Bradykinin binding to B2 kinin receptors and stimulation of phosphoinositide turnover and arachidonic acid release in primary cultures of cells from late pregnant rat myometrium.

Primary cultures of cells from late pregnant rat myometrium contain B2 kinin receptors through which bradykinin (BK) stimulates inositol phosphate (InsP) formation and arachidonic acid (20:4) release. Equilibrium binding at 4 degrees C revealed that [3H]BK identified a maximal number of cell surface B2 kinin receptor binding sites on rat myometrial cells of 308 +/- 78 fmol/10(6) cells with apparently a single equilibrium dissociation constant of 1.8 +/- 0.2 nM. At 37 degrees C, [3H]BK binding was associated with a time-dependent decrease in the reversibility of the binding. This decrease was due in part to formation of slowly dissociating cell surface receptor [3H]BK binding and in part to internalization of the receptor-bound [3H]BK. Exposure of labeled cells to BK resulted in dose-dependent increases in [3H]InsP3, [3H]InsP2 ([3H]Ins(1,4)P2), and [3H]InsP1 ([3H]Ins(1)P1) formation and [3H]20:4 release. Pretreatment with 100 ng/mL pertussis toxin did not perturb BK stimulation of [3H]InsP formation but partially (approximately 30%) inhibited BK stimulation of [3H]20:4 release. BK stimulation of [3H]20:4 release was directly proportional to the number of receptor sites occupied by BK. In contrast, stimulation of [3H]InsP formation required a threshold level of receptor occupancy, which decreased as a function of time of BK exposure. These results show that BK interacts with B2 kinin receptors on rat myometrial cells with apparently a single affinity through which BK stimulates [3H]InsP formation and [3H]20:4 release. BK stimulation of [3H]InsP formation requires a threshold BK concentration, which decreases with time, and we suggest that the decrease is due to a time-dependent formation of a BK receptor binding state from which BK slowly dissociates.     

Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5.

We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.     

Bradykinin-induced internalization of the human B2 receptor requires phosphorylation of three serine and two threonine residues at its carboxyl tail

The binding of bradykinin (BK) to B2 receptor triggers the internalization of the agonist-receptor complex. To investigate the mechanisms and the receptor structures involved in this fundamental process of receptor regulation, the human B2 receptor was mutated within its cytoplasmic tail by complementary strategies of truncation, deletion, and amino acid substitution. Ligand binding, signal transduction, internalization as well as phosphorylation were studied for the mutated receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor's cytoplasmic tail corresponding to positions 321-364 did not alter the kinetics of BK binding and the receptor coupling to phospholipase C and phospholipase A2. By contrast, truncations after positions 320 and 334, deletions within the segment covering positions 335-351, as well as alanine substitution of serine and threonine residues within segment 335-351 diminished the internalization capacity of the mutant receptors. Mutants with a markedly reduced internalization potential failed to produce BK-induced receptor phosphorylation suggesting that phosphorylation may be involved in receptor internalization. The mutagenesis approaches converged at the conclusion that three serines in positions 339, 346, and 348 and two threonines in positions 342 and 345, contained in a sequence segment that is highly conserved between species, have a critical role in the ligand-dependent internalization and phosphorylation of kinin receptors and can intervene in these processes in an alternative manner. However, mutants lacking these residues were still sensitive to dominant-negative forms of beta-arrestin and dynamin, suggesting the existence of additional receptor structure(s) involved in the receptor sequestration through clathrin-coated vesicles.     

Quantification of G-Protein Coupled Receptor Internatilization Using G-Protein Coupled Receptor-Green Fluorescent Protein Conjugates with the ArrayScantrade mark High-Content Screening System

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologous desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intracellular endosomal compartment. In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method for the identification of receptor mimetics. A series of studies were undertaken to evaluate the expression, functionality, and ligand-dependent trafficking of GPCR-green fluorescent protein (GFP) fusion conjugates stably transfected into HEK 293 cells. These GPCR-GFP expressing cells were then utilized in the validation of the ArrayScantrade mark (Cellomicstrade mark, Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular quantitation of fluorescence in whole cells. These studies demonstrated our ability to measure the internalization of a parathyroid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH. Similar results were obtained for a beta(2)-adrenergic receptor (beta(2) AR)-GFP conjugate stably expressed in HEK 293 cells. The internalized GFP-labeled receptors were visualized as numerous punctate 3spots2 within the cell interior. An algorithm has been developed that identifies and collects information about these spots, allowing quantification of the internalization process. Variables such as the receptor-GFP expression level, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development of this assay. The method represents a valuable tool to screen for receptor mimetics and antagonists of receptor internalization in whole cells rapidly.     

