oxyhaemoglobin is presented and the hypothesis discussed in relation to earlier work, in which the indirect interconversions of reduced cytochrome d and d650 have been explained by proposing the existence of an 'invisible' form. It is suggested that this form could be the oxidized enzyme.     

Oxantel is an N-type (methyridine and nicotine) agonist not an L-type (levamisole and pyrantel) agonist: classification of cholinergic anthelmintics in Ascaris

Three pharmacological subtypes of cholinergic receptors have been distinguished in Ascaris suum using a muscle contraction assay and classical pharmacological techniques. The receptor subtypes are: a B-subtype (sensitive to bephenium); an L-subtype (sensitive to levamisole and pyrantel); and an N-subtype (sensitive to nicotine and methyridine). Oxantel is a cholinergic anthelmintic that was first introduced for the treatment of whipworm, Trichuris, infections in children. Here, we compare the subtype selectivity of oxantel with thenium and other cholinergic anthelmintics. We used the A. suum assay to derive pA(2) values for the agonists: oxantel, thenium, bephenium, levamisole, pyrantel, nicotine and methyridine with the antagonists: paraherquamide, 2-desoxyparaherquamide and methyllycaconitine. pA(2) values, rather than pK(B) values, were determined for all agonists when it was found that Schild slopes for some agonists were significantly less than 1.0. The pA(2) of oxantel was 6.58+/-0.25 for paraherquamide; 5.39+/-0.28 for 2-desoxyparaherquamide; 7.01+/-0.19 for methyllycaconitine. Comparison of pA(2) values using cluster analysis showed that oxantel was grouped with nicotine and methyridine, the N-subtype agonists. Thenium had pA(2)s of 7.84+/-0.41 for paraherquamide; 5.52+/-0.50 for 2-desoxyparaherquamide; 6.33+/-0.19 for methyllycaconitine. Cluster analysis placed thenium between the L-subtype agonists and the B-subtype agonist. The therapeutic significance of classification of cholinergic anthelmintics is discussed. Combination of oxantel and pyrantel would have therapeutic advantages, covering N- and L-subtypes, and so increasing spectrum of action and reducing the potential for development of resistance. Our results predict that oxantel may remain effective in some nematode isolates that have become levamisole- and pyrantel-resistant.     

The CC chemokine eotaxin (CCL11) is a partial agonist of CC chemokine receptor 2b

Despite sharing considerable homology with the members of the monocyte chemoattractant protein (MCP) family, the CC chemokine eotaxin (CCL11) has previously been reported to signal exclusively via the receptor CC chemokine receptor 3 (CCR3). Using the monocyte cell line THP-1, we investigated the relative abilities of eotaxin and MCPs 1-4 to induce CCR2 signaling, employing assays of directed cell migration and intracellular calcium flux. Surprisingly, 1 microm concentrations of eotaxin were able to recruit THP-1 cells in chemotaxis assays, and this migration was sensitive to antagonism of CCR2 but not CCR3. Radiolabeled eotaxin binding assays performed on transfectants bearing CCR2b or CCR3 confirmed eotaxin binding to CCR2 with a K(d) of 7.50 +/- 3.30 nm, compared with a K(d) of 1.68 +/- 0.91 nm at CCR3. In addition, whereas 1 microm concentrations of eotaxin were able to recruit CCR2b transfectants, substimulatory concentrations of eotaxin inhibited MCP-1-induced chemotaxis of CCR2b transfectants and also inhibited MCP-1-induced intracellular calcium flux of THP-1 cells. Collectively, these findings suggest that eotaxin is a partial agonist of the CCR2b receptor. A greater understanding of the interaction of CCR2 with all of its ligands, both full and partial agonists, may aid the rational design of specific antagonists that hold great promise as future therapeutic treatments for a variety of inflammatory disorders.     

Structural comparison of metarhodopsin II, metarhodopsin III, and opsin based on kinetic analysis of Fourier transform infrared difference spectra.

