Increased AT(1) receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness.

Several examples of functional G-protein-coupled receptor heterodimers have been identified. However, it is not known whether receptor heterodimerization is involved in the pathogenesis of human disorders. Here we show that in preeclamptic hypertensive women, a significant increase in heterodimerization occurs between the AT(1)-receptor for the vasopressor angiotensin II and the B(2)-receptor for the vasodepressor bradykinin. AT(1)-B(2)-receptor heterodimerization in preeclampsia correlated with a 4-5-fold increase in B(2)-receptor protein levels. Expression of the AT(1)-B(2) heterodimer increased the responsiveness to angiotensin II and conferred resistance in AT(1)-receptors to inactivation by reactive oxygen species raised in normotensive and preeclamptic pregnancies. We suggest that AT(1)-B(2) heterodimers contribute to angiotensin II hypersensitivity in preeclampsia. Moreover, we identify preeclampsia as the first disorder associated with altered G-protein-coupled receptor heterodimerization.

Binding of Gbetagamma subunits to cRaf1 downregulates G-protein-coupled receptor signalling.

Receptors of the seven transmembrane domain family are coupled to heterotrimeric G proteins [1]. Binding of ligand to these receptors induces dissociation of the heterotrimeric complex into free GTP-Galpha and Gbetagamma subunits, which then interact with their respective effector molecules to stimulate specific cellular responses. In some cases, these cellular responses involve mitogenic signalling [2]. The mitogen-activated protein (MAP) kinase cascade is initiated by the protein kinase cRaf1 and links growth factor receptor signalling to cell growth and differentiation [3]. The main activator of cRaf1 is the small GTP-binding protein Ras [4], and the binding of cRaf1 to GTP-Ras translocates cRaf1 to the plasma membrane, where it is activated [5]. It has been reported that cRaf1 associates directly with the beta subunit of heterotrimeric G proteins in vitro, and with the betagamma subunit complex in vivo [6], but the role of this association is not yet understood. Here, we show that cRaf1 associates with Gbeta1gamma2, and that this association in mammalian cells is significantly enhanced when active p21(Ras) is present or when cRaf1 is otherwise targeted to the membrane. Association with Gbeta1gamma2 has no effect on the kinase activity of cRaf1, but cRaf1 can affect Gbetagamma-mediated signalling events. Thus, membrane-localised cRaf1 inhibits G-protein-coupled receptor (GPCR)-stimulated activation of phospholipase Cbeta (PLCbeta) by sequestration of Gbetagamma subunits, an effect also observed with endogenous levels of cRaf1. Our data suggest that cRaf1 may be an important regulator of signalling by Gbetagamma, particularly in those GPCR systems that stimulate the MAP kinase cascade through the activation of p21(Ras).

Investigation of the extracellular accessibility of the connecting loop between membrane domains I and II of the bradykinin B2 receptor.

In analogy to the structure of rhodopsin, the seven hydrophobic segments of G-protein-coupled receptors are supposed to form seven membrane-spanning alpha-helices. To analyze the topology of the bradykinin B2 receptor, we raised site-directed antibodies to peptides corresponding to the loop regions and the amino and carboxyl terminus of this receptor. We found that a segment with predicted intracellular orientation according to the rhodopsin model, the connecting loop between membrane domains I and II of the bradykinin B2 receptor, was accessible to site-directed antibodies on intact fibroblasts, A431 cells, or COS cells expressing human B2 receptors. Extracellular orientation of this loop was further confirmed by the substituted cysteine accessibility method which showed that exchange of cysteine 94 for serine on this loop by point mutagenesis suppressed the effect of thiol modification by a membrane impermeant maleimide. In addition, this segment seemed to be involved in B2 receptor activation, since (i) thiol modification of cysteine 94 partially suppressed B2 receptor activation, and (ii) site-directed antibodies to the connecting loop between membrane domains I and II were agonists. The agonistic activity of the antibodies was suppressed by the B2 antagonist HOE140 confirming the B2 specificity of the antibody-generated signal. The extracellular orientation of the connecting loop between membrane domains I and II suggests a topology of the B2 receptor different from rhodopsin, consisting of five (instead of seven) transmembrane domains and two hydrophobic segments with both ends facing the extracellular side.

AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration.

