Reconstitution of human 5-hydroxytryptamine5A receptor--G protein coupling in E. coli and Sf9 cell membranes with membranes from Sf9 cells expressing mammalian G proteins.

The human 5-hydroxytryptamine5A (h5-ht5A) receptor was expressed in Escherichia coli (h5-ht5A-E. coli) to verify its pharmacological profile in the absence of G proteins. In addition, the ability of the h5-ht5A receptor to interact with mammalian Gi/o and Gs proteins was investigated by a new reconstitution approach. Agonists displayed lower affinities for h5-ht5A-E. coli than for stably transfected h5-ht5A-HEK 293 cells, due to the absence of G protein coupling in E. coli. Lysergic acid diethylamide behaved as a neutral antagonist, showing equal affinities for the G protein-coupled and the uncoupled receptor. To analyze the G protein coupling behavior of the h5-ht5A receptor, h5-ht5A-E. coli membranes or h5-ht5A-Sf9 insect cell membranes were fused by vortexing to membranes from baculovirus-infected Sf9 cells expressing mammalian G proteins. The ability of the h5-ht5A receptor to differentiate between Gi/Go/Gz and Gs proteins was explored by investigation of agonist binding affinities and agonist-induced stimulation of [35S]GTP gamma S binding. The h5-ht5A receptor failed to interact with Gz and Gs proteins and coupled equally well to Gj and Go proteins to form a complex with high affinity for agonists. Under the applied conditions, however, Gi proteins were found to be better activated than Go proteins in the [35S]GTP gamma S binding assay.

G-protein sensitivity of ligand binding to human dopamine D(2) and D(3) receptors expressed in Escherichia coli: clues for a constrained D(3) receptor structure.

Human dopamine D(2) and D(3) receptors were expressed in Chinese hamster ovary (CHO) and Escherichia coli cells to compare their ligand binding properties in the presence or absence of G-proteins and to analyze their ability to interact with G(i/o)-proteins. Binding affinities of agonists (dopamine, 7-OH-DPAT, PD128907, lisuride) and antagonists/inverse agonists (haloperidol, risperidone, domperidone, spiperone, raclopride, nemonapride), measured using [(125)I]iodosulpride and [(3)H]7-OH-DPAT, were similar for hD(3) receptors in E. coli and CHO cell membranes. Both agonists and antagonists showed 2- to 25-fold lower binding affinities at hD(2) receptors in E. coli versus CHO cell membranes (measured with [(3)H]spiperone), but the rank order of potencies remained similar. Purported inverse agonists did not display higher affinities for G-protein-free receptors. In CHO membranes, GppNHp decreased high affinity agonist ([(3)H]7-OH-DPAT) binding at hD(2) receptors but not at hD(3) receptors. Also, [(3)H]7-OH-DPAT (nanomolar concentration range) binding was undetectable at hD(2) but clearly measurable at hD(3) receptors in E. coli membranes. Addition of a G(i/o)-protein mix to E. coli membranes increased high affinity [(3)H]7-OH-DPAT binding in a concentration-dependent manner at hD(2) and hD(3) receptors; this effect was reversed by addition of GppNHp. The potency of the G(i/o)-protein mix to reconstitute high affinity binding was similar for hD(2) and hD(3) receptors. Thus, agonist binding to D(3) receptors is only slightly affected by G-protein uncoupling, pointing to a rigid receptor structure. Furthermore, we propose that the generally reported lower signaling capacity of D(3) receptors (versus D(2) receptors) is not due to its lower affinity for G-proteins but attributed to its lower capacity to activate these G-proteins.

Reconstitution of the human 5-HT(1D) receptor-G-protein coupling: evidence for constitutive activity and multiple receptor conformations.