The "membrane bilayer" pathway (Rhodes, D. G., J. G. Sarmiento, and L. G. Herbette. 1985. Mol. Pharmacol. 27:612-623.) for 1,4-dihydropyridine calcium channel drug (DHP) binding to receptor sites in cardiac sarcolemmal membranes has been extended to include the interaction of amphiphiles within the lipid bilayer. These studies focused on the ability of the Class III antiarrhythmic agents bretylium and clofilium to nonspecifically inhibit DHP-receptor binding in canine cardiac sarcolemma. Clofilium was found to inhibit nimodipine binding with an inhibition constant of approximately 5 microM, whereas bretylium had no effect on nimodipine binding. Small angle x-ray diffraction was then used to examine the differential ability of these two Class III agents to inhibit DHP-receptor binding. The time-averaged locations of bretylium, clofilium, and nimodipine in bovine cardiac phosphatidylcholine (BCPC) bilayers (supplemented with 13 mol% cholesterol) were determined to a resolution of 9 A. The location of bretylium as dominated by its phenyl ring in BCPC bilayers was found to be at the hydrocarbon core/water interface, similar to that of the dihydropyridine ring of nimodipine. The location of clofilium as dominated by its phenyl ring was found to be below the hydrocarbon/core water interface within the hydrocarbon chain region of the bilayer, similar to that of the phenyl ring of nimodipine. The location of the dihydropyridine ring portion of nimodipine has previously been shown by neutron diffraction to be located at the hydrocarbon core/water interface of native sarcoplasmic reticulum, consistent with the small angle x-ray data from model membranes in this paper. Therefore, we speculate that the nonspecific inhibition arises from the interaction of clofilium's phenyl ring with the site on the calcium channel receptor where the phenyl ring portion of nimodipine must interact. The DHP-receptor binding pathway would then involve both nonspecific (membrane) and specific (protein) binding components, both of which are necessary for receptor binding.     

Arotinolol is a weak partial agonist on beta 3-adrenergic receptors in brown adipocytes

Arotinolol, a clinically used alpha/beta-adrenergic blocker, has been demonstrated to be an anti-obesity agent. The anti-obesity effect of arotinolol was suggested to be the result of direct activation of thermogenesis in brown-fat cells. We tested the ability of arotinolol to stimulate thermogenesis (oxygen consumption) in isolated brown-fat cells and in intact animals. Arotinolol stimulated thermogenesis in brown-fat cells isolated from mouse and hamster. A relatively low sensitivity to the beta-adrenergic antagonist propranolol (pK(B) approximately 6) indicated that arotinolol interacted with the beta3-adrenergic receptor. On the beta3-receptor, arotinolol was a very weak (EC50 approximately 20 microM) and only partial (approximately 50%) agonist, but arotinolol also demonstrated the properties of being a beta3-receptor antagonist with a pK(B) of 5.7. In intact animals, only the antagonistic action of arotinolol could be observed. Because arotinolol is only a very weak and partial agonist on the beta3-receptors, direct stimulation of thermogenesis in brown adipose tissue is unlikely to be sufficient to cause significant weight loss. It may be necessary to invoke additional pathways to explain the anti-obesity effects of chronic treatment with arotinolol.     

The photochemistry and photophysics of all-trans-1,4-diindanylidenyl-2-butene, a rigid analogue of all-trans-1,6-diphenyl-1,3,5-hexatriene.