Kallikrein activates bradykinin B2 receptors in absence of kininogen

Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the cultured medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected Chinese hamster ovary (CHO) cells that express human B2 receptors (CHO B2) and cells that coexpress angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1-10 nM) both significantly increased release of arachidonic acid beyond unstimulated baseline level. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. We next tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analog as a control for these experiments. Enalaprilat (1 microM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B2 cells that do not bear ACE. We concluded that kallikrein activated B2 receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors.     

Inhibition of sequestration of human B2 bradykinin receptor by phenylarsine oxide or sucrose allows determination of a receptor affinity shift and ligand dissociation in intact cells

Depending on their interaction with intracellular proteins, G protein-coupled receptors (GPCR) often display different affinities for agonists at 37 degrees C. Determining the affinity at that temperature is often difficult in intact cells as most GPCRs are internalized after activation. When sequestration of the B2 bradykinin receptor (B2R) was inhibited by either 0.5 M sucrose or phenylarsine oxide (PAO), a shift in the affinity was detected when the incubation temperature was raised from 4 degrees C to 37 degrees C or lowered from 37 degrees C to 4 degrees C. In contrast, binding of the antagonist [3H]NPC 17731 was temperature-independent. B2R mutants displayed different affinity shifts allowing conclusions on the role of the involved amino acids. By inhibiting receptor sequestration it was possible to determine also dissociation of [3H]BK and of [3H]NPC 17731 from intact cells at 37 degrees C. Surprisingly, both dissociation rates were markedly enhanced by the addition of unlabeled ligand, most likely via prevention of reassociation of dissociated [3H]ligand. This suggests that dissociated [3H]ligand cannot move freely away from the receptor. In summary, our data demonstrate that inhibition of receptor internalization either by PAO or sucrose provides an excellent method to study receptor function and the effects of mutations in intact cells.     

Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers

Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ET(B)-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ET(A) receptor and CFP (ET(A)myc.CFP) and a fusion protein consisting of an ET(B) receptor and YFP (ET(B).YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ET(B) and myc-tagged ET(A) receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ET(A)myc.CFP and ET(B).YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ET(B) receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A.dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ET(A)myc.CFP/ET(B)flag.YFP receptors revealed a slower sequestration of the ET(B)flag.YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ET(B)flag.YFP receptors alone. The data suggest that ET(A) and ET(B) receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ET(B)-selective stimulation.     

beta-arrestins regulate interleukin-8-induced CXCR1 internalization.

The functional role of neutrophils during acute inflammatory responses is regulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) that are rapidly desensitized and internalized upon binding their cognate chemokine ligands. The efficient re-expression of CXCR1 on the surface of neutrophils following agonist-induced internalization suggests that CXCR1 surface receptor turnover may involve regulatory pathways and intracellular factors similar to those regulating beta2-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligand-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two different cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimulation promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalization in HEK 293 cells required co-expression of G protein-coupled receptor kinase 2 and beta-arrestin proteins. The importance of beta-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative beta-arrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalization in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Agonist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin-coated vesicles. Our data provides a direct link between agonist-induced internalization of CXCR1 and a requirement for G protein-coupled receptor kinase 2, beta-arrestins, and dynamin during this process.     

Agonist-induced internalization and recycling of the human A(3) adenosine receptors: role in receptor desensitization and resensitization

A(3) adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen-dependent nature of the therapeutic effects probably related to receptor desensitization and down-regulation. Here we studied the agonist-induced internalization of human A(3) adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine-5'-N-methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A(3) adenosine receptor showed a profile typical of these receptors in other cell lines (K:(D) = 1.3+/-0.08 nM; B(max) = 400+/-28 fmol/mg of proteins). The iodinated agonist, bound at 4 degrees C to whole transfected cells, was internalized by increasing the temperature to 37 degrees C with a rate constant of 0.04+/-0.034 min(-1). Agonist-induced internalization of A(3) adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short-term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02+/-0.0017 min(-1). Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.     