The 5-hydroxytryptamine (5-HT) 1D/1B receptors have gained particular interest as potential targets for treatment of migraine and depression. G-protein coupling and other intrinsic properties of the human 5-HT(1D) receptor were studied using a baculovirus-based expression system in Sf9 cells. Coexpression of the human 5-HT(1D) receptor with Galpha(i1), alpha(i2), alpha(i3), or Galpha(o)-proteins and Gbeta(1)gamma(2)-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [(3)H]5-HT to this receptor, whereas the Galpha(q)beta(1)gamma(2) heterotrimer was ineffective in this respect. Competition of [(3)H]5-HT binding by various compounds confirmed that coexpression of the human 5-HT(1D) receptor with Galpha(i/o)beta(1)gamma(2) reconstitutes the receptor in a high affinity agonist binding state, having the same pharmacological profile as the receptor expressed in mammalian cells. Binding of the antagonist ocaperidone to the human 5-HT(1D) receptor in coupled or noncoupled state was analyzed. This compound competed with [(3)H]5-HT binding more potently on the human 5-HT(1D) receptor in the noncoupled state, showing its inverse agonistic character. Ocaperidone acted as a competitive inhibitor of [(3)H]5-HT binding when tested with the coupled receptor form but not so when tested with the noncoupled receptor preparation. Finally, [(35)S]GTPgammaS binding experiments using the inverse agonist ocaperidone revealed a high level of constitutive activity of the human 5-HT(1D) receptor. Taken together, the reconstitution of the human 5-HT(1D) receptor-G-protein coupling using baculovirus-infected Sf9 cells made possible the assessment of coupling specificity and the detection of different binding states of the receptor induced by G-protein coupling or ligand binding.

Human 5-hydroxytryptamine(5A) receptors activate coexpressed G(i) and G(o) proteins in Spodoptera frugiperda 9 cells.

The ability of the human 5-hydroxytryptamine serotonin type 5A (h5-ht(5A)) receptor to couple to G proteins from distinct families was investigated through the simultaneous infection of Spodoptera frugiperda 9 insect cells with recombinant baculoviruses encoding the various proteins. Expression of G proteins was demonstrated in immunoblots. Receptor-G protein coupling was monitored by high-affinity agonist binding and agonist-induced stimulation of [(35)S]guanosine-5'-O-(3-thio) triphosphate binding to membranes. Receptors expressed alone displayed low-affinity agonist binding, and endogenous G proteins were only poorly stimulated on the addition of 5-hydroxytryptamine. When receptors were coexpressed with mammalian G(i)/G(o) proteins (Galpha(i) or Galpha(o) plus Gbeta(1)gamma(2)), the coupled phenotype was achieved: agonists bound with high affinity in a guanosine-5'-(beta, gamma-imido)triphosphate-sensitive manner and stimulated [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to high levels. These effects were not observed on coexpression with G(z)/G(s)/G(q/11/16) or G(12/13). Various ligands were evaluated for their agonistic, antagonistic, or inverse agonistic behavior in both receptor binding and activation assays. Although G(o) displayed different receptor coupling characteristics than G(i) proteins, no clear coupling preference was evident. Coexpression of receptors and Galpha(i) subunits without Gbeta(1)gamma(2) produced increases in both agonist affinity and maximum G protein activation that were smaller than those in the presence of Gbeta(1)gamma(2), suggesting that Gbeta(1)gamma(2) coexpression improves receptor-G protein coupling. Similarly, coexpression of receptors with Gbeta(1)gamma(2) alone resulted in an improved interaction with endogenous G proteins. Our results demonstrate that h5-ht(5A) receptors expressed in Spodoptera frugiperda 9 cells selectively and functionally couple to coexpressed mammalian G(i) and G(o) proteins.

Acute transcriptional response of the honeybee peptide-antibiotics gene repertoire and required post-translational conversion of the precursor structures.

The cell-free immune repertoire of honeybees (Apis mellifera) consists of four polypeptides that are induced by bacterial infection and, through complementarity, provide broad-spectrum antibacterial defense. apidaecin is overproduced by a combination of low threshold transcriptional activation and a unique, genetically encoded amplification mechanism. In contrast, sizable experimental infections are required for induction of the normally silent hymenoptaecin, abaecin, and bee defensin genes; even so, bee defensin transcription is minimal and delayed, and only minute quantities of corresponding peptide are produced. The specific, temporal organization of the multi-component immune response in bees has therefore likely been selected to cope with infection of prevalent, plant-associated Gram-negative bacteria. Post-translational processing and modifications are substantially different for each of the four antibacterial peptides. While no similarities were observed among precursor structures of the various bee peptides, surprisingly, the signal sequences of abaecin (bee) and drosocin (Drosophila) shared unmistakable homology, possibly indicating common ancestral secretion/processing mechanisms. Finally, we report that bee defensin contains a typical disulfide-rich structure (40 amino acids) but also a unique, amphipathic, putatively amidated carboxyl-terminal tail (10 amino acids). We speculate that this structure is a "co-drug," assembled by fusing "disulfide-rich" and "alpha-helical" class peptide antibiotics, a novel concept in naturally occurring antibacterials.

