Stimulated Inositol Phosphate Accumulation Research Articles

RF9 Acts as a KISS1R Agonist In Vivo and In Vitro.

RF9, a reported antagonist of the mammalian gonadotropin-inhibitory hormone receptor, stimulates gonadotropin secretion in mammals. Recent studies have suggested that the stimulatory effect of RF9 on gonadotropin secretion relies on intact kisspeptin receptor (KISS1R) signaling, but the underlying mechanisms remain to be elucidated. Using Chinese Hamster Ovary cells stably transfected with KISS1R, we show that RF9 binds specifically to KISS1R, with a Kd of 1.6 × 10(-5)M, and stimulates an increase in intracellular calcium and inositol phosphate accumulation in a KISS1R-dependent manner, with EC50 values of 3.0 × 10(-6)M and 1.6 × 10(-7)M, respectively. RF9 also stimulated ERK phosphorylation, with a time course similar to that of kisspeptin-10. RFRP-3, the putative endogenous ligand for NPFFR1, did not stimulate inositol phosphate accumulation or pERK, nor did it alter responses to of kisspeptin-10 or RF9. In agreement with these in vitro data, we found that RF9 stimulated a robust LH increase in Npffr1(-/-) mice, similar to that in wild-type littermates, whereas the stimulatory effect of RF9 was markedly reduced in Kiss1r(-/-) and double Kiss1r(-/-)/Npfrr1(-/-) mice. The stimulatory effect of RF9 on LH secretion was restored by the selective rescue of Kiss1r expression in GnRH neurons, in Kiss1r(-/-T) mice. Taken together, our study demonstrates that RF9 acts primarily as a KISS1R agonist, but not as an allosteric modulator, to stimulate LH secretion. Our findings raise questions regarding the utility of RF9 for assessing NPFF1R function and de-emphasize a predominant role of this signaling system in central regulation of reproduction.

Mefloquine and psychotomimetics share neurotransmitter receptor and transporter interactions in vitro

Mefloquine is used for the prevention and treatment of chloroquine-resistant malaria, but its use is associated with nightmares, hallucinations, and exacerbation of symptoms of post-traumatic stress disorder. We hypothesized that potential mechanisms of action for the adverse psychotropic effects of mefloquine resemble those of other known psychotomimetics. Using in vitro radioligand binding and functional assays, we examined the interaction of (+)- and (-)-mefloquine enantiomers, the non-psychotomimetic anti-malarial agent, chloroquine, and several hallucinogens and psychostimulants with recombinant human neurotransmitter receptors and transporters. Hallucinogens and mefloquine bound stereoselectively and with relatively high affinity (K i = 0.71-341 nM) to serotonin (5-HT) 2A but not 5-HT1A or 5-HT2C receptors. Mefloquine but not chloroquine was a partial 5-HT2A agonist and a full 5-HT2C agonist, stimulating inositol phosphate accumulation, with similar potency and efficacy as the hallucinogen dimethyltryptamine (DMT). 5-HT receptor antagonists blocked mefloquine's effects. Mefloquine had low or no affinity for dopamine D1, D2, D3, and D4.4 receptors, or dopamine and norepinephrine transporters. However, mefloquine was a very low potency antagonist at the D3 receptor and mefloquine but not chloroquine or hallucinogens blocked [(3)H]5-HT uptake by the 5-HT transporter. Mefloquine, but not chloroquine, shares an in vitro receptor interaction profile with some hallucinogens and this neurochemistry may be relevant to the adverse neuropsychiatric effects associated with mefloquine use by a small percentage of patients. Additionally, evaluating interactions with this panel of receptors and transporters may be useful for characterizing effects of other psychotropic drugs and for avoiding psychotomimetic effects for new pharmacotherapies, including antimalarial quinolines.

δ-Opioid Receptors Stimulate the Metabolic Sensor AMP-Activated Protein Kinase through Coincident Signaling with Gq/11-Coupled Receptors

AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-D-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dominant-negative mutant of AMPKα1, and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems, DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with G(q/11)-coupled receptors, potentiating Ca(2+) release and CaMKKβ-dependent AMPK phosphorylation. In CHO cells, this coincident signaling mechanism is involved in DOR-induced glucose uptake.