Cloning and functional expression of the hNPY Y5 receptor in human endometrial cancer (HEC-1B) cells

Aiming to develop a functional assay for the human NPY Y5 receptor based on adenylyl cyclase activity, HEC-1B cells, in which cAMP synthesis can be efficiently stimulated with forskolin, were selected for the transfection with the pcDNA3-Y5-FLAG and the pcDEF3-Y5 vectors. After optimization of the transfection procedure, the binding of [3H]propionyl-NPY to transiently and stably expressed Y5 receptors was determined. The affinities of NPY, NPY derivatives, and rPP (pNPY > or = p(Leu31Pro34)NPY = p(2-36)NPY > or = p(D-Trp32)NPY > p(13-36)NPY > rPP) were in accordance with the NPY Y5 receptor subtype. For [3H]propionyl-pNPY approximately 1.7 x 10(5) and 1 x 10(6) binding sites per transiently and stably transfected cell, respectively, were determined. The KD values were 2.4 +/- 0.4 and 1.7 +/-0.2 nM, respectively. Due to the high expression of the receptor protein, both stably and transiently transfected cells can be conveniently used in routine radioligand binding studies. By contrast, functional assays were only feasible with HEC-1B cells stably expressing the Y5 receptor. In these cells, 10 nM pNPY inhibited the forskolin-stimulated cAMP synthesis by 75%. This effect was partially antagonized by the Y5 antagonist N-?trans-[4-(2-naphthylmethylamino)-methyl]cyclohexylmethyl) naphthalene-2-sulfonamide. Although the genetic variability of cancer cells is in principle incompatible with a stable phenotype, both ligand binding characteristics and functionality of the Y5 receptor remained unchanged for more than 30 passages.     

Visualization of agonist-induced association and trafficking of green fluorescent protein-tagged forms of both beta-arrestin-1 and the thyrotropin-releasing hormone receptor-1.

A fusion protein (beta-arrestin-1-green fluorescent protein (GFP)) was constructed between beta-arrestin-1 and a modified form of the green fluorescent protein from Aequorea victoria. Expression in HEK293 cells allowed immunological detection of an 82-kDa cytosolic polypeptide with antisera to both beta-arrestin-1 and GFP. Transient expression of this construct in HEK293 cells stably transfected to express the rat thyrotropin-releasing hormone receptor-1 (TRHR-1) followed by confocal microscopy allowed its visualization evenly distributed throughout the cytoplasm. Addition of thyrotropin-releasing hormone (TRH) caused a profound and rapid redistribution of beta-arrestin-1-GFP to the plasma membrane followed by internalization of beta-arrestin-1-GFP into distinct, punctate, intracellular vesicles. TRH did not alter the cellular distribution of GFP transiently transfected into these cells nor the distribution of beta-arrestin-1-GFP following expression in HEK293 cells lacking the receptor. To detect potential co-localization of the receptor and beta-arrestin-1 in response to agonist treatment, beta-arrestin-1-GFP was expressed stably in HEK293 cells. A vesicular stomatitis virus (VSV)-tagged TRHR-1 was then introduced transiently. Initially, the two proteins were fully resolved. Short term exposure to TRH resulted in their plasma membrane co-localization, and sustained exposure to TRH resulted in their co-localization in punctate, intracellular vesicles. In contrast, beta-arrestin-1-GFP did not relocate or adopt a punctate appearance in cells that did not express VSV-TRHR-1. Reciprocal experiments were performed, with equivalent results, following transient expression of beta-arrestin-1 into cells stably expressing VSVTRHR-1-GFP. These results demonstrate the capacity of beta-arrestin-1-GFP to interact with the rat TRHR-1 and directly visualizes their recruitment from cytoplasm and plasma membrane respectively into overlapping, intracellular vesicles in an agonist-dependent manner.     