Biodiversity of apidaecin-type peptide antibiotics. Prospects of manipulating the antibacterial spectrum and combating acquired resistance.

Insects have a unique repertoire of peptide antibiotics but, to date, prospects of clinical applications are not clear. Apidaecin, a small peptide isolated from honeybees, inhibits viability of Gram-negative bacteria; lethal activity is near immediate, independent of a conventional "lytic" mechanism, and involves stereoselective recognition of target molecules. Here we report structural analysis of 14 naturally occurring apidaecin-type peptides and the existence of evolutionarily conserved ("constant") regions. By detailed analysis of activities against clinically relevant bacteria, we demonstrate that the diversity of the intervening ("variable") regions confers specificity to the antibacterial spectrum of each homolog. As a result, apidaecin-homolog-based antibiograms (using 16 peptides) differ markedly between bacterial strains, contrasting the most between Yersinia enterocolitica and Campylobacter jejuni. Furthermore, in at least one instance, acquired resistance to apidaecin could be negated by minor substitutions in the variable regions. The delineation in a short peptide of constant and variable regions, responsible for, respectively, general antibacterial capacity and specificity of the antibacterial spectrum, is unprecedented. Taken together, we provide evidence that antibacterial spectra of apidaecin-type peptides can be manipulated, and that, in some cases, resistance can be countered and perhaps prevented. The current findings will guide rational design of second generation peptide antibiotics for clinical trials.

Costs of reproduction in a historical perspective.

Costs of reproduction constitute a core assumption of life history theory. After reformulation by G.C. Williams, the cost hypothesis soon became a major foundation of phenotypic life history models. More-recent studies have approached reproductive costs from the perspective of quantitative genetics. Here, we present a brief historical perspective to the development of the cost of reproduction hypothesis. We evaluate the status and heuristic value of the different approaches, and outline how the approaches have originated.

Apidaecin multipeptide precursor structure: a putative mechanism for amplification of the insect antibacterial response.

Apidaecins are the most prominent components of the honeybee humoral defense against microbial invasion. Our analysis of cDNA clones indicated that up to 12 of these short peptides (2 kDa) can be generated by processing of single precursor proteins; different isoforms are hereby linked in one promolecule. Assembly of the multipeptide precursors and the putative three-step maturation are strongly reminiscent of yeast alpha-mating factor. Bioactive apidaecins are flanked by the two 'processing' sequences, EAEPEAEP (or variants) and RR; joined together, they form a single unit that is repeated numerous times. The number of such repeats is variable and was reflected in the observed diversity of transcript lengths. Each such transcript is likely to be encoded by a different gene, forming a tight gene cluster. While transcriptional activation upon bacterial challenge is not exceptionally fast, the multigene and multipeptide precursor nature of the apidaecin genetic information allows for amplification of the response, resulting in a real overproduction of peptide antibiotic. Enhanced efficiency of the 'immune' response to bacterial infection through such a mechanism is, to our knowledge, unique among insects.

Lactobacillus hilgardii plasmid pLAB1000 consists of two functional cassettes commonly found in other gram-positive organisms.

A Lactobacillus hilgardii plasmid, pLAB1000, was studied to understand the organization of autonomous replicons from lactobacilli. Two cassettes could be identified. First, the replication region consisted of a sequence coding for a replication protein (Rep) and its corresponding target site, similar to those from plasmids pUB110, pC194 (Staphylococcus aureus), pFTB14, pBAA1 (Bacillus sp.), and pLP1 (Lactobacillus sp.). Sequence analysis indicated the possible synthesis of an antisense RNA that might regulate Rep production. The results also suggested that pLAB1000 replicates via a single-stranded DNA intermediate, and a putative lagging-strand initiation site was found that had similarities to those of alpha 3, St-1, and G4 isometric bacteriophages. The second cassette of pLAB1000 consisted of a sequence coding for a putative mobilization protein (Mob) and its corresponding RSA site. This cassette was similar to those found in pT181, pUB110, pE194 (S. aureus), and pG12 (Bacillus sp.), and it was found to be conserved among different Lactobacillus plasmid replicons. The origin and evolution of these functional cassettes are also discussed.

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii.

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.