Identification and characterization of mesotocin and V1a-like vasotocin receptors in a urodele amphibian, Taricha granulosa

The cDNA sequences encoding the mesotocin receptor (MTR) and vasotocin 1a receptor (VTR-1a) were identified in a urodele amphibian, the rough-skinned newt, Taricha granulosa. Saturation binding of [ 3H]oxytocin (OT) to the Taricha MTR (tMTR) was best fit by a two-state model; a high affinity-low abundance site and a lower affinity-high abundance site. Competition-binding studies found the following rank-order affinities for the tMTR: mesotocin (MT) > OT ≈ vasotocin (VT) > vasopressin (VP) > isotocin (IT). Inositol phosphate (IP) accumulation studies demonstrated functional activity of both the tMTR and Taricha VTR-1a (tVTR-1a) in a heterologous cell culture system. The rank-order potencies for the tMTR were MT > OT > VT ≈ VP > IT. The combined binding and IP results indicate that VT may act as a partial agonist of the tMTR. Rank-order potencies for the tVTR-1a were VT > VP > MT ≈ OT > IT. For both receptors, stimulation of IP accumulation was blocked by d(CH 2) 5[Tyr(Me) 2]AVP (Manning compound) and d(CH 2) 5[Tyr(Me) 2,Thr 4,Tyr-NH 2]OVT (OTA). OTA was a more potent antagonist for the transiently expressed tMTR while Manning compound was relatively more potent at inhibiting IP accumulation in tVTR-1a expressing cells. In contradiction to earlier assumptions, the absolute IC 50 of Manning compound was lower for the tMTR (27 nM ± 13) than the tVTR-1a (586 nM ± 166) indicating its potential higher affinity for the tMTR, a finding with special relevance to interpretation of comparative studies investigating the behavioral and physiological actions of neurohypophysial peptides in non-mammalian species.

Action of Pasteurella multocida toxin on Gαq is persistent and independent of interaction with G-protein-coupled receptors

Pasteurella multocida toxin (PMT) activates Gαq and facilitates stimulation of inositol phosphate accumulation induced by agonists via Gq-coupled membrane receptors. Here, we studied the effects of PMT on agonist-induced GTPγS binding to Gq in cell membranes and a role of G-protein-coupled receptors in the action of PMT. Pre-treatment of Swiss 3T3 cells with PMT increased bombesin or vasopressin-induced GTPγS-binding in cell membranes by about 50 to 150%. Increase in agonist-stimulated GTPγS-binding caused by PMT pretreatment was specific for Gαq and not observed with Gα11. PMT-induced effects on GTPγS-binding were persistent after removing the toxin or in the presence of anti-PMT antibody. Stimulation of agonist-induced GTPγS-binding by PMT was independent of phosphorylation of the C-terminal tyrosine356 of Gαq. Activation of phospholipase C by PMT occurred via Gαq which was fused to the α1b-adrenoceptor and also with a C-terminally deleted Gαq, which is not able to interact with G protein-coupled membrane receptors. The data indicate that activation of Gαq by PMT is persistent and independent of a functional interaction of Gq with G-protein-coupled receptors.

The calcium-sensing receptor (CaR) is involved in strontium ranelate-induced osteoblast proliferation

