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

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 beta-adrenergic receptor (beta-AR) and the transiently transfected human bradykinin (BK) B(2) receptor (B(2)R). When beta-AR and B(2)R are costimulated, we found two different cross talk mechanisms. First, the predominantly G(q) protein-coupled B(2)R is enabled to activate a G(i) protein and, subsequently, type II adenylate cyclase. This results in augmentation of beta-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 beta-AR leads to protein kinase A-mediated blockade of phospholipase C activation by BK. Thereby, the pathway from B(2)R 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.

Defining the location and function of domains of McrB by deletion mutagenesis.

The GTP-dependent restriction endonuclease McrBC of E. coli K12, which recognizes cytosine-methylated DNA, consists of two protein subunits, McrB and McrC. We have investigated the structural assignment and interdependence of the McrB subunit functions, namely (i) specific DNA recognition and (ii) GTP binding and hydrolysis. Extending earlier work, we have produced McrB variants comprising N- and C-terminal fragments. The variants McrB1-162 and McrB1-170 are still capable of specific DNA binding. McrB169-465 shows GTP binding and hydrolysis characteristics indistinguishable from full-length McrB as well as wild-type like interaction with McrC. Thus, DNA and GTP binding are spatially separated on the McrB molecule, and the respective domains function quite independently.

The GTP-binding domain of McrB: more than just a variation on a common theme?

The methylation-dependent restriction endonuclease McrBC from Escherichia coli K12 cleaves DNA containing two R(m)C dinucleotides separated by about 40 to 2000 base-pairs. McrBC is unique in that cleavage is totally dependent on GTP hydrolysis. McrB is the GTP binding and hydrolyzing subunit, whereas MrC stimulates its GTP hydrolysis. The C-terminal part of McrB contains the sequences characteristic for GTP-binding proteins, consisting of the GxxxxGK(S/T) motif (position 201-208), followed by the DxxG motif (position 300-303). The third motif (NKxD) is present only in a non-canonical form (NTAD 333-336). Here we report a mutational analysis of the putative GTP-binding domain of McrB. Amino acid substitutions were initially performed in the three proposed GTP-binding motifs. Whereas substitutions in motif 1 (P203V) and 2 (D300N) show the expected, albeit modest effects, mutation in the motif 3 is at variance with the expectations. Unlike the corresponding EF-Tu and ras -p21 variants, the D336N mutation in McrB does not change the nucleotide specificity from GTP to XTP, but results in a lack of GTPase stimulation by McrC. The finding that McrB is not a typical G protein motivated us to perform a search for similar sequences in DNA databases. Eight microbial sequences were found, mainly from unfinished sequencing projects, with highly conserved sequence blocks within a presumptive GTP-binding domain. From the five sequences showing the highest homology, 17 invariant charged or polar residues outside the classical three GTP-binding motifs were identified and subsequently exchanged to alanine. Several mutations specifically affect GTP affinity and/or GTPase activity. Our data allow us to conclude that McrB is not a typical member of the superfamily of GTP-binding proteins, but defines a new subfamily within the superfamily of GTP-binding proteins, together with similar prokaryotic proteins of as yet unidentified function.

Does the restriction endonuclease EcoRV employ a two-metal-Ion mechanism for DNA cleavage?

Two models for the catalytic mechanism of the restriction endonuclease EcoRV exist which differ in the number and function of metal ions proposed to be directly involved in catalysis. In one model, two metal ions bound by Glu45, Asp74, and Asp90 are assumed to have a direct catalytic function; in the other, only one metal ion bound by Asp74 and Asp90. We show here that in the presence of Mn2+, the catalytic activity of an EcoRV-E45A mutant is only slightly reduced (1.8-fold) as compared to wild type EcoRV and that the single-turnover rate constant of DNA cleavage by E45A is reduced only 39-fold, whereas the D74A and D90A mutants are catalytically inactive under all conditions. These findings make an important catalytic function of Glu45, like binding of an essential divalent metal ion, unlikely. In addition, we have analyzed the dependence of the DNA cleavage rate by EcoRV and EcoRV mutants on the concentration of Mg2+ and Mn2+. We found for the wild type enzyme a sigmoidal dependence of the rate of DNA cleavage on the concentration of Mg2+ or Mn2+, indicative of at least two metal ions involved in DNA binding and catalysis. This, however, does not mean that EcoRV follows a two-metal-ion mechanism in DNA cleavage, because also for the E45A mutant a sigmoidal dependence of the rate of DNA cleavage on the Mg2+ concentration was found, making metal ion binding to the E45/D74 site unlikely. In contrast, the Y219C mutant shows a hyperbolic dependence. In agreement with results obtained earlier, these findings demonstrate binding of a Mg2+ ion at a site influenced by Tyr219, an amino acid residue that is far away from the active site. Metal binding at this site does not have a catalytic role but rather supports specific DNA binding. We conclude that on the basis of our data a two-metal-ion mechanism of DNA cleavage is unlikely for EcoRV and that the complex metal ion effects observed are due to metal ion binding at sites that are not directly involved in catalysis.