Claus Liebmann

Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity.

G protein-coupled receptors (GPCRs) can stimulate the mitogen-activated protein kinase (MAPK) cascade and thereby induce cellular proliferation like receptor tyrosine kinases (RTKs). Work over the past 5 years has established several models which reduce the links of G(i)-, G(q)-, and G(s)-coupled receptors to MAPK on few principle pathways. They include (i) Ras-dependent activation of MAPK via transactivation of RTKs such as the epidermal growth factor receptor (EGFR), (ii) Ras-independent MAPK activation via protein kinase C (PKC) that converges with the RTK signalling at the level of Raf, and (iii) activation as well as inactivation of MAPK via the cAMP/protein kinase A (PKA) pathway in dependency on the type of Raf. Most of these generalizing hypotheses are founded on experimental data obtained from expression studies and using a limited set of individual receptors. This review will compare these models with pathways to MAPK found for a great variety of peptide hormone and neuropeptide receptor subtypes in various cells. It becomes evident that under endogenous conditions, the transactivation pathway is less dominant as postulated, whereas pathways involving isoforms of PKC and, especially, phosphoinositide 3-kinase (PI-3K) appear to play a more important role as assumed so far. Highly cell-specific and unusual connections of signalling proteins towards MAPK, in particular tumour cells, might provide points of attacks for new therapeutic concepts.

Bradykinin B 2 Receptor-Mediated Mitogen-Activated Protein Kinase Activation in COS-7 Cells Requires Dual Signaling via Both Protein Kinase C Pathway and Epidermal Growth Factor Receptor Transactivation

ABSTRACT The signaling routes linking G-protein-coupled receptors to mitogen-activated protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-kinase γ (PI3Kγ), and protein kinase C (PKC). To characterize the mitogenic pathway of bradykinin (BK), COS-7 cells were transiently cotransfected with the human bradykinin B2receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK is mediated via the α subunits of a Gq/11 protein. Both activation of Raf-1 and activation of MAPK in response to BK were blocked by inhibitors of PKC as well as of the epidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 cells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-Kγ or Src kinase failed to diminish MAPK activation by BK. BK-induced translocation and overexpression of PKC isoforms as well as coexpression of inactive or constitutively active mutants of different PKC isozymes provided evidence for a role of the diacylglycerol-sensitive PKCs α and ɛ in BK signaling toward MAPK. In addition to PKC activation, BK also induced tyrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. Inhibition of PKC did not alter BK-induced transactivation, and blockade of EGF receptor did not affect BK-stimulated phosphatidylinositol turnover or BK-induced PKC translocation, suggesting that PKC acts neither upstream nor downstream of the EGF receptor. Comparison of the kinetics of PKC activation and EGF receptor transactivation in response to BK also suggests simultaneous rather than consecutive signaling. We conclude that in COS-7 cells, BK activates MAPK via a permanent dual signaling pathway involving the independent activation of the PKC isoforms α and ɛ and transactivation of the EGF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.

A novel mitogenic signaling pathway of bradykinin in the human colon carcinoma cell line SW-480 involves sequential activation of a Gq/11 protein, phosphatidylinositol 3-kinase beta, and protein kinase Cepsilon.

The signaling routes connecting G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) pathway reveal a high degree of complexity and cell specificity. In the human colon carcinoma cell line SW-480, we detected a mitogenic effect of bradykinin (BK) that is mediated via a pertussis toxin-insensitive G protein of the Gq/11 family and that involves activation of MAPK. Both BK-induced stimulation of DNA synthesis and activation of MAPK in response to BK were abolished by two different inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY 294002, as well as by two different inhibitors of protein kinase C (PKC), bisindolylmaleimide and Ro 31-8220. Stimulation of SW-480 cells by BK led to increased formation of PI3K lipid products (phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate) and to enhanced translocation of the PKCε isoform from the cytosol to the membrane. Both effects of BK were inhibited by wortmannin, too. Using subtype-specific antibodies, only the PI3K subunits p110β and p85, but not p110α and p110γ, were detected in SW-480 cells. Finally, p110β was found to be co-immunoprecipitated with PKCε. Our data suggest that in SW-480 cells, (i) dimeric PI3Kβ is activated via a Gq/11 protein; (ii) PKCε is a downstream target of PI3Kβ mediating the mitogenic signal to the MAPK pathway; and (iii) PKCε associates with the p110 subunit of PI3Kβ. Thus, these results add a novel possibility to the emerging picture of multiple pathways linking G protein-coupled receptors to MAPK.