The vasopressor angiotensin II regulates vascular contractility and blood pressure through binding to type 1 angiotensin II receptors (AT1; refs 1, 2). Bradykinin, a vasodepressor, is a functional antagonist of angiotensin II (ref. 3). The two hormone systems are interconnected by the angiotensin-converting enzyme, which releases angiotensin II from its precursor and inactivates the vasodepressor bradykinin. Here we show that the AT1 receptor and the bradykinin (B2) receptor also communicate directly with each other. They form stable heterodimers, causing increased activation of G alpha(q) and G alpha(i) proteins, the two major signalling proteins triggered by AT1. Furthermore, the endocytotic pathway of both receptors changed with heterodimerization. This is the first example of signal enhancement triggered by heterodimerization of two different vasoactive hormone receptors.

Crosstalk between Galpha(i)- and Galpha(q)-coupled receptors is mediated by Gbetagamma exchange.

Activation of Galpha(i)-coupled receptors often causes enhancement of the inositol phosphate signal triggered by Galpha(q)-coupled receptors. To investigate the mechanism of this synergistic receptor crosstalk, we studied the Galpha(i)-coupled adenosine A(1) and alpha(2C) adrenergic receptors and the Galpha(q)-coupled bradykinin B(2) and a UTP-preferring P2Y receptor. Stimulation of either Galpha(i)-coupled receptor expressed in COS cells increased the potency and the efficacy of inositol phosphate production by bradykinin or UTP. Likewise, overexpression of Gbeta(1)gamma(2) resulted in a similar increase in potency and efficacy of bradykinin or UTP. In contrast, these stimuli did not affect the potency of direct activators of Galpha(q); a truncated Gbeta(3) mutant had no effect on the receptor-generated signals whereas signals generated at the G-protein level were still enhanced. This suggests that the Gbetagamma-mediated signal enhancement occurs at the receptor level. Almost all possible combinations of Gbeta(1-3) with Ggamma(2-7) were equally effective in enhancing the signals of the B(2) and a UTP-preferring P2Y receptor, indicating a very broad specificity of this synergism. The enhancement of the bradykinin signal by (i) Galpha(i)-activating receptor ligands or (ii) cotransfection of Gbetagamma was suppressed when the B(2) receptor was replaced by a B(2)Gbeta(2) fusion protein. Gbetagamma enhanced the B(2) receptor-stimulated activation of G-proteins as determined by GTPgammaS-induced decrease in high affinity agonist binding and by B(2) receptor-enhanced [(35)S]GTPgammaS binding. These findings support the concept that Gbetagamma exchange between Galpha(i)- and Galpha(q)-coupled receptors mediates this type of receptor crosstalk.

Paradoxical block of parathormone secretion is mediated by increased activity of G alpha subunits.

The paradox of blunted parathormone (PTH) secretion in patients with severe hypomagnesemia has been known for more than 20 years, but the underlying mechanism is not deciphered. We determined the effect of low magnesium on in vitro PTH release and on the signals triggered by activation of the calcium-sensing receptor (CaSR). Analogous to the in vivo situation, PTH release from dispersed parathyroid cells was suppressed under low magnesium. In parallel, the two major signaling pathways responsible for CaSR-triggered block of PTH secretion, the generation of inositol phosphates, and the inhibition of cAMP were enhanced. Desensitization or pertussis toxin-mediated inhibition of CaSR-stimulated signaling suppressed the effect of low magnesium, further confirming that magnesium acts within the axis CaSR-G-protein. However, the magnesium binding site responsible for inhibition of PTH secretion is not identical with the extracellular ion binding site of the CaSR, because the magnesium deficiency-dependent signal enhancement was not altered on CaSR receptor mutants with increased or decreased affinity for calcium and magnesium. By contrast, when the magnesium affinity of the G alpha subunit was decreased, CaSR activation was no longer affected by magnesium. Thus, the paradoxical block of PTH release under magnesium deficiency seems to be mediated through a novel mechanism involving an increase in the activity of G alpha subunits of heterotrimeric G-proteins.

The N-terminal amino group of [Tyr8]bradykinin is bound adjacent to analogous amino acids of the human and rat B2 receptor.