Strontium ranelate has several beneficial effects on bone and reduces the risk of vertebral and hip fractures in women with postmenopausal osteoporosis. We investigated whether Sr2+ acts via a cell surface calcium-sensing receptor (CaR) in HEK293 cells stably transfected with the bovine CaR (HEK-CaR) and rat primary osteoblasts (POBs) expressing the CaR endogenously. Elevating Cao2+ or Sr2+ concentration-dependently activated the CaR in HEK-CaR but not in non-transfected cells, but the potency of Sr2+ varied depending on the biological response tested. Sr2+ was less potent than Cao2+ in stimulating inositol phosphate accumulation and in increasing Cai2+, but was comparable to Cao2+ in stimulating ERK phosphorylation and a non-selective cation channel, suggesting that Ca2+ and Sr2+ have differential effects on specific cellular processes. With physiological concentrations of Cao2+, Sr2+-induced further CaR activation. Neither Sr2+ nor Cao2+ affected the four parameters just described in non-transfected cells. In POB, Sr2+ stimulated cellular proliferation. This effect was CaR-mediated, as transfecting the cells with a dominant negative bovine CaR significantly attenuated Cao2+-stimulated POB proliferation. Finally, Sr2+ significantly increased the mRNA levels of the immediate early genes, c-fos and egr-1, which are involved in POB proliferation, and this effect was attenuated by overexpressing the dominant negative CaR. In conclusion, Sr2+ is a full CaR agonist in HEK-CaR and POB, and, therefore, the anabolic effect of Sr2+ on bone in vivo could be mediated, in part, by the CaR.

G Protein-coupled Receptor (GPCR) Kinase 2 Regulates Agonist-independent Gq/11 Signaling from the Mouse Cytomegalovirus GPCR M33

The mouse cytomegalovirus M33 protein is highly homologous to mammalian G protein-coupled receptors (GPCRs) yet functions in an agonist-independent manner to activate a number of classical GPCR signal transduction pathways. M33 is functionally similar to the human cytomegalovirus-encoded US28 GPCR in its ability to induce inositol phosphate accumulation, activate NF-kappaB, and promote smooth muscle cell migration. This ability to promote cellular migration suggests a role for viral GPCRs like M33 in viral dissemination in vivo, and accordingly, M33 is required for efficient murine cytomegalovirus replication in the mouse. Although previous studies have identified several M33-induced signaling pathways, little is known regarding the membrane-proximal events involved in signaling and regulation of this receptor. In this study, we used recombinant retroviruses to express M33 in wild-type and Galpha(q/11)(-/-) mouse embryonic fibroblasts and show that M33 couples directly to the G(q/11) signaling pathway to induce high levels of total inositol phosphates in an agonist-independent manner. Our data also show that GRK2 is a potent regulator of M33-induced G(q/11) signaling through its ability to phosphorylate M33 and sequester Galpha(q/11) proteins. Taken together, the results from this study provide the first genetic evidence of a viral GPCR coupling to a specific G protein signaling pathway as well as identify the first viral GPCR to be regulated specifically by both the catalytic activity of the GRK2 kinase domain and the Galpha(q/11) binding activity of the GRK2 RH domain.

Muscarinic M2Receptors Directly Activate Gq/11and GsG-Proteins

Muscarinic M(2) receptors preferentially couple with the G(i/o) class of G-proteins to inhibit cAMP synthesis. However, they can also stimulate net synthesis of cAMP and inositol phosphate (IP) accumulation. We investigated in intact Chinese hamster ovary (CHO) cells expressing human M(2) receptors (CHO-M(2) cells) whether direct interaction of M(2) receptors with G(s) and G(q/11) G-proteins is responsible for the latter effects. Suppression of the G(s)alpha subunit using RNA interference abolished stimulation of cAMP synthesis induced by 1 mM carbachol in both control and pertussis toxin-treated CHO-M(2) cells but had no effect on the inhibition of forskolin-stimulated cAMP synthesis. Carbachol stimulated accumulation of IP with an EC(50) of 79 microM. Removal of the G(q),G(11), or both alpha subunits reduced this response by 78, 54, and 92%, respectively, whereas suppression of the G(s)alpha subunit had no effect. Similar results obtained in CHO cells expressing M(1) receptors that preferentially couple with G(s) and G(q/11) G-proteins confirmed the efficiency of siRNA treatments. Stimulation of M(2) receptors in control and pertussis toxin-treated cells by a series of full agonists with respect to inhibition of adenylyl cyclase displayed different efficacies in stimulating IP accumulation. Carbachol, acetylcholine, and oxotremorine-M [N,N,N-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium] behaved as full agonists, furmethide (N,N,N-trimethyl-2-furanmethammonium) and methylfurmethide [(5-methyl-2-furyl)methyltrimethylammonium] were partial agonists, and oxotremorine (1-[4-(1-pyrrolidinyl)-2-butynyl]-2-pyrrolidinone) had no effect. Our results provide direct evidence of M(2) receptor coupling with the alpha subunits of G(s) and G(q/11) G-proteins and demonstrate induction of multiple receptor conformational states dependent on both the concentration and the nature of the agonist used.

Gα12/13- and Rho-Dependent Activation of Phospholipase C-ϵ by Lysophosphatidic Acid and Thrombin Receptors

Because phospholipase C epsilon (PLC-epsilon) is activated by Galpha(12/13) and Rho family GTPases, we investigated whether these G proteins contribute to the increased inositol lipid hydrolysis observed in COS-7 cells after activation of certain G protein-coupled receptors. Stimulation of inositol lipid hydrolysis by endogenous lysophosphatidic acid (LPA) or thrombin receptors was markedly enhanced by the expression of PLC-epsilon. Expression of the LPA(1) or PAR1 receptor increased inositol phosphate production in response to LPA or SFLLRN, respectively, and these agonist-stimulated responses were markedly enhanced by coexpression of PLC-epsilon. Both LPA(1) and PAR1 receptor-mediated activation of PLC-epsilon was inhibited by coexpression of the regulator of G protein signaling (RGS) domain of p115RhoGEF, a GTPase-activating protein for Galpha(12/13) but not by expression of the RGS domain of GRK2, which inhibits Galpha(q) signaling. In contrast, activation of the G(q)-coupled M1 muscarinic or P2Y(2) purinergic receptor was neither enhanced by coexpression with PLC-epsilon nor inhibited by the RGS domain of p115RhoGEF but was blocked by expression of the RGS domain of GRK2. Expression of the Rho inhibitor C3 botulinum toxin did not affect LPA- or SFLLRN-stimulated inositol lipid hydrolysis in the absence of PLC-epsilon but completely prevented the PLC-epsilon-dependent increase in inositol phosphate accumulation. Likewise, C3 toxin blocked the PLC-epsilon-dependent stimulatory effects of the LPA(1), LPA(2), LPA(3), or PAR1 receptor but had no effect on the agonist-promoted inositol phosphate response of the M1 or P2Y(2) receptor. Moreover, PLC-epsilon-dependent stimulation of inositol phosphate accumulation by activation of the epidermal growth factor receptor, which involves Ras- but not Rho-mediated activation of the phospholipase, was unaffected by C3 toxin. These studies illustrate that specific LPA and thrombin receptors promote inositol lipid signaling via activation of Galpha(12/13) and Rho.

Probing the Molecular Mechanism of Interaction between 4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the Muscarinic M1Receptor: Direct Pharmacological Evidence That AC-42 Is an Allosteric Agonist

4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M(1) receptor previously suggested to interact with an "ectopic" site on this receptor. However, the pharmacological properties of this site (i.e., whether it overlaps to any extent with the classic orthosteric site or represents a novel allosteric site) remain undetermined. In the present study, atropine or pirenzepine significantly inhibited the ability of either carbachol or AC-42 to stimulate inositol phosphate accumulation or intracellular calcium mobilization in Chinese hamster ovary (CHO) cells stably expressing the human M(1) receptor. However, the interaction between either of these antagonists and AC-42 was characterized by Schild slopes significantly less than unity. Increasing the concentrations of atropine revealed that the Schild regression was curvilinear, consistent with a negative allosteric interaction. More direct evidence for an allosteric mode of action of AC-42 was obtained in [(3)H]N-methylscopolamine ([(3)H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [(3)H]NMS binding in a manner that was quantitatively described by an allosteric model applied to both modulator data sets. Furthermore, AC-42 and gallamine significantly retarded the rate of [(3)H]NMS dissociation from CHO-hM(1) cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M(1) receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M(1) receptors in the absence of the orthosteric agonist.

