Multiple functions of polypeptides mediated by distinct domains interacting with different receptors

There exist multiple functions of polypeptide molecules. Both a polypeptide molecule interacting with one receptor, and distinct domains of the molecule interacting with different receptors could induce different intracellular signal transduction to elicit multiple functions. This review highlights the distinct domains of the polypeptide molecule interacting with different receptors to elicit multiple functions. It includes distinct domains, different receptor mechanisms, and different signal transduction of the polypeptide molecule.     

Opioid antinociception and positive reinforcement are mediated by different types of opioid receptors

Fentanyl (FEN) and diprenorphine's (DIPR) potentials for analgesia and reinforcement were assayed using rats. Analgesia was measured by the classic tail-flick test. The test germane to opioid reinforcement involved measuring pressing rates for direct electrical stimulation of the lateral hypothalamus and ventral tegmental area. FEN, as does morphine and heroin, produced strong analgesia and enhanced pressing rates for brain stimulation. DIPR produced no analgesia and antagonized FEN's analgesia. DIPