Chinese Hamster Ovary Cell Membranes Research Articles

Functional characterization of a human receptor for neuropeptide FF and related peptides

1. Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein-coupled receptors (GPCR). We describe here, using an aequorin-based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR. 2. Binding experiments were performed with a new radioiodinated probe, [(125)I]-EYF, derived from the EFW-NPSF sequence of the rat NPFF precursor. Chinese hamster ovary (CHO) cell membranes expressing NPFFR bound [(125)I]-EYF with a K(d) of 0.06 nM. Various NPFF analogues and related peptides inhibited [(125)I]-EYF specific binding with the following rank order (K(i)): human NPAF (0.22 nM), SQA-NPFF (0.29 nM), NPFF (0.30 nM), 1DMe (0.31 nM), EYW-NPSF (0.32 nM), QFW-NPSF (0.35 nM), 3D (1.12 nM), Met-enk-RF-NH(2) (3.25 nM), FMRF-NH(2) (10.5 nM) and NPSF (12.1 nM). 3. The stimulatory activity of the same set of peptides was measured by a functional assay based on the co-expression of NPFFR, G(alpha 16) and apoaequorin. The rank order of potency was consistent with the results of the binding assay. 4. Membranes from NPFFR expressing CHO cells bound GTP gamma[(35)S] in the presence of SQA-NPFF. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of G(i) family members. 5. SQA-NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre-treatment. 6. RT -- PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.

Activation of Guanosine 5′-[γ-35S]thio-triphosphate Binding through M1Muscarinic Receptors in Transfected Chinese Hamster Ovary Cell Membranes: 2. Testing the “Two-States” Model of Receptor Activation

I suggested in the accompanying article [Mol Pharmacol 2001;59:875-885] that muscarinic receptors catalyzed G protein activation. Acetylcholine or carbamylcholine recognition facilitated not only the GDP release from receptor-coupled inactive G proteins but also the release of G from the (unstable) HRG complex. The two effects facilitated [(35)S]GTP gamma S binding in the presence of GDP, but could be studied separately by comparing [(35)S]GTP gamma S binding in the absence and presence of GTP. Guanyl nucleotides affected the efficiency of receptor-G protein coupling. The relative efficacies of partial agonists in the absence and presence of GTP should remain nonlinearly correlated if all agonists stabilize (to different extents) the same active receptor conformation. The correlation between M(1) muscarinic agonists' efficacy in accelerating [(35)S]GTP gamma S binding in the absence of other nucleotides and their in vivo efficacy (inositol phosphate accumulation) was in fact very poor. This probably reflected the presence of GTP in intact cells: pertussis toxin pretreatment (which inactivates the G(i/o) proteins) did not affect the agonists' efficacy profile (evaluated in the absence of spare receptors), but the addition of GTP to the [(35)S]GTP gamma S binding medium did. These results did not support the allosteric "two states" model of receptor activation, but suggested that different agonists induced different receptor conformations ("induced fit").

Activation of Guanosine 5′-[γ-35S]thio-triphosphate Binding through M1Muscarinic Receptors in Transfected Chinese Hamster Ovary Cell Membranes: 1. Mathematical Analysis of Catalytic G Protein Activation

I analyzed in this work the effect of agonists and unlabeled guanyl nucleotides on [(35)S]GTP gamma S and [(3)H]NMS binding to transfected CHO cells expressing hM(1) muscarinic receptors. I was unable to explain my kinetic results by "traditional" (one-site, two-site, or two-step) bimolecular binding models. I therefore examined the equations that describe catalytic G protein activation. My results were fully consistent with the following interpretation: G protein-coupled receptors either interacted with GDP-bound G proteins and facilitated the GDP release or recognized empty G proteins, depending on the incubation conditions. The receptor-coupled empty G proteins (RG) then recognized GTP gamma S, and the occupied G protein (G) dissociated reversibly from the receptor. Agonists accelerated the GDP release from receptor-coupled G proteins and accelerated the G dissociation: both effects accelerated synergically the G protein-GTP gamma S association reaction in the presence of GDP. GTP gamma S-bound G proteins, G, competed efficiently with inactive (empty or GDP-bound) G proteins for receptor recognition, and were able, therefore, at low concentrations, to quench the [(35)S]GTP gamma S binding reaction.

Constitutive activity at serotonin 5-HT 1D receptors: detection by homologous GTPγS versus [ 35S]-GTPγS binding isotherms

Although many G-protein-coupled receptors (GPCRs) may display constitutive activity, their detection has, to date, depended on the use of inverse agonists. The present study exploited a novel procedure to investigate constitutive activity at recombinant human (h) serotonin (5-HT) 5-HT 1D receptors stably expressed in Chinese hamster ovary (CHO) cells. 5-HT modestly stimulated guanosine-5′-O-(3-[ 35S]thio)-triphosphate ([ 35S]-GTPγS) binding to CHO-h5-HT 1D membranes whereas methiothepin and the 5-HT 1B/1D-selective ligand, SB224,289, exerted robust inhibition of basal [ 35S]-GTPγS binding (inverse agonism). These actions were specific inasmuch as they were reversed by the novel, selective 5-HT 1B/1D ligand, S18127. Constitutive activity was investigated by homologous inhibition of [ 35S]-GTPγS binding to CHO-h5-HT 1D membranes with unlabelled GTPγS. Under ‘basal’ conditions (absence of receptor ligand), biphasic isotherms were observed. Most (80%) [ 35S]-GTPγS binding sites were in the high affinity (HA) versus low affinity (LA) component of the isotherms. HA binding was augmented by 5-HT (to 155%; relative to basal values=100%), but decreased by methiothepin (to 23%) and by SB224,289 (to 67%). In contrast, LA binding was not altered. Further, membranes of untransfected CHO cells exhibited only LA binding sites, indicating that the latter are not related to h5-HT 1D receptor-G-protein coupling. Thus, at 5-HT 1D receptors expressed in this CHO cell line, HA binding detected in homologous inhibition experiments (GTPγS versus [ 35S]-GTPγS) under basal conditions provides a measure of constitutive G-protein activation. Thus, it is suggested that for h5-HT 1D receptors and, possibly, other GPCRs, inverse agonists will be detectable by [ 35S]-GTPγS binding if a HA component is present under basal conditions.

GTP differentially affects antagonist radioligand binding to adenosine A 1 and A 2A receptors in human brain

The effect of guanosine triphosphate (GTP) on the interaction of antagonists with human adenosine A 1 and A 2A receptors was studied using whole-hemisphere sections from human brain and membranes from Chinese hamster ovary (CHO) cells expressing human A 1 and A 2A receptors. Adenosine A 1 receptors, studied using [ 3 H] 1,3-dipropyl-8-cyclopentylxanthine ( [ 3 H] DPCPX) as radioligand, showed the expected regional distribution in human brain. Addition of 500 μM GTP significantly increased (23–55%) [ 3 H] DPCPX binding in all regions measured. In CHO cells transfected with human adenosine A 1 receptor cDNA, the number of receptors, B max, increased from 401 (359–442) to 667 (592–743) fmol/mg protein upon addition of GTP. [ 3 H] 5-Amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]-pyrimidine ( [ 3 H] SCH 58261), a selective adenosine A 2A receptor ligand, showed saturable binding to membranes from CHO cells transfected with adenosine A 2A receptor cDNA and was localized to striatum and globus pallidus in human brain sections. Addition of GTP did not significantly change [ 3 H] SCH 58261 binding to brain sections or CHO cell membranes. These results indicate that human A 1 and A 2A receptors are not substantially different from those of the rat as regards regulation by GTP and interactions with endogenous adenosine in binding experiments. However, the relative abundance of the receptors differs between species, and this may be related to the differences observed in the potency of the endogenous agonist.

Inhibition of acetylcholine muscarinic M(1) receptor function by the M(1)-selective ligand muscarinic toxin 7 (MT-7).

MT-7 (1 - 30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M(1) receptor, inhibited the acetylcholine (ACh)-stimulated [(35)S]-guanosine-5'-O-(3-thio)triphosphate ([(35)S]-GTPgammaS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M(1) receptor subtype. MT-7 failed to affect the ACh-stimulated [(35)S]-GTPgammaS binding in membranes of CHO cells expressing either the M(2), M(3) or M(4) receptor subtype. In N1E-115 neuroblastoma cells endogenously expressing the M(1) and M(4) receptor subtypes, MT-7 (0.3 - 3.0 nM) inhibited the carbachol (CCh)-stimulated inositol phosphates accumulation, but failed to affect the CCh-induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38-stimulated cyclic AMP accumulation. In both CHO/M(1) and N1E-115 cells the MT-7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC(50) value. In CHO/M(1) cell membranes, MT-7 (0.05 - 25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [(3)H]-N-methylscopolamine ([(3)H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [(3)H]-NMS by about 5 fold. CHO/M(1) cell membranes preincubated with MT-7 (10 nM) and washed by centrifugation and resuspension did not recover control [(3)H]-NMS binding for at least 8 h at 30 degrees C. It is concluded that MT-7 acts as a selective noncompetitive antagonist of the muscarinic M(1) receptors by binding stably to an allosteric site.

Effect of serum on in vitro electrically mediated gene delivery and expression in mammalian cells

In many cell systems, electric pulses can efficiently mediate gene transfer with a high level of expression in vitro. In vivo results have been reported where decrease in efficiency was obtained. The mechanisms involved in the process are unknown. Since, in vivo, the efficiency of non-viral methods of gene transfer is generally limited by the presence of serum, we report here the effect of serum on in vitro electrically mediated chinese hamster ovary cell membrane permeabilization, viability, gene transfer and expression. The results indicate that permeabilization and gene transfer are not inhibited by serum. By acting as a protector of cell viability, serum indeed increases gene transfer and expression.

Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

After short preincubations with N-[(3)H]methylscopolamine ([(3)H]NMS) or R(-)-[(3)H]quinuclidinyl benzilate ([(3)H]QNB), radioligand dissociation from muscarinic M(1) receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [(3)H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [(3)H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M(1) receptors. Covalently binding [(3)H]propylbenzilylcholine mustard detected substantially more binding sites than [(3)H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M(1) receptors may include Asp-99. Experimental data on [(3)H]NMS and [(3)H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.

O-1057, a potent water-soluble cannabinoid receptor agonist with antinociceptive properties.

Cannabinoids have low water solubility, necessitating the use of a solubilizing agent. In this paper we investigated whether a novel water-soluble cannabinoid, 3-(5'-cyano-1', 1'-dimethylpentyl)-1-(4-N-morpholinobutyryloxy)-Delta(8)- tetrahydroca nnabinol hydrochloride (O-1057), would interact with cannabinoid receptors when water or saline were used as the only vehicle. O-1057 displaced [(3)H]-CP55940 from specific binding sites on Chinese hamster ovary (CHO) cell membranes expressing CB(1) or CB(2) cannabinoid receptors, with pK(i) values of 8.36 and 7.95 respectively. It also displaced [(3)H]-CP55940 from specific binding sites on rat brain membranes (pK(i) = 7.86). O-1057 inhibited forskolin-stimulated cyclic AMP production by both CB(1)- and CB(2)-transfected CHO cells (pEC(50) = 9.16 and 9.72 respectively), its potency matching that of CP55940 and exceeding that of Delta(9)-tetrahydrocannabinol. In the mouse isolated vas deferens, O-1057 inhibited electrically-evoked contractions with pEC(50) and E(max) values of 9.73 and 76.84% respectively. It was antagonized by 100 nM SR141716A, the pK(B) of SR141716A against O-1057 (8.90) approximating to that against CP55940 (8.97). O-1057 also behaved as a CB(1) receptor agonist in vivo, reducing mouse spontaneous activity and rectal temperature when injected intravenously and inducing antinociception in the mouse tail flick test when given intravenously (ED(50) = 0.02 mg kg(-1)), intrathecally, intracerebroventricularly or by gavage. In all these assays, O-1057 was more potent than Delta(9)-tetrahydrocannabinol and, at 0.1 mg kg(-1) i.v., was antagonized by SR141716A (3 mg kg(-1) i.v.). These data demonstrate the ability of the water-soluble cannabinoid, O-1057, to act as a potent agonist at CB(1) and CB(2) receptors and warrant investigation of the clinical potential of O-1057 as an analgesic.

Modulation of [ 35S]GTPγS binding to Chinese hamster ovary cell membranes by D 2(short) dopamine receptors

Rat dopamine D 2short expressed in Chinese hamster ovary (CHO) cells were characterized by means of activation of [ 35S]-guanosine 5′-O-( γ-thiotriphosphate) ([ 35S]GTP γS) binding and inhibition of [ 3H]raclopride binding. Among 18 dopaminergic ligands studied dopamine, NPA, apomorphine and quinpirole were full agonists in activation of [ 35S]GTP γS binding, while seven ligands were partial agonists with efficacies from 16 to 69% of the effect of dopamine and seven ligands were antagonists having no effect on the basal level of [ 35S]GTP γS binding, but inhibited dopamine-dependent activation in a dose-response manner. Despite the different efficacies, the potencies of all 18 ligands to modulate [ 35S]GTP γS binding revealed a good correlation with their potencies to inhibit [ 3H]raclopride binding in the CHO cell membranes. This indicates that the binding of the ligand to the receptor determines its potency, but has no direct correlation with its intrinsic activity.

Structural determinants of the partial agonist-inverse agonist properties of 6'-azidohex-2'-yne-delta8-tetrahydrocannabinol at cannabinoid receptors.

1. We have extended previous investigations of four analogues of Delta8-tetrahydrocannabinol (Delta8-THC): 6'-azidohex-2'-yne-Delta8-THC (O-1184), 6'-azidohex-cis-2'-ene-Delta8-THC (O-1238) and octyl-2'-yne-Delta8-THC (O-584) and its 1-deoxy-analogue (O-1315). 2. O-1184, O-1238 and O-584 displaced [3H]-CP55940 from specific binding sites on Chinese hamster ovary (CHO) cell membranes expressing CB1 or CB2 cannabinoid receptors, with pKi values of 8.28 to 8.45 (CB1) and 8.03 to 8.13 (CB2). The pKi values of O-1315 were significantly less, 7.63 (CB1) and 7.01 (CB2). 3. All the analogues inhibited forskolin-stimulated cyclic AMP production by CB1-transfected CHO cells (pEC50=9.16 to 9.72). Only O-1238 behaved as a full agonist in this cell line. 4. In mouse vasa deferentia, O-1238 inhibited electrically-evoked contractions (pEC50=10.18 and Emax=70.5%). Corresponding values for O-1184 were 9.08 and 21.1% respectively. At 1 nM, O-1184 produced surmountable antagonism of the cannabinoid receptor agonist, CP55940. However, at 0.1 nM, O-1184 did not attenuate CP55940-induced inhibition of cyclic AMP production by CB1-transfected CHO cells. 5. In CB2-transfected CHO cells, cyclic AMP production was inhibited by CP55940 (pEC50=8.59), enhanced by O-1184 and O-584 (pEC50=8.20 and 6.86 respectively) and not significantly affected by O-1238 or O-1315. 6. At 100 nM, O-1184 and O-1238 produced surmountable antagonism of CP55940 in CB2 cells, decreasing the pEC50 of CP55940 from 8.61 to 7.42 (O-1184) or from 8. 54 to 7.44 (O-1238). 7. These data support the hypothesis that increasing the degree of unsaturation of the aliphatic side-chain of Delta8-THC analogues has little effect on CB1 or CB2 receptor affinity but can reduce CB1 receptor efficacy and reverse the direction of responses elicited at CB2 receptors.

Activation of Transfected M1or M3Muscarinic Acetylcholine Receptors Induces Cell–Cell Adhesion of Chinese Hamster Ovary Cells Expressing Endogenous Cadherins

Expression of endogenous cadherins by Chinese hamster ovary (CHO) cells has not been previously reported. However, we observed that CHO cells adhere to one another upon activation of transfected muscarinic acetylcholine receptors (mAChR), suggesting that the cells express endogenous cadherins. A 160-base pair RT-PCR product with 100% homology to the cytoplasmic domain of human E-cadherin was amplified from CHO cells. A second RT-PCR product amplified from these cells has 92% homology to the cytoplasmic domain of human cadherin-9 and 86% homology to the cytoplasmic domain of human cadherin-6. Western blotting indicates that CHO cells express a 165-kDa protein recognized by E-cadherin antibodies and a 120-kDa protein recognized by an antibody to the cadherin C-terminus sequence. The ability of transfected mAChR subtypes to regulate cadherin-mediated adhesion of CHO cells was tested by measuring the permeation of horseradish peroxidase across confluent CHO cell monolayers, by microscopic examination of the cells, and by aggregation assays. Cell–cell adhesion is induced within 15 min of activating transfected M1or M3mAChR which functionally couple to protein kinase C (PKC). In contrast, CHO cell adhesion is not affected by activating transfected M2mAChR which functionally couple to other effectors. Activation of PKC with phorbol esters also induces cell–cell adhesion of all CHO sublines tested. Immunofluorescence assays reveal that endogenous cadherins redistribute on the plasma membrane of CHO cells following mAChR or PKC activation. Inactivation of cadherins by removal of extracellular Ca2+abrogates adhesion induced by mAChR or PKC activation. Our demonstration that activation of only odd-numbered mAChR subtypes induces cadherin-mediated adhesion suggests that the unique responses of cells to M1or M3mAChR stimulation may involve cadherin activation.

Sialic Acid-Specific Lectin-Mediated Adhesion of Tritrichomonas foetus and Tritrichomonas mobilensis

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract producing infection associated with inflammatory changes, abortion, and infertility, Tritrichomonas mobilensis was isolated from squirrel monkey colon, and symptoms involve diarrheal complications. Both tritrichomonads produced hemagglutinins with the properties of sialic acid-specific lectins. Assays on the adherence of these protozoans to Chinese hamster ovary (CHO) cells and to bovine cervical and monkey colon mucus were performed to assess the function of the lectins in adhesion. Sialic acid at concentration as low as 2 mM inhibited the adhesion to CHO cells, less effectively to the mucus. Predigestion with Clostridium perfringens sialidase prevented the adhesion to both epithelial cells and the mucus. Inhibition of endogenous sialidases with 2,3-dehydro-2-deoxy-NeuAc increased the adhesion of T. mobilensis to CHO cells. Specific anti-T. foetus lectin (TFL) and anti-T. mobilensis lectin (TML) antibodies inhibited adhesion of the trichomonads to the epithelial cells and to the mucus. TFL histochemistry disclosed the presence of lectin ligands on keratinized vaginal epithelia, cervical mucosa, and mucin and on endometrial glands and their secretions. TML histochemistry showed reactivity with the luminal membranes of colonic glandular epithelium and less with the colonic mucin. Both lectins bound to the surface membrane of CHO cells. Anti-lectin antibodies showed granular cytoplasmic and strong membrane localization of the lectins in both tritrichomonads. Although the 2 tritrichomonads have different habitats, the results indicate that both these protozoa use lectins with sialic acid specificity for adhesion to mucosal surfaces.

Development of Flashplate™ Technology to Measure [35S]GTPγS Binding to Chinese Hamster Ovary Cell Membranes Expressing the Cloned Human 5-HT1B Receptor

With the exponential rate at which proposed drugs are being produced for disease therapy, it is now essential to automate assays used to screen these compounds and increase throughput. This has been rapidly adopted for simple radioligand binding assays but is less amenable for certain functional screens. [35S]GTPγS binding represents a convenient method for screening ligands that bind to G protein-coupled receptors and, ultimately, stimulate G-protein activation. In this study we have investigated the use of 96-well FlashPlates™ (NEN DuPont, Stevenage, England) to measure [35S]GTPγS binding to human 5-HT1B receptors expressed in Chinese hamster ovary cells. The cells were added to the individual wells of the FlashPlate and incubated with [35S]GTPγS in the presence or absence of test drug and bound radioactivity measured in a 96-well spectrometer. 5-HT produced a stimulation of basal [35S]GTPγS binding, which was robust within and between experiments, with pEC50= 8.1. The 5-HT1Bpartial agonist GR127935 (2′n-methyl-4′-5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide) caused a partial stimulation (pEC5o = 8.3, intrinsic activity = 0.7), and the selective 5-HTIB receptor antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2′-methyl-4′-[(5-methyl-1,2,4-oxadiazole-3-yl)biphenyl-4-yl]carbonyl}furo[2,3-flindole-3-spiro-4′-piperidine oxalate) displayed inverse agonism with pEC50 = 7.6. These results are similar to those obtained using the conventional filtration method and indicate that FlashPlate technology can provide a rapid method for measuring [35S]GTPγS binding.

Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor.

1. Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells. 2. In rat striatum, clozapine (1 nM-10 microM) caused a slight inhibition of forskolin-stimulated adenylyl cyclase activity, which was not counteracted by 10 microM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA2 value of 7.51. Moreover, clozapine (1 microM) failed to inhibit dopamine D1 receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [3H]-N-methylscopolamine ([3H]-NMS) bound to striatal M4 receptors with a monophasic inhibitory curve and a pKi value of 7.69. The clozapine inhibition was not affected by the addition of guanosine-5'-O-(thio)triphosphate (GTPgammaS). 3. In intact CHO cells, clozapine inhibited forskolin-stimulated cyclic AMP accumulation with an EC50 of 31 nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 microM (EC50=50 nM) and stimulating at higher concentrations (EC50 = 7 microM). Clozapine (0.3-5 microM) antagonized the CCh stimulation of cyclic AMP with a pKi value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes. 4. In CHO cells pretreated with the receptor alkylating agent 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10 microM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum. 5. As in rat striatum, in CHO cell membranes the displacement of [3H]-NMS binding by clozapine yielded a monophasic curve which was not affected by GTPgammaS. 6. Clozapine (10 nM-10 microM) had a small stimulant effect (approximately 20%) on the binding of [35S]-GTPgammaS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50 nM-5 microM) antagonized the CCh-stimulated [35S]-GTPgammaS binding with a pA2 value of 7.48. 7. These results show that at the striatal M4 receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.

Mapping Peptide-binding Domains of the Human V1a Vasopressin Receptor with a Photoactivatable Linear Peptide Antagonist

The study of antagonist-binding domains of the human V1a vasopressin receptor was performed using a radioiodinated photoreactive peptide antagonist. This ligand displayed a high affinity for the receptor expressed in Chinese hamster ovary cell membranes, and specifically labeled two protein bands with apparent molecular mass at 85-90 and 46 kDa. Our results clearly show that the V1a receptor is degraded during incubation with the ligand and that the 46-kDa species is probably the result of the 85-90-kDa species proteolytic cleavage. Truncation of the receptor was then confirmed by deglycosylation with N-glycosidase F. A monoclonal antibody directed against a c-Myc epitope added at the receptor NH2 terminus allowed immunoprecipitation of the 85-90-kDa photolabeled species. The 46-kDa photolabeled protein never immunoprecipitated, indicating that the truncated form of the receptor lacks the NH2 terminus region. To localize photolabeled domains of the receptor, the 46-kDa protein was cleaved with V8 and/or Lys-C endoproteinases. The identity of the smallest photolabeled fragment, observed at approximately 6 kDa, was then confirmed by mutation of the potential V8 cleavage sites. Our results indicate that covalent labeling of the vasopressin V1a receptor with the photoreactive antagonist occurs in a region including transmembrane domain VII (residues Asn327-Lys370).

In Vitro Properties of a High Affinity Selective Antagonist of the VIP 1 Receptor

Gourlet, P., P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. In vitro properties of a high affinity selective antagonist of the VIP 1 receptor. Peptides 18(10) 1555–1560, 1997.—A selective high affinity VIP 1 receptor antagonist [Acetyl-His 1, D-Phe 2, Lys 15, Arg 16, Leu 17] VIP(3-7)/GRF(8-27) or PG 97-269 was synthesized, by analogy with recently obtained selective VIP 1 receptor agonists. The properties of the new peptide were evaluated on Chinese hamster ovary (CHO) cell membranes expressing either the rat VIP 1-, rat VIP 2- or the human VIP 2- recombinant receptors and on LoVo cell membranes expressing exclusively the human VIP 1 receptor. The IC 50 values of 125I-VIP binding inhibition by PG 97-269 were 10, 2000, 2 and 3000 nM on the rat VIP 1-, rat VIP 2-, human VIP 1- and human VIP 2 receptors, respectively. PG 97-269 had a negligible affinity for the PACAP I receptor type. It did not stimulate adenylate cyclase activity, but inhibited competitively effect of VIP on the VIP 1 receptor mediated stimulation of adenylate cyclase activity. The K i values were respectively of 15 ± 5 nM and 2 ± 1 nM for the rat and human VIP 1 receptors. Thus the described molecule in the first reported VIP antagonist with an affinity in the nM range and with a high selectivity for the VIP 1 receptor subclass. It may be useful for evaluation of the physiological role of VIP in rat and human tissues.