Expression of the Third Intracellular Loop of the δ-Opioid Receptor Inhibits Signaling by Opioid Receptors and Other G Protein-Coupled Receptors

To explore the feasibility of developing inhibitors of signaling by opioid receptors and other G protein-coupled receptors (GPCRs) that use the same G protein pool, we investigated the capacity of a minigene encoding the third intracellular loop of the delta-opioid receptor (delta-i3L) to act as competitive antagonist of the receptor-G protein interface interaction. In delta-i3L-expressing cells, the peptide blocked high-affinity agonist binding to both the delta- and the mu-opioid (delta-OR and mu-OR) and attenuated opioid and alpha2-adrenergic receptor (alpha2AR)-dependent [35S]guanosine-5'-O-(3-thio)triphosphate binding. Furthermore, delta-i3L expression resulted in inhibition of delta-, mu-OR-, and alpha2AR-receptor-mediated cAMP accumulation, whereas the cAMP response produced by activation of the beta2-adrenergic receptor was unaffected, suggesting that the inhibitory effects of delta-i3L expression were selective for Gi/Go proteins. Moreover, although delta-i3L expression also attenuated drastically phospholipase C accumulation and Ca2+ release following mu- and delta-OR stimulation, it failed to inhibit carbachol-mediated stimulation of inositol phosphate accumulation in M1-muscarinic receptor-expressing human embryonic kidney 293 cells. Finally, we also examined the effects of delta-i3L expression on the regulation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway. Our results demonstrate that, although ERK activation by mu- and delta-ORs is attenuated by the presence of delta-i3L, ERK activation mediated by alpha2AR remained unaffected. Collectively, our data demonstrate that the delta-i3L can be used as potent inhibitor of G protein signaling for various GPCRs that use a common pool of G proteins.

Addition of a signal peptide sequence to the α1D‐adrenoceptor gene increases the density of receptors, as determined by [3H]‐prazosin binding in the membranes

1. Both in mammalian tissues and in transfected cells, only low levels of alpha1D-adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of the alpha1D-adrenoceptor is responsible for the inefficient surface expression of the receptor. 2. In the present study, we created gene constructs for six N-terminally truncated variants of the human alpha1D-adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density of alpha1D-adrenoceptors, observed by [3H]-prazosin binding, gradually increased with longer truncations of the N-terminus. This seems to indicate that the long N-terminal tail nonspecifically interferes with receptor translocation to the plasma membrane. 3. The addition of a 16 amino acids long signal peptide to the N-terminus of the wild-type alpha1D-adrenoceptor increased the density of receptor binding sites 10-fold in Neuro2A and COS-7 cells. This indicates that, after the addition of a signal peptide, the long N-terminal tail of the alpha1D-adrenoceptor does not interfere with proper translocation of the receptor to the plasma membrane. This, in turn, indicates that the N-terminal tail of the wild-type alpha1D-adrenoceptor, merely by its long length, hinders the first transmembrane helix of the receptor from being a signal anchor. 4. Neither the wild-type alpha1D-adrenoceptor (for which the expression level of [3H]-prazosin binding sites is low) nor the truncated alpha1D-adrenoceptor variant (for which the expression level of [3H]-prazosin binding sites is high) showed any constitutive activity in stimulating inositol phosphate accumulation. This indicates that the low expression level of [3H]-prazosin binding sites, after transfection with the wild-type alpha1D-adrenoceptor, is not caused by constitutive activity of the receptor and subsequent receptor downregulation.

Effects of (±)-huprine Y and (±)-huprine Z, two new anticholinesterasic drugs, on muscarinic receptors

The cholinergic profile of (±)-huprine Y and (±)-huprine Z on muscarinic receptors has been determined. Displacement of [ 3H]-pirenzepine and [ 3H]-QNB plus pirenzepine was performed in rat hippocampus. Both compounds showed a higher degree of affinity to M 1 muscarinic receptors ( P < 0.01) than to M 2 muscarinic receptors. To determine the M 1 agonist or antagonist role of the two huprines, studies of inositol phosphates (IP) production were performed. Both huprines significantly stimulated IP accumulation in a concentration-dependent manner. The reversion of this effect by different antagonists showed that M 1 muscarinic receptors were activated by (±)-huprine Y and (±)-huprine Z, but some other mechanisms, such as α 1-adrenoceptors or nicotinic receptors, were involved.