Opioid receptor affinities of the blood-derived tetrapeptides hemorphin and cytochrophin

Hemorphin-4 and cytochrophin-4 displayed affinities for mu- and delta-opioid receptors that were in the same range as those observed for the structurally related beta-casomorphins. However, they showed markedly higher affinities at kappa-opioid binding sites when compared to the beta-casomorphins. These blood-derived peptides could be involved in blood pressure regulation.

Tyrosine Phosphorylation of Gsα and Inhibition of Bradykinin-induced Activation of the Cyclic AMP Pathway in A431 Cells by Epidermal Growth Factor Receptor

An increasing amount of experimental data suggest that cross-talk exists between pathways involving tyrosine kinases and heterotrimeric G proteins. In a previous study, we demonstrated that bradykinin (BK) increases the intracellular accumulation of cAMP in the human epidermoid carcinoma cell line A431 by stimulating adenylate cyclase activity via a stimulatory G protein (Gsα) (Liebmann, C., Graneß, A., Ludwig, B., Adomeit, A., Boehmer, A., Boehmer, F.-D., Nürnberg, B., and Wetzker, R. (1996) Biochem. J. 313, 109-118). Here, we present several lines of evidence indicating the ability of epidermal growth factor (EGF) to suppress BK-induced activation of the cAMP pathway in A431 cells via tyrosine phosphorylation of Gsα. Gsα was specifically immunoprecipitated from A431 cells using the anti-αs antiserum AS 348. Tyrosine phosphorylation of Gsα was detectable in EGF-pretreated cells with monoclonal anti-phosphotyrosine antibodies. Additionally, A431 cells were labeled with [32P]orthophosphate in vivo and treated with EGF, and the resolved immunoprecipitates were subjected to amino acid analysis. The results clearly indicate that EGF induces tyrosine phosphorylation of Gsα in A431 cells. Treatment of A431 cells with EGF decreased BK-induced cAMP accumulation in intact cells as well as the stimulation of adenylate cyclase by BK, NaF, and guanyl nucleotides, but not by forskolin. Also, EGF treatment abolished both the BK- and isoprenaline-induced stimulation of guanosine 5′-O-(3-[35S]thiotriphosphate) binding to Gsα. In contrast, the BK-evoked, Gq-mediated stimulation of inositol phosphate formation in A431 cells was not affected by EGF pretreatment. Thus, EGF-induced tyrosine phosphorylation of Gsα is accompanied by a loss of its susceptibility to G protein-coupled receptors and its ability to stimulate adenylate cyclase via guanyl nucleotide exchange. We propose that Gsα may represent a key regulatory protein in the cross-talk between the signal transduction pathways of BK and EGF in A431 cells.

Cross talk between beta-adrenergic and bradykinin B(2) receptors results in cooperative regulation of cyclic AMP accumulation and mitogen-activated protein kinase activity.

ABSTRACT Costimulation of G protein-coupled receptors (GPCRs) may result in cross talk interactions between their downstream signaling pathways. Stimulation of GPCRs may also lead to cross talk regulation of receptor tyrosine kinase signaling and thereby to activation of mitogen-activated protein kinase (MAPK). In COS-7 cells, we investigated the interactions between two particular mitogenic receptor pathways, the endogenously expressed β-adrenergic receptor (β-AR) and the transiently transfected human bradykinin (BK) B2receptor (B2R). When β-AR and B2R are costimulated, we found two different cross talk mechanisms. First, the predominantly Gq protein-coupled B2R is enabled to activate a Gi protein and, subsequently, type II adenylate cyclase. This results in augmentation of β-AR-mediated cyclic AMP (cAMP) accumulation by BK, which alone is unable to increase the cAMP level. Second, independently of BK-induced superactivation of the cAMP system, costimulation of β-AR leads to protein kinase A-mediated blockade of phospholipase C activation by BK. Thereby, the pathway from B2R to MAPK, which essentially involves protein kinase C activation, is selectively switched off. The MAPK activation in response to isoproterenol was not affected due to costimulation. Furthermore, in the presence of isoproterenol, BK lost its ability to stimulate DNA synthesis in COS-7 cells. Thus, our findings might establish a novel paradigm: cooperation between simultaneously activated mitogenic pathways may prevent multiple stimulation of MAPK activity and increased cell growth.