To obtain data of the bradykinin B2 receptor's agonist binding site, we used a combined approach of affinity labeling and "immunoidentification" of receptor fragments generated by cyanogen bromide cleavage. Domain-specific antibodies to the various extracellular receptor domains were applied to detect receptor fragments with covalently attached [125I-Tyr8]bradykinin. As a cross-linker we used the homobifunctional reagent disuccinimidyl tartarate (DST), which reacts preferentially with primary amines. With this technique a [125I-Tyr8]bradykinin-labeled receptor fragment derived from the third extracellular domain was identified. The epsilon-amino group of lysine (Lys172) of the human B2 receptor provides the only primary amino group within this receptor fragment. This strongly suggests that DST attached the N-terminal amino group of [Tyr8]bradykinin to Lys172 of the human B2 receptor. Next we asked whether DST attaches [Tyr8]bradykinin to the analogous residue, Lys174 of the rat B2 receptor, which is 81% identical to the human B2 receptor, and we attempted to label the wild-type rat B2 receptor and a rat B2 receptor mutant where Lys174 had been exchanged for alanine. Affinity labeling of the wild-type rat B2 receptor worked efficiently, whereas DST did not attach detectable amounts of [125I-Tyr8]bradykinin to the K174A rat B2 receptor mutant. Taken together these observations indicate that the N-terminal amino group of [Tyr8]bradykinin is bound to analogous positions of the rat and of the human B2 receptor, i.e. [Tyr8]bradykinin's N terminus is bound adjacent to Lys172 of the human and Lys174 of the rat B2 receptor.

Two distinct Ca2+ influx pathways activated by the bradykinin B2 receptor.

The hormone-induced depletion of cellular Ca stores provides a signal for the Ca2+ influx into electrically non-excitable cells; however, the underlying molecular mechanisms remain elusive. Therefore, we analyzed bradykinin-activated Ca2+ influx into human foreskin fibroblast cells, HF-15, by fura-2 and 45Ca labeling to discriminate between Ca2+ influx into the fura-sensitive compartment and Ca uptake into fura-insensitive Ca stores. Bradykinin-activated Ca2+ influx into the fura-sensitive compartment was blocked by inhibitors of NO synthases. These inhibitors also suppressed bradykinin-activated increases in cGMP, indicating that the NO-dependent increase in cGMP is involved in the activation of the Ca2+ influx into the fura-sensitive compartment. The cGMP-dependent kinase inhibitors KT5823 and Rp-8-(parachlorophenylthio)-cGMP (Rp-8-pCPT-cGMPS) blocked bradykinin-activated Ca2+ influx into the fura-sensitive compartment, suggesting that a cGMP-dependent kinase step participates in the activation of this Ca2+ influx pathway. In addition to the NO/cGMP-mediated Ca2+ influx into the fura-sensitive compartment, bradykinin enhanced 45Ca uptake into Ca stores that were not accessible to fura-2. This enhanced 45Ca uptake was insensitive to blockers of the NO/cGMP pathway, indicating that the 45Ca uptake pathway is distinct from the NO-dependent Ca2+ influx into the fura-sensitive compartment. Furthermore, bradykinin enhanced 45Ca uptake into proliferating but not into quiescent HF-15 fibroblasts. Hence, bradykinin stimulates two distinct Ca2+ influx pathways in HF-15 cells, (a) Ca2+ influx into the fura-sensitive compartment which is NO/cGMP-dependent and (b) Ca uptake into Ca stores which bypasses the cytoplasm, which is NO insensitive and which is linked to cell proliferation.

Na+ ions binding to the bradykinin B2 receptor suppress agonist-independent receptor activation.

Control of the balance between receptor activation and inactivation is a prerequisite for seven transmembrane domain (7TM) receptor function. We asked for a mechanism to stabilize the inactive receptor conformation which prevents agonist-independent receptor activation. Na+ ions have reciprocal effects on agonist versus antagonist interaction with various 7TM receptors. To investigate the Na+ dependence of receptor activation we chose the bradykinin B2 receptor as a prototypic 7TM receptor. Decrease of the intracellular Na+ content from 40 mM to 10 mM of COS-1 cells transiently expressing rat B2 receptors activated the B2 receptor in the absence of agonist as shown by a 3-fold increase in the basal release of inositolphosphates and increased the intrinsic activity of bradykinin to 1.2. In contrast, under increased intracellular Na+ (148 mM) the intrinsic activity of bradykinin decreased to 0.72. When the interaction of Na+ with the B2 receptor was prevented by exchanging a conserved aspartate in transmembrane domain II for asparagine the B2 receptor was also constitutively-activated in the absence of agonist. Agonist-independence B2 receptor activation under decreased intracellular Na+ was similarly observed with primary human fibroblasts endogenously expressing human B2 receptors by a 2.5-fold increase in basal inositolphosphates. Activation of human B2 receptors in the absence of agonist under decreased intracellular Na+ was further evident by an increased basal phosphorylation of the B2 receptor protein. Thus our data suggest that the interaction of Na+ ions with the B2 receptor stabilizes or induces an inactive receptor conformation thereby providing a mechanism to suppress agonist-independent receptor activation in vivo.

Involvement of the amino terminus of the B(2) receptor in agonist-induced receptor dimerization.

The mechanisms and the functional importance of G-protein-coupled receptor dimerization are poorly understood. We therefore analyzed dimerization of the bradykinin B(2) receptor. The binding of the agonist bradykinin to the B(2) receptor endogenously expressed on PC-12 cells led to the formation of receptor dimers, whereas the B(2) antagonist HOE140 did not induce dimerization, suggesting that B(2) receptor dimerization was linked to receptor activation. Addition of a peptide corresponding to the amino terminus of the receptor reduced the amount of detected B(2) receptor dimers, whereas peptides derived from the extracellular loops had no effect. To further analyze the role of the amino terminus of the receptor in receptor dimerization, we created two different rat B(2) receptor variants with truncated amino termini, B(2)(53) and B(2)(65), starting at amino acids 53 and 65. In contrast to the wild-type B(2) receptor and to B(2)(53), bradykinin did not induce dimerization of the B(2)(65) receptor. Both receptor variants were similar to the wild-type B(2) receptor with respect to agonist binding and signal generation. However, B(2)(65) was not phosphorylated, did not desensitize, and was not downregulated upon bradykinin stimulation. Likewise, antibodies directed to the amino terminus of the receptor partially reduced internalization of [(3)H]bradykinin on PC-12 cells. These findings suggest that the amino terminus of the B(2) receptor is necessary for triggering agonist-induced B(2) receptor dimerization, and receptor dimers are involved in receptor-mediated signal attenuation.

Effects of bradykinin and endothelin-1 on the calcium homeostasis of mammalian cells.

Ca2+ mobilization from intracellular stores is a major event in the signaling cascade triggered by peptide hormone receptors. The transient rise in intracellular free Ca2+ concentration ([Ca2+]i) is well characterized, but little is known about alterations of total cell Ca. Therefore we established a technique to determine changes in total cell Ca during hormone stimulation of 45Ca-loaded cells. Bradykinin and endothelin-1 reduced total cell Ca by up to 56% in HF-15 cells, COS-7 cells, and CHO K1 cells transfected with the rat B2 receptor cDNA. In Rat-1 cells and PC-12 cells, stimulation with endothelin-1 or bradykinin did not result in a net decrease in total cell Ca at physiological extracellular Ca2+ concentration. Decrease in total cell Ca was preceded by an increase in [Ca2+]i and blunting of the transient rise in [Ca2+]i by a Ca2+ chelator prevented the hormone-induced decrease in total cell Ca. Previous reduction of total cell Ca by one hormone suppressed the transient rise in [Ca2+]i induced by another. The data present evidence that the hormones bradykinin and endothelin-1 are capable of switching off the Ca(2+)-mobilizing signal transduction pathway in a cell by depleting intracellular Ca stores. This process is accompanied by a significant reduction of total cell Ca.

Phosphorylation-independent inhibition of parathyroid hormone receptor signaling by G protein-coupled receptor kinases.