Quantitative analysis of the formation and diffusion of A1-adenosine receptor-antagonist complexes in single living cells.

The A1-adenosine receptor (A1-AR) is a G protein-coupled receptor that mediates many of the physiological effects of adenosine in the brain, heart, kidney, and adipocytes. Currently, ligand interactions with the A1-AR can be quantified on large cell populations only by using radioligand binding. To increase the resolution of these measurements, we have designed and characterized a previously undescribed fluorescent antagonist for the A1-AR, XAC-BY630, based on xanthine amine congener (XAC). This compound has been used to quantify ligand-receptor binding at a single cell level using fluorescence correlation spectroscopy (FCS). XAC-BY630 was a competitive antagonist of A1-AR-mediated inhibition of cAMP accumulation [log10 of the affinity constant (pKb) = 6.7)] and stimulation of inositol phosphate accumulation (pKb = 6.5). Specific binding of XAC-BY630 to cell surface A1-AR could also be visualized in living Chinese hamster ovary (CHO)-A1 cells by using confocal microscopy. FCS analysis of XAC-BY630 binding to the membrane of CHO-A1 cells revealed three components with diffusion times (tauD) of 62 micros (tauD1, free ligand), 17 ms (tauD2, A1-AR-ligand), and 320 ms (tauD3). Confirmation that tauD2 resulted from diffusion of ligand-receptor complexes came from the similar diffusion time observed for the fluorescent A1-AR-Topaz fusion protein (15 ms). Quantification of tauD2 showed that the number of receptor-ligand complexes increased with increasing free ligand concentration and was decreased by the selective A1-AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine. The combination of FCS with XAC-BY630 will be a powerful tool for the characterization of ligand-A1-AR interactions in single living cells in health and disease.

Direct Activation of Phospholipase C-ϵ by Rho

Unique among the phospholipase C isozymes, the recently identified phospholipase C-epsilon (PLC-epsilon) contains an amino-terminal CDC25 domain capable of catalyzing nucleotide exchange on Ras family GTPases as well as a tandem array of Ras-associating (RA) domains near its carboxyl terminus that are effector binding sites for activated H-Ras and Rap. To determine whether other small GTPases activate PLC-epsilon, we measured inositol phosphate accumulation in COS-7 cells expressing a broad range of GTPase-deficient mutants of Ras superfamily proteins. RhoA, RhoB, and RhoC all markedly stimulated inositol phosphate accumulation in PLC-epsilon-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-epsilon with the known regulators Ras, Galpha12/13, or Gbeta1gamma2. In contrast, little effect was observed with the other Rho family members Rac1, Rac2, Rac3, and Cdc42. Truncation of the two carboxyl-terminal RA domains caused loss of responsiveness to H-Ras but not to Rho. Truncation of PLC-epsilon to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Galpha12/13, or Gbeta1gamma2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique approximately 65 amino acid insert within the catalytic core of PLC-epsilon not present in PLC-beta, gamma, delta, or zeta. A PLC-epsilon construct lacking this region was no longer activated by Rho or Galpha12/13 but retained regulation by Gbetagamma and H-Ras. GTP-dependent interaction of Rho with PLC-epsilon was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-epsilon construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-epsilon by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-epsilon imparts responsiveness to Rho, which may signal downstream of Galpha12/13 in the regulation of PLC-epsilon, because activation by both Rho and Galpha12/13 is lost in the absence of this sequence.

A fusion protein of the human P2Y(1) receptor and NTPDase1 exhibits functional activities of the native receptor and ectoenzyme and reduced signaling responses to endogenously released nucleotides.