Mutation of tyrosine in the conserved NPXXY sequence leads to constitutive phosphorylation and internalization, but not signaling, of the human B2 bradykinin receptor

Although the G protein-coupled receptors (GPCRs) share a similar seven-transmembrane domain structure, only a limited number of amino acid residues is conserved in their protein sequences. One of the most highly conserved sequences is the NPXXY motif located at the cytosolic end of the transmembrane region-7 of many GPCRs, particularly of those belonging to the family of the rhodopsin/beta-adrenergic-like receptors. Exchange of Tyr(305) in the corresponding NPLVY sequence of the bradykinin B(2) receptor (B(2)R) for Ala resulted in a mutant, termed Y305A, that internalized [(3)H]bradykinin (BK) almost as rapidly as the wild-type (wt) B(2)R. However, receptor sequestration of the mutant after stimulation with BK was clearly reduced relative to the wt B(2)R. Confocal fluorescence microscopy revealed that, in contrast to the B(2)R-enhanced green fluorescent protein chimera, the Y305A-enhanced green fluorescent protein chimera was predominantly located intracellularly even in the absence of BK. Two-dimensional phosphopeptide analysis showed that the mutant Y305A constitutively exhibited a phosphorylation pattern similar to that of the BK-stimulated wt B(2)R. Ligand-independent Y305A internalization was demonstrated by the uptake of rhodamine-labeled antibodies directed to a tag sequence at the N terminus of the mutant receptor. Co-immunoprecipitation revealed that Y305A is precoupled to G(q/11) without activating the G protein because the basal accumulation rate of inositol phosphate was unchanged as compared with wt B(2)R. We conclude, therefore, that the Y305A mutation of B(2)R induces a receptor conformation which is prone to ligand-independent phosphorylation and internalization. The mutated receptor binds to, but does not activate, its cognate heterotrimeric G protein G(q/11), thereby limiting the extent of ligand-independent receptor internalization.     

Heterologous inhibition of G protein-coupled receptor endocytosis mediated by receptor-specific trafficking of beta-arrestins

We have observed an unexpected type of nonreciprocal "cross-regulation" of the agonist-induced endocytosis of G protein-coupled receptors by clathrin-coated pits. Isoproterenol-dependent internalization of beta2-adrenergic receptors in stably transfected HEK293 cells was specifically blocked (>65% inhibition) by vasopressin-induced activation of V2 vasopressin receptors co-expressed at similar levels. In contrast, activation of beta2 receptors caused no detectable effect on V2 receptor internalization in the same cells. Several pieces of evidence suggest that this nonreciprocal inhibition of endocytosis is mediated by receptor-specific intracellular trafficking of beta-arrestins. First, previous studies showed that the activation of V2 but not beta2 receptors caused pronounced recruitment of beta-arrestins to endocytic membranes (Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron, M. G. (1999) J. Biol. Chem. 274, 32248-32257). Second, overexpression of arrestin 2 or 3 (beta-arrestin 1 or 2) abolished the V2 receptor-mediated inhibition of beta2 receptor internalization. Third, mutations of the V2 receptor that block endomembrane recruitment of beta-arrestins eliminated the V2 receptor-dependent blockade of beta2 receptor internalization. These results identify a novel type of heterologous regulation of G protein-coupled receptors, define a new functional role of receptor-specific intracellular trafficking of beta-arrestins, and suggest an experimental method to rapidly modulate the functional activity of beta-arrestins in intact cells.     

Spontaneous human B2 bradykinin receptor activity determines the action of partial agonists as agonists or inverse agonists. Effect of basal desensitization.

In this report, we show that desensitization regulates ligand-independent, spontaneous activity of the human B2 bradykinin (BK) receptor, and the level of spontaneous receptor activity determines the action of the BK antagonists and partial receptor agonists NPC17731 and HOE140 as agonists or inverse agonists. Spontaneous receptor activity was monitored by measuring basal cellular phosphoinositide (PI) hydrolysis as a function of the density of the receptor in transiently transfected HEK293 cells. Minimal spontaneous activity of the wild-type B2 receptor was detected in these cells. Mutating a cluster of serines and threonines within the fourth intracellular domain of the receptor, which is critical for agonist-promoted desensitization, significantly increased the spontaneous receptor activity. BK, the natural B2 receptor ligand and, consequently, a full agonist, stimulated PI hydrolysis at high and low levels of spontaneous receptor activity. On the other hand, the partial agonists NPC17731 and HOE140 were stimulatory, or agonists, at the lower level of receptor activity but inhibitory, or inverse agonists, at the higher level of activity. These results show that receptors are desensitized in response to their spontaneous activity. Furthermore, these results, which refute traditional theories, show that the capacity of a drug to modulate a receptor response is not intrinsic to the drug but is also dependent on the cellular environment in which the drug acts.     