Acylation of Glucosaminyl Phosphatidylinositol Revisited: PALMITOYL-CoA DEPENDENT PALMITOYLATION OF THE INOSITOL RESIDUE OF A SYNTHETIC DIOCTANOYL GLUCOSAMINYL PHOSPHATIDYLINOSITOL BY HAMSTER MEMBRANES PERMITS EFFICIENT MANNOSYLATION OF THE GLUCOSAMINE RESIDUE

Two critical steps in the assembly of yeast and mammalian glycosylphosphatidylinositol (GPI) anchor precursors are palmitoylation of the inositol residue and mannosylation of the glucosamine residue of the glucosaminyl phosphatidylinositol (GlcNalpha-PI) intermediate. Palmitoylation has been reported to be acyl-CoA dependent in yeast membranes (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 267, 8599-8603) but strictly acyl-CoA independent in rodent membranes (Stevens, V. L., and Zhang, H. (1994) J. Biol. Chem. 269, 31397-31403), and thus poorly conserved. In addition, it was suggested that acylation must precede mannosylation in both yeast (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 276, 8599-8603) and rodent (Urakaze, M., Kamitani, T., DeGasperi, R., Sugiyama, E., Chang, H.-M., Warren, C. D., and Yeh, E. T. H. (1992) J. Biol. Chem. 267, 6459-6462) cells because GlcNalpha-acyl-PI accumulates in vivo when mannosylation is blocked. However, GlcNalpha-acyl-PI accumulation would also be expected if mannosylation and acylation were independent of each other. These issues were addressed by the use of a synthetic dioctanoyl GlcNalpha-PI analogue (GlcNalpha-PI(C8)) as an in vitro substrate for GPI-synthesizing enzymes in Chinese hamster ovary cell membranes. GlcNalpha-PI(C8) was acylated in an manner requiring acyl-CoA. Thus, the process involving acyl-CoA reported for yeast has been conserved in mammals. Furthermore, both GlcNalpha-PI(C8) and GlcNalpha-acyl-PI(C8) could be mannosylated in vitro, but mannosylation of the latter was significantly more efficient. This provides direct support for the earlier suggestion that acylation precedes mannosylation in rodents cells. A similar result was also observed with the Saccharomyces cerevisiae mannosyltransferase. In contrast, it has been reported that mannosylation of endogenous GlcNalpha-PI by Trypansoma brucei membranes occurs without prior acylation. The same result was obtained with GlcNalpha-PI(C8), confirming that the mannosyltransferase of trypanosomes is divergent from those in yeasts and rodents.

The affinity of adenosine for the high- and low-affinity states of the human adenosine A 1 receptor

The affinity of adenosine for the human adenosine A 1 receptor expressed on Chinese hamster ovary cell membranes has been measured in the presence and absence of GTP. The competitive effect of endogenous adenosine on the binding properties of adenosine A 1 receptors was estimated from differences in the binding of N 6-cyclohexyladenosine measured in the absence and presence of adenosine deaminase. From these data, the affinity of adenosine for the high- and low-affinity states of the human adenosine A 1 receptor (7 × 10 7 and 1.3 × 10 5 M −1, respectively) was calculated.

Selective enhancement of antagonist ligand binding at muscarinic M 2 receptors by heparin due to receptor uncoupling

The selectivity of heparin in inducing potentiation of binding of antagonist ligands to muscarinic receptors was investigated at the five known subtypes of muscarinic receptors. The effects of heparin on binding of [ 3H] N-methylscopolamine at equilibrium was studied in Chinese hamster ovary (CHO) cells which express each of the individual muscarinic receptor subtypes and in membranes prepared from these cells. Heparin markedly increased equilibrium binding of subsaturating concentrations of the ligand only in membranes of CHO cells which express muscarinic M 2 receptors. These effects of heparin were qualitatively similar to those obtained in heart membranes. In contrast, heparin did not influence ligand binding to muscarinic M 2 receptors in intact cells. The positive cooperative effects of heparin at muscarinic receptors were abolished following treatment of cells with pertussis toxin. The latter treatment by itself resulted in a significant increase in [ 3H] N-methylscopolamine binding. Taken together with previous reports of heparin-induced uncoupling of receptors and G-proteins, these data suggest that the effects of heparin on ligand binding to muscarinic M 2 receptors might be due to disruption of receptor-G-protein interactions which results in enhancement of binding of antagonist ligands to the receptor.