Mapping the interaction site for ß-arrestin-2 in the prokineticin 2 receptor.

G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the ß-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with ß-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for ß-arrestin-2 binding and to investigate the role of ß-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to ß-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to ß-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the ß-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the ß-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.

Lipodepsipeptides from Pseudomonas syringae are partially proteolyzed and are not absorbed by humans: an in vitro study.

There are some concerns about the use of Pseudomonas-based products as biocontrol agents because of the hemolytic activity shown by their metabolites. The effects of Pseudomonas lipodepsipeptides (LDPs) on mammals via ingestion and the LDP degradation during the digestion and intestinal permeability have not been evaluated. In this research, the susceptibility of different LDPs to degradation was assayed with enzymatic gastrointestinal digestion, and intestinal permeability to LDPs was investigated in an in vitro system based on an intestinal cell layer system. Results demonstrated that trypsin and chymotrypsin hydrolyze up to 50% of the various LDPs, and that proteolysis was further increased by pronase E treatment. A decrease in LDP hemolytic activity matched LDP degradation during the various steps of the digestion process. Moreover, it was shown that syringomycin E (SRE), the main known LDP, was not able to cross the intestinal cell layer, suggesting that SRE does not reach the bloodstream in vivo. It was concluded that the Pseudomonas-based biocontrol products do not represent a serious risk for consumer health. In fact, LDPs possibly present on biocontrol-treated agricultural commodities would likely be partially digested by gastrointestinal enzymes and would not be absorbed at the intestinal level.

Fusicoccin effect on the in vitro interaction between plant 14-3-3 proteins and plasma membrane H+-ATPase.

A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H+-ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H+-ATPase. The binding was dependent on Mg2+ and was strongly increased by fusicoccin. Controlled trypsin digestion of H+-ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the C-terminal domain of H+-ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.

The H+-ATPase purified from maize root plasma membranes retains fusicoccin in vivo activation.

The activity of 'P-type' ATPases is modulated through the C-terminal autoinhibitory domain. The molecular bases of the regulation are unknown. Their understanding demands functional and structural studies on the activated purified enzyme. In this paper the plasma membrane H+-ATPase from maize roots activated in vivo by fusicoccin was solubilised and fractionated by anion-exchange HPLC. Results showed that the H+-ATPase separated from fusicoccin receptors retained fusicoccin activation and that it was more evident after enzyme insertion into liposomes. These data suggest that fusicoccin stimulation does not depend on a direct action of the fusicoccin receptor on the H+-ATPase, but rather, fusicoccin brings about a permanent modification of the H+-ATPase which very likely represents a general regulatory mechanism for 'P-type' ATPases.

The fungal H(+)-ATPase from Neurospora crassa reconstituted with fusicoccin receptors senses fusicoccin signal.

Fusicoccin affects several physiological processes regulated by the plasma membrane H(+)-ATPase in higher plants while other organisms having P-type H(+)-ATPases (e.g., fungi) are fusicoccin-insensitive. We have previously shown that fusicoccin binding to its receptor is necessary for H(+)-ATPase stimulation and have achieved the functional reconstitution into liposomes of fusicoccin receptors and the H(+)-ATPase from maize. In this paper we show that fusicoccin sensitivity can be conferred on the H(+)-ATPase from Neurospora crassa, a fungus insensitive to fusicoccin. In fact, H+ pumping by purified H(+)-ATPase from Neurospora crassa reconstituted into liposomes containing crude or partially purified fusicoccin receptors from maize was markedly enhanced by fusicoccin. The stimulation of H+ pumping by fusicoccin is dependent upon pH, fusicoccin, and protein concentration, as was reported for the system reconstituted with both proteins from maize.

The 30-kilodalton protein present in purified fusicoccin receptor preparations is a 14-3-3-like protein.