Homologous desensitization of G protein-coupled receptors is thought to occur in several steps: binding of G protein-coupled receptor kinases (GRKs) to receptors, receptor phosphorylation, kinase dissociation, and finally binding of beta-arrestins to phosphorylated receptors. It generally is assumed that only the last step inhibits receptor signaling. Investigating the parathyroid hormone (PTH) receptor --> inositol phosphate pathway, we report here that GRKs can inhibit receptor signaling already under nonphosphorylating conditions. GRKs phosphorylated the PTH receptor in membranes and in intact cells; the order of efficacy was GRK2>GRK3>GRK5. Transient transfection of GRKs with the PTH receptor into COS-1 cells inhibited PTH-stimulated inositol phosphate generation. Such an inhibition also was seen with the kinase-negative mutant GRK2-K220R and also for a C-terminal truncation mutant of the PTH receptor that could not be phosphorylated. Several lines of evidence indicated that this phosphorylation-independent inhibition was exerted by an interaction between GRKs and receptors: (a) this inhibition was not mimicked by proteins binding to G proteins, phosducin, and GRK2 C terminus, (b) GRKs caused an agonist-dependent inhibition (= desensitization) of receptor-stimulated G protein GTPase-activity (this effect also was seen with the kinase-inactive GRK2-mutant and the phosphorylation-deficient receptor mutant), and (c) GRKs bound directly to the PTH receptor. These data suggest that signaling by the PTH receptor already is inhibited by the first step of homologous desensitization, the binding of GRKs to the receptors.

Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2.

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gbetagamma subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 +/- 0.1 and 0.83 +/- 0.09 mol of P(i)/mol of protein, respectively). The phosphorylation reactions exhibit apparent K(m) values in the range of 40-100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with G beta gamma subunits. Phosphorylation of phosducin by GRK2 markedly reduces its G beta gamma binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.

The angiotensin II AT2 receptor is an AT1 receptor antagonist.

The vasopressor angiotensin II activates AT(1) and AT(2) receptors. Most of the known in vivo effects of angiotensin II are mediated by AT(1) receptors while the biological functions of AT(2) receptors are less clear. We report here that the AT(2) receptor binds directly to the AT(1) receptor and thereby antagonizes the function of the AT(1) receptor. The AT(1)-specific antagonism of the AT(2) receptor was independent of AT(2) receptor activation and signaling, and it was effective on different cells and on human myometrial biopsies with AT(1)/AT(2) receptor expression. Thus, the AT(2) receptor is the first identified example of a G-protein-coupled receptor which acts as a receptor-specific antagonist.

Structure of the bradykinin B2 receptors' amino terminus.

The peptide hormone bradykinin exerts important biological functions by binding to and activating bradykinin B2 receptors. B2 receptors belong to the seven transmembrane domain (7TM) receptor family. Cloning of the cDNA sequences for the rat, human, and mouse bradykinin B2 receptor revealed several in-frame AUG triplets as potential initiation sites for translation. Due to "Kozak-like" consensus nucleotides, the AUG codon closest to transmembrane domain 1 was assumed the preferred initiation site for translation, but the real amino terminus of the B2 receptor protein was unknown. The amino terminus of several 7TM receptors has been shown to be essentially involved in receptor activation and/or ligand binding. Therefore we determined the amino terminus of the human and of the rat B2 receptor using domain-specific antipeptide antibodies, amino acid sequence analysis, and in vitro transcription/translation. We report that the human and rat B2 receptor protein start with the methionine which is translated from the first in-frame AUG. This start site extends the known amino terminus of the human and rat B2 receptors by 27 or 30 amino acid residues, respectively. Antibodies raised against a peptide of the initial 27 amino acids of the human B2 receptor stained B2 receptors on intact cells. This finding excludes the existence of a signal sequence for this receptor.

Structural features of the human bradykinin B2 receptor probed by agonists, antagonists, and anti-idiotypic antibodies.