To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y(1) receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y(1) receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1:0.4 for both the wild-type NTPDase1 and P2Y(1)-NTPDase1 fusion protein. Stable expression of the P2Y(1)-NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca(2+) response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPbetaS to stimulate inositol phosphate accumulation was similar, and the EC(50) of ADPbetaS was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y(1) and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y(1) receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y(1) receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y(1) receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.

Involvement of 5-HT1B and 5-HT1D receptors in sumatriptan mediated vasocontractile response in rabbit common carotid artery.

1. In this study we examined the involvement of 5-HT(1B) and 5-HT(1D) receptors in the vasocontractile response induced by 5-HT(1B/D)-receptor agonist sumatriptan in rabbit common carotid artery (CCA). 2. Immunoblotting experiments using specific antisera against 5-HT(1B) or 5-HT(1D) receptors revealed the presence of one weak (at 93 kD for 5-HT(1B) or at 105 kD for 5-HT(1D)) and one strong band (at 46 kD for 5-HT(1B) or at 52 kD for 5-HT(1D)) in CCA. 3. Sumatriptan-mediated vasocontractile response was antagonized by SB216641 with an apparent pKb value of 8.6, which was consistent with its affinity for 5-HT(1B) receptor. Antagonism by BRL15572 was weak and calculated apparent pKb (6.0) value was consistent with its affinity for 5-HT(1B) subtype (but not for 5-HT(1D) subtype). This result indicates insignificant or no involvement of 5-HT(1D) receptor in the vasocontractile response. 4. The vasocontractile response induced by sumatriptan was highly sensitive to pertussis toxin treatment of CCA. Nicardipine, a calcium channel blocker, also potently antagonized vasocontractile response induced by sumatriptan. 5. 5-HT, but not sumatriptan, stimulated inositol phosphate accumulation in CCA. 6. These results indicate that stimulation of 5-HT(1B) subtype activate a pertussis toxin (PTX) sensitive G protein (Go/Gi) and mediate vasocontraction, in which L-type voltage dependent calcium channels are involved.

Pharmacological characterization of F-180: a selective human V(1a) vasopressin receptor agonist of high affinity.

1. The pharmacological properties of F-180, a vasopressin (VP) structural analogue, were determined on CHO cells expressing the different human vasopressin and oxytocin (OT) receptor subtypes. Binding experiments revealed that F-180 exhibited a high affinity for the human V(1a) receptor subtype (K(i)=11 nM) and was selective for this receptor subtype. 2. Functional studies performed on CHO cells expressing human V(1a) receptors indicate that similarly to AVP, F-180 can stimulate the accumulation of inositol phosphate. The activation constant (K(act)) for both F-180 and AVP was 1.7 nM. F-180 was also an agonist for the human V(2) and V(1b) receptor subtypes and an antagonist for the human OT receptor. 3. Since marked species pharmacological differences for vasopressin receptors have been described, we studied the properties of F-180 on various mammalian species. F-180 showed high affinity and good selectivity for human and bovine V(1a) receptors, but weak affinity and non selective properties for rat V(1a) receptors. 4. To assess the functional properties of F-180 on a native biological model, we performed studies on primary cultures of cells from bovine zona fasciculata (ZF). As AVP, F-180 stimulated inositol phosphate accumulation and cortisol secretion with similar efficiency. 5. In conclusion, we demonstrate that F-180 is the first selective V(1a) agonist described for human and bovine vasopressin receptors. Therefore F-180 can be used as a powerful pharmacological tool to characterize the actions of vasopressin that are mediated by V(1a) receptor subtypes.