Involvement of mitogen-activated protein kinase in agonist-induced phosphorylation of the mu-opioid receptor in HEK 293 cells

Agonist exposure of many G protein-coupled receptors stimulates an activation of extracellular signal-regulated protein kinases (ERKs) 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. Here, we show that treatment of human embryonic kidney (HEK) 293 cells stably transfected to express the rat micro-opioid receptor (MOR1) with [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) stimulated a rapid and transient (3-5-min) activation and nuclear translocation of MAPK. Exposure of these cells to the MAPK kinase 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the mu-opioid receptor. We have therefore determined the effect of PD98059 on agonist-induced mu-receptor phosphorylation. DAMGO stimulated a threefold increase in MOR1 phosphorylation within 20 min that could be reversed by the antagonist naloxone. PD98059 produced a dose-dependent inhibition of agonist-promoted mu-receptor phosphorylation with an IC50 of 20 microM. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO-induced MOR1 internalization was also largely inhibited in the presence of PD98059. U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on mu-receptor phosphorylation and desensitization. MOR1 itself, however, appears to be a poor substrate for MAPK because mu-receptors immunoprecipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK activation but did not induce MOR1 internalization indicates that receptor internalization was not required for MOR1-mediated mitogenic signaling. We conclude that MOR1 stimulates a rapid and intemalization-independent MAPK activation. Activation of the MAPK cascade in turn may not only relay mitogenic signals to the nucleus but also trigger initial events leading to phosphorylation and desensitization of the mu-opioid receptor.     

Guanosine nucleotides regulate B2 kinin receptor affinity of agonists but not of antagonists: discussion of a model proposing receptor precoupling to G protein

The effect of nucleotides on binding of the B2 kinin (BK) receptor agonist [3H]BK and the antagonist [3H]NPC17731 to particulate fractions of human foreskin fibroblasts was studied. At 0 degrees C, particulate fractions exhibited a single class of binding sites with a Kd of 2.3 nM for [3H]BK and a Kd of 3.8 nM for the antagonist [3H]NPC17731. Incubation with radioligands at 37 degrees C for 5 min gave a reduction of agonist, as well as antagonist, binding that was between 0-40% depending on the preparation, even in the absence of guanosine nucleotides. As shown by Scatchard analysis, this reduction in specific binding was due to a shift in the affinity of at least a fraction of the receptors. The presence at 37 degrees C of the guanine nucleotides GTP, GDP and their poorly hydrolyzable analogs left [3H]NPC17731 binding unaffected, but reduced the receptor affinity for [3H]BK to a Kd of about 15 nM. The maximal number of receptors, however, was unchanged. This affinity change was strongly dependent on the presence of bivalent cations, in particular Mg2+. It was reversed by incubation at 0 degrees C. The rank order of the guanosine nucleotides for [3H]BK binding reduction was GTP[gammaS] = Gpp[NH]p > GTP = GDP > GDP[betaS]. GMP, ATP, ADP and AMP showed no influence on agonist binding. A model for the interaction of the B2 kinin receptor with G proteins is discussed.     

Decreased agonist, but not antagonist, binding to the naturally occurring Thr92Lys variant of the h5-HT7(a) receptor