The human bradykinin B2 receptor belongs to the family of G-protein-coupled receptors. To characterize the receptor protein, we have solubilized the membranes of cultured human foreskin fibroblasts bearing the B2 receptor. Affinity cross-linking of the solubilized receptor with the labeled agonist, 125I-Tyr0-bradykinin, or the labeled antagonist, 125I-(4-hydroxy-phenyl-propionyl)-HOE140, revealed major bands of apparent molecular mass of 69 kDa in SDS-polyacrylamide gel electrophoresis under reducing conditions, and of 59 kDa under non-reducing conditions. A 1000-fold molar excess of each of the unlabeled ligands quenched the specific labeling suggesting that the agonist and the antagonist compete for overlapping binding site(s). Covalent coupling of the receptor to bradykinin or HOE140, followed by Western blotting and immunoprinting with specific anti-ligand antibodies confirmed that the major ligand-binding form of the receptor is of 69 kDa. Anti-idiotypic antibodies which bear the internal image of bradykinin (Haasemann, M., Buschko, J., Faussner, A., Roscher, A.A., Hoebeke, J., Burch, R.M., and Müller-Esterl, W. (1991) J. Immunol. 147, 3882-3892) immunoprecipitated the 125I-labeled receptor as a major band of 68 kDa and a minor band of 47 kDa indicative of partial proteolysis. Chemical deglycosylation of the 125I-labeled receptor shifted the apparent molecular mass from 69 to 44 kDa demonstrating that the receptor is heavily glycosylated. Two-dimensional electrophoresis of the affinity-purified receptor revealed overlapping spots of 69 kDa and of pI 6.8-7.1 pointing to a microheterogeneity of the carbohydrate moiety. Elucidation of the key structural features of the B2 receptor protein will aid in understanding the structure-function relationships governing this prototypic peptide receptor.

Extracellular domains of the bradykinin B2 receptor involved in ligand binding and agonist sensing defined by anti-peptide antibodies.

Many of the physiological functions of bradykinin are mediated via the B2 receptor. Little is known about binding sites for bradykinin on the receptor. Therefore, antisera against peptides derived from the putative extracellular domains of the B2 receptor were raised. The antibodies strongly reacted with their corresponding antigens and cross-reacted both with the denatured and the native B2 receptor. Affinity-purified antibodies to the various extracellular domains were used to probe the contact sites between the receptor and its agonist, bradykinin or its antagonist HOE140. Antibodies to extracellular domain 3 (second loop) efficiently interfered, in a concentration-dependent manner, with agonist and antagonist binding and vice versa. Antibodies to extracellular domain 4 (third loop) blocked binding of the agonist but not of the antagonist, whereas antibodies to extracellular domains 1 and 2 or to intracellular domains failed to block ligand binding. Antibodies to ectodomain 3 competed with agonistic anti-idiotypic antibodies for B2 receptor binding. Further, affinity-purified antibodies to the amino-terminal portion of extracellular domain 3 transiently increased intracellular free Ca2+ concentration and thus are agonists. The Ca2+ signal was specifically blocked by the B2 antagonist HOE140. By contrast, antibodies to the carboxyl-terminal segment of extracellular domain 4 failed to trigger Ca2+ release. The specific effects of antibodies to the amino-terminal portion of extracellular domain 3 suggest that this portion of the B2 receptor may be involved in ligand binding and in agonist function.

Bradykinin induces translocation of the protein kinase C isoforms alpha, epsilon, and zeta.

Bradykinin exerts a broad spectrum of cellular effects on different tissues. It is believed that these effects are predominantly mediated by the recently cloned B2 receptor. The mechanism of post-receptor signal transduction is not known in detail. Involvement of protein kinase C (PKC) was suggested and activation of the classical PKC isoforms alpha and beta was recently demonstrated. The aim of the present study was to investigate whether the B2 receptor also activates new (delta, epsilon) and atypical (zeta) PKC isoforms. To investigate this, chinese hamster ovary (CHO) cells, stably transfected with human B2 receptor, were used. In these cells the PKC isoforms alpha, delta, epsilon and zeta were detected by immunoblotting with specific antibodies. To monitor hormone-induced PKC translocation plasma membranes were prepared. Stimulation of the cells with bradykinin resulted in a rapid (30-60 s) translocation of the PKC isoforms alpha, epsilon, and zeta. Translocation of PKC delta was not detected. The effect of bradykinin was reduced by simultaneous addition of the receptor antagonist HOE 140, a bradykinin-related decapeptide. The data show that the B2 receptor in this cell model is able to activate, in addition to the classical PKC isoform alpha, the new PKC isoform epsilon and the atypical PKC isoform zeta. To test whether these effects are as well observed in a non-transfected cell, the experiments were repeated in human foreskin fibroblasts which naturally express high levels of B2 receptors. In this cell system similar results on PKC alpha, epsilon, and zeta were observed, suggesting that all three PKC isoforms are involved in signal transduction of the B2 receptor.