Serine 232 of the alpha(2A)-adrenergic receptor is a protein kinase C-sensitive effector coupling switch.

alpha(2)-adrenergic receptors (alpha(2)AR) couple to multiple effectors including adenylyl cyclase and phospholipase C. We hypothesized that signaling selectivity to these effectors is dynamically directed by kinase-sensitive domains within the third intracellular loop of the receptor. Substitution of Ala for Ser232, which is in the N-terminal region of this loop in the alpha(2A)AR, resulted in a receptor that was markedly uncoupled ( approximately 82% impairment) from stimulation of inositol phosphate accumulation while the capacity to inhibit adenylyl cyclase remained relatively intact. In S232A alpha(2A)AR transfected cell membranes, agonist-promoted [(35)S]GTPgammaS binding was reduced by approximately 50%. Coexpression of modified G proteins rendered insensitive to pertussis toxin revealed that the S232A receptor was uncoupled from both G(i) and G(o). S232 is a potential PKC phosphorylation site, and whole cell phosphorylation studies showed that the mutant had depressed phosphorylation compared to wild type (1.3- vs 2.1-fold/basal). Consistent with S232 directing coupling to phospholipase C, PMA exposure resulted in approximately 67% desensitization of agonist-promoted inositol phosphate accumulation without significantly affecting inhibition of adenylyl cyclase. The dominant effect of mutation or phosphorylation at this site on inositol phosphate as compared to cAMP signaling was found to most likely be due to the low efficiency of signal transduction via phospholipase C vs adenylyl cyclase. Taken together, these results indicate that S232 acts as a selective, PKC-sensitive, modulator of effector coupling of the alpha(2A)AR to inositol phosphate stimulation. This represents one mechanism by which cells route stimuli directed to multifunctional receptors to selected effectors so as to attain finely targeted signaling.

Two novel mutations in the gonadotropin-releasing hormone receptor gene in Brazilian patients with hypogonadotropic hypogonadism and normal olfaction.

Several point mutations in the GnRH receptor gene have been described in an autosomal recessive form of congenital isolated hypogonadotropic hypogonadism (HH). We investigated 17 Brazilian patients (10 males and 7 females) from 14 different families, with HH and normal olfaction. The diagnosis of HH was based on absent or incomplete sexual development after 17 yr of age associated with low or normal levels of LH in both sexes and low levels of testosterone in males and of estradiol in females. All patients presented with a normal sense of smell in an olfactory specific test. The coding region of the GnRH receptor gene was amplified by PCR and directly sequenced. A novel missense mutation, Arg(139)His, located in the conserved DRS motif at the junction of the third transmembrane and the second intracellular loop of the GnRH receptor was identified in the homozygous state in one female with complete HH. The Arg(139)His mutation completely eliminated detectable GnRH-binding activity and prevented GnRH-induced stimulation of inositol phosphate accumulation in vitro. In another family, a new compound heterozygous mutation (Asn(10)Lys and Gln(106)Arg) was identified in four siblings (two males and two females) with partial HH. The Gln(106)Arg mutation, located in the first extracellular loop, has been previously described, and in vitro analysis indicated that the mutant receptor was able to bind GnRH, but with a reduced affinity. The Asn(10)Lys mutation in the extracellular amino-terminal domain of the receptor also reduced the affinity for GnRH in vitro. In this family we also identified a previously described silent polymorphism at amino acid residue 151 in the second intracellular loop that segregated with the two inactivating mutations of the GnRH receptor. This polymorphism was also found in two unrelated patients with sporadic HH without GnRH receptor loss of function mutations. No mutations were identified in the remaining cases. A good correlation between genotype and phenotype was found in our patients. The woman, who is homozygous for the completely inactivating Arg(139)His mutation, has complete HH with undetectable serum basal LH and FSH levels that failed to respond to GnRH stimulation. In addition, the affected patients who are compound heterozygotes for the Asn(10)Lys/Gln(106)Arg mutations, have partial HH with low serum basal LH levels that were responsive to GnRH stimulation. No clinical or hormonal differences were found between HH patients with and without mutations in the GnRH receptor gene, indicating that these data do not contribute to the identification of HH patients with GnRH receptor mutations. In conclusion, we report the first naturally occurring mutation within the conserved DRS motif of the GnRH receptor in a female with complete HH and a novel compound heterozygous mutation (Asn(10)Lys and Gln(106)Arg) in a family with partial HH, increasing the repertoire of the inactivating mutations of the GnRH receptor.