Bradykinin signalling to MAP kinase: cell-specific connections versus principle mitogenic pathways.

Abstract Mitogenic signalling pathways from G protein-coupled receptors (GPCRs) to the mitogen-activated protein kinase (MAPK) cascade may involve ?- or ??-subunits of heterotrimeric G proteins, receptor or nonreceptor tyrosine kinases, adaptor molecules, phosphoinositide 3-kinases, protein kinase C, and probably other proteins. The majority of models describing the connection of different signalling proteins within a mitogenic pathway are based on experimental data obtained by co- and overexpression of epitope-tagged MAPK together with the respective GPCR and other signalling proteins of interest in transfectable cell lines. Here the link of the bradykinin B2 receptor (B2R) to MAPK in the COS-7 cell expression system is compared with mitogenic signalling pathways of bradykinin in various tumour cell lines. It becomes evident that in natural or tumour cells expressing individual amounts and different isoforms of signalling proteins completely other relations between B2R and MAPK may exist than in COS-7 cells, suggesting a high degree of cellular specificity in mitogenic signalling.

Antagonist binding reveals two heterogenous B2 bradykinin receptors in rat myometrial membranes

In rat myometrial membranes, two bradykinin binding sites with K1 values of 18 pM and 5.6 nM were identified. Three potent bradykinin antagonists were tested for their ability to compete for [3H]bradykinin binding. Two of them, D-Arg[Hyp3,Thi5.8,D-Phe7]bradykinin and [Hyp3,Thi5.8,D-Phe7]bradykinin, also bound to both the high- (KH) and the low-affinity (KL) site whereas [Thi5.8,D-Phe7]bradykinin identified only the low-affinity bradykinin receptor. There is a close correlation between the antagonistic potencies and the KL site affinities.

Opiate receptor binding affinities of some D-amino acid substituted β-casomorphin analogs

beta-Casomorphin-(5) and some analogs modified by the introduction of some D-amino acids and D-pipecolic acid as well as by C-terminal amidation were tested for their affinities to mu- and delta-binding sites in rat brain membranes. The binding affinities of these compounds are compared with the known activities in the guinea pig ileum (GPI) and mouse vas deferens (MVD) test and their antinociceptive potencies in rats. The substitution of D-proline for proline in position 4 in beta-casomorphin-(5) and beta-casomorphin-(4)amide (morphiceptin) results in derivatives with very high mu-binding affinity and mu-selectivity. These affinities correspond to the respective analgesic potencies. Both binding to mu-receptors and analgesic potency are also enhanced by the introduction of D-Phe in position 3. Testing D-Ala2 substituted derivatives with respect to their ability to compete for 3H-naloxone, we observed apparent differences between the pentapeptide amides (biphasic displacement curves) and the tetrapeptide amides (monophasic displacement curves). The substitution of L-Pro2 by D-pipecolic acid yields an analog with preferential delta-receptor affinity in the organ preparations (MVD) but preferential mu-receptor affinity in brain membranes. This finding suggests a possible difference between peripheral and central mu-binding sites.

Expression and Functional Characterization of a pHis-Tagged Human Bradykinin B2 Receptor in COS-7Cells

Abstract A polyHistagged bradykinin (BK) B[2] receptor (pHis BKR) cDNA was constructed and expressed in COS-7 cells. The pHisBKR is suitable for both immunoprecipitation and immunoblotting with antipolyHis antibodies and can be easily purified using NiNTA columns. Immunochemical detection revealed a molecular mass of approximately 66 kDa. The pHisBKR is capable of mediating BKinduced stimulation of inositol phosphate formation as well as of mitogenactivated protein kinase (MAPK) activity. Compared with the wildtype receptor (WTBKR) the tagged receptor showed a slightly enhanced affinity towards BK but a reduced expression level. Despite these modified pharmacological properties the pHistagged BKR may be a useful tool for studying BKR modifications and signaling.