We have recently reported on the purification of the fusicoccin (FC) receptor from corn (Zea mays L.) and its identification by photoaffinity labeling (P. Aducci, A. Ballio, V. Fogliano, M.R. Fullone, M. Marra, N. Proietti [1993] Eur J Biochem 214: 339-345). Pure preparations of FC receptors, obtained under nondenaturing conditions, showed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis two doublets of proteins with apparent molecular masses of 30 and 90 kD. In the present paper we describe the isolation and identification of the primary structure of the 30-kD doublet proteins. Sequencing studies of peptides resulting from the digestion of the 30-kD protein showed a full identity with a 14-3-3-like protein from corn, named GF14. The 14-3-3 family is a class of proteins that is widely distributed in eukaryotes and is known to play various regulatory roles. The 30-kD protein has been immunologically identified by specific antibodies prepared against a synthetic peptide based on the determined amino acid sequence. A similar protein is recognized in partially purified FC receptor preparations from bean and spinach leaves.

Purification and photoaffinity labeling of fusicoccin receptors from maize.

Crude soluble proteins from plasma membranes of maize shoots were purified (following the increase of fusicoccin-binding specificity) by using an original multi-step HPLC procedure. The method, based on a combination of adsorption, ion-exchange and gel-filtration chromatographies, is quick, efficient and does not damage the binding activity. It allows a 5000-fold increase of specific activity; SDS/PAGE of purified fractions shows two doublets that correspond to proteins with apparent molecular masses of 90 kDa and 30 kDa. Crude or partially purified material was irradiated for various periods in the presence of a tritiated azido analogue of fusicoccin. The electrophoretic analysis of the irradiated material shows that with a short irradiation time only the 90-kDa band is radiolabeled, whereas, as the irradiation time increases, a 30-kDa band becomes radiolabeled and less radioactivity is detected in the 90-kDa band. Irradiation of the crude material in the absence of the analogue results in a decrease of the binding capability of fusicoccin. The irradiated preparation also shows a decrease of photolabeling of the 90-kDa band. Our data suggest that the 90-kDa protein is the functional fusicoccin receptor. This conclusion is at variance with results of other authors who suggest the 30-kDa protein as the true receptor.

Phospholipase A2 affects the activity of fusicoccin receptors.

Biochemical properties of fusicoccin receptors are strongly influenced by the phospholipid environment. In this report we have studied the effect of different exogenous phospholipases on fusicoccin binding ability of both plasma membrane and solubilised receptors. Among the phospholipases tested only phospholipase A2 showed an inhibitory effect on fusicoccin binding. In particular, the influence of this enzyme on the time course and reversibility of the fusicoccin binding reaction was studied. The inhibitory effect of phospholipase A2 was the consequence of fatty acid release. The usual fatty acids of plasma membrane phospholipids were active in inhibiting the interaction of fusicoccin with its receptors. It is concluded that a phospholipid associated to the fusicoccin receptor might play a significant role in the modulation of binding.

Some Properties of a Functional Reconstituted Plasmalemma H-ATPase Activated by Fusicoccin.

Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H(+)-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V(max) of the enzyme. Fusicoccin stimulation of maize H(+)-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).

Properties of proteoliposomes containing fusicoccin receptors from maize.

We have recently described a fusicoccin (FC)-sensitive system reconstituted by inserting into liposomes FC-receptors and H(+)-ATPase-enriched preparations from maize tissues. While the proteoliposomes of maize H(+)-ATPase had been already investigated, those of FC-receptors required a careful characterization before use in the dual system. In particular, the influence of the phospholipid environment on time-course, reversibility, and pH-dependence of the FC-binding reaction has been studied by comparing these properties in microsome-bound, solubilized, and liposome-entrapped receptors. Similarities and differences between the results of this investigation and those previously obtained with FC-receptors from spinach leaves suggest that functionally similar binding proteins from monocot and dicot plants have distinct structural features.

Functional reconstitution of a proton-translocating system responsive to fusicoccin.

Crude fusicoccin binding proteins and a partially purified plasma membrane H+-transporting ATPase (EC 3.6.1.34), both solubilized from maize tissues, were simultaneously inserted into liposomes by the freeze-thaw method. ATP-driven intravesicular acidification in the proteoliposomes, measured by the fluorescence quenching of the dye 9-amino-6-chloro-2-methoxyacridine, markedly increased upon addition of fusicoccin to the reconstituted system. This effect could not be observed when binding sites and ATPase preparations were separately reconstituted into the proteoliposomes, thus demonstrating that fusicoccin binding to its receptor is a prerequisite for ATPase stimulation.