In the present study on transfected human embryonic kidney (HEK)293 cells, we aimed at establishing whether expression of the naturally occurring Thr92Lys variation of the Gs-coupled h5-HT7(a) receptor leads to changes of ligand binding properties, of agonist-evoked cAMP formation and/or of antagonist-mediated blockade of the latter. Binding of [3H]5-carboxamidotryptamine ([3H]5-CT) to membranes and stimulated [3H]cAMP accumulation in whole cells were determined. Saturation binding experiments in membranes of transiently transfected cells expressing either the wild-type or the variant receptor revealed a single binding site in both cases and no difference in Bmax between both receptor isoforms. In competition binding experiments in membranes of stably transfected cells, the Thr92Lys variant exhibited a 2.8-11 times lower binding affinity of the ligands 5-hydroxytryptamine (5-HT), 5-CT, 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4yl)-1H-indole (RU24969), (+/-)-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) and sumatriptan compared to the wild-type receptor. However, the variant did not differ from the wild-type with respect to the binding properties of the antagonists (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)-pyrrolodine-1-sulfonyl)phenol hydrochloride (SB-269970), risperidone, mesulergine and clozapine. In agreement with the decreased binding affinity of 5-HT, 5-CT, RU24969 and 8-OH-DPAT for the variant receptor, these agonists were less potent in stimulating [3H]cAMP accumulation in cells stably expressing the Thr92Lys h5-HT7(a) receptor. Sumatriptan did not stimulate cAMP accumulation in spite of its affinity for both receptor isoforms pointing to a putative weak antagonistic property of this drug at the h5-HT7 receptor. SB-269970 and clozapine were equipotent at both the variant and the wild-type receptor in producing a rightward shift of the 5-HT concentration-response curve for its stimulant effect on [3H]cAMP accumulation. In view of, e.g., the purported involvement of the 5-HT7 receptor in the regulation of circadian rhythm, it may be concluded that the decrease in affinity of 5-HT and other 5-HT receptor agonists at the (Thr92Lys) h5-HT7 receptor may be associated with changes of sleep physiology and of actions of new 5-HT7 receptor agonists designed to treat circadian dysregulation.     

Synthesis, radiolabeling, and preliminary biological evaluation of [3H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine, a potent antagonist radioligand for the P2X7 receptor

The design, synthesis, and preliminary biological evaluation of the first potent radioligand antagonist for the P2X(7) receptor, named [(3)H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine (compound 13), are reported. This compound bound to human P2X(7) receptors expressed in HEK transfected cells with K(D) and B(max) value of 3.46+/-0.1 nM and 727+/-73 fmol/mg of protein, respectively. The high affinity and facile labeling makes it a promising radioligand for a further characterization of P2X(7) receptor subtype.     

The prostacyclin receptor is isoprenylated. Isoprenylation is required for efficient receptor-effector coupling.

The prostacyclin receptor (IP), a G protein-coupled receptor, mediates the actions of the prostanoid prostacyclin and its mimetics. IPs from a number of species each contain identically conserved putative isoprenylation CAAX motifs, each with the sequence CSLC. Metabolic labeling of human embryonic kidney (HEK) 293 cells stably overexpressing the hemagluttinin epitope-tagged IP in the presence of [(3)H]mevalonolactone established that the mouse IP is isoprenylated. Studies involving in vitro assays confirmed that recombinant forms of the human and mouse IP are modified by carbon 15 farnesyl isoprenoids. Disruption of isoprenylation, by site-directed mutagenesis of Cys(414) to Ser(414), within the CAAX motif, abolished isoprenylation of IP(SSLC) both in vitro and in transfected cells. Scatchard analysis of the wild type (IP) and mutant (IP(SSLC)) receptor confirmed that each receptor exhibited high and low affinity binding sites for [(3)H]iloprost, which were not influenced by receptor isoprenylation. Whereas stable cell lines overexpressing IP generated significant agonist (iloprost and cicaprost)-mediated increases in cAMP relative to nontransfected cells, cAMP generation by IP(SSLC) cells was not significantly different from the control, nontransfected HEK 293 cells. Moreover, co-expression of the alpha (alpha) subunit of Gs generated significant augmentations in cAMP by IP but not by IP(SSLC) cells. Whereas IP also demonstrated significant, dose-dependent increases in [Ca(2+)](i) in response to iloprost or cicaprost compared with the nontransfected HEK 293 cells, mobilization of [Ca(2+)](i) by IP(SSLC) was significantly impaired. Co-transfection of cells with either Galpha(q) or Galpha(11) resulted in significant augmentation of agonist-mediated [Ca(2+)](i) mobilization by IP cells but not by IP(SSLC) cells or by the control, HEK 293 cells. In addition, inhibition of isoprenylation by lovastatin treatment significantly reduced agonist-mediated cAMP generation by IP in comparison to the nonisoprenylated beta(2) adrenergic receptor or nontreated cells. Hence, isoprenylation of IP does not influence ligand binding but is required for efficient coupling to the effectors adenylyl cyclase and phospholipase C.