all-trans-1,4-Diindanylidenyl-2-butene (ttt-stiff-5-DPH), a torsionally constrained analogue of all-trans-1,6-diphenyl-1,3,5-hexatriene (ttt-DPH), was synthesized and studied in order to evaluate the role of phenyl-vinyl torsional motions in the photophysical and photochemical responses of the DPH chromophore. Spectroscopic and photoisomerization measurements reveal that the behavior of the rigid DPH analogue is very similar to that of the parent DPH. This similarity is obtained despite the fact that the alkyl substitution from the five-membered rings selectively lowers the energy of the 1 (1)B(u)* state, leading to inversion of the order of the 1 (1)B(u)* and 2 (1)A(g)* energy levels in hydrocarbon solvents. In stiff-5-DPH, as in DPH, an increase in solvent polarity enhances terminal over central bond photoisomerization. Analyses of fluorescence and photoisomerization quantum yields show that, as in DPH, the torsional relaxation channel on the singlet excited state manifold is inefficient, falling far short of accounting for all radiationless decay. Significant ( approximately 50 and 80% of all singlet decay in Bz and AN, respectively), photochemically unproductive, radiationless decay channels exist in both molecules. Competing one bond photoisomerizations give the two major photoproducts: tct-stiff-5-DPH and ctt-stiff-5-DPH. They were isolated in pure form and were spectroscopically characterized. Biacetyl-sensitization was used to study the behavior of the stiff-5-DPH triplet state. As in the parent DPH, stiff-5-DPH triplets undergo relatively efficient concentration dependent geometric photoisomerization.     

UTP is not a biased agonist at human P2Y11 receptors

Biased agonism describes a multistate model of G protein-coupled receptor activation in which each ligand induces a unique structural conformation of the receptor, such that the receptor couples differentially to G proteins and other intracellular proteins. P2Y receptors are G protein-coupled receptors that are activated by endogenous nucleotides, such as adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP). A previous report suggested that UTP may be a biased agonist at the human P2Y₁₁ receptor, as it increased cytosolic [Ca²⁺], but did not induce accumulation of inositol phosphates, whereas ATP did both. The mechanism of action of UTP was unclear, so the aim of this study was to characterise the interaction of UTP with the P2Y₁₁ receptor in greater detail. Intracellular Ca²⁺ was monitored in 1321N1 cells stably expressing human P2Y₁₁ receptors using the Ca²⁺-sensitive fluorescent indicator, fluo-4. ATP evoked a rapid, concentration-dependent rise in intracellular Ca²⁺, but surprisingly, even high concentrations of UTP were ineffective. In contrast, UTP was slightly, but significantly more potent than ATP in evoking a rise in intracellular Ca²⁺ in 1321N1 cells stably expressing the human P2Y₂ receptor, with no difference in the maximum response. Thus, the lack of response to UTP at hP2Y₁₁ receptors was not due to a problem with the UTP solution. Furthermore, coapplying a high concentration of UTP with ATP did not inhibit the response to ATP. Thus, contrary to a previous report, we find no evidence for an agonist action of UTP at the human P2Y₁₁ receptor, nor does UTP act as an antagonist.     

Activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to an agonist

Several lines of evidence indicate that constraining intramolecular interactions between transmembrane domains are required to maintain G protein-coupled receptors in an inactive conformation in the absence of agonist. For the glycoprotein hormone receptors, which harbor a long amino-terminal ectodomain responsible for hormone binding, it has been suggested that the ectodomain could contribute to these negative constraints. To test this hypothesis, we expressed at the surface of COS-7 cells mutants of the TSH receptor in which variable portions of the amino-terminal ectodomain are replaced by a 19-residue tag from bovine rhodopsin. Whereas none of the rhodopsin-tagged truncated mutants could be activated by saturating concentrations of TSH, the constructs with the shortest amino-terminal extension displayed increased constitutive activity toward the cAMP pathway, when compared with the wild-type holoreceptor. The shortest truncated construct was strongly activated by the introduction of mutations in transmembrane segment VI (D633A), or in the third intracellular loop (A623I) of the receptor. The magnitude of the stimulation was similar to that observed when the same mutations were introduced in the intact wild-type receptor. On the contrary, the shortest truncated construct was unaffected by activating mutations affecting residues of the extracellular loop region (I486F, I568T) or the top of transmembrane segment VII (del658-661). Together, our results are compatible with a model in which activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to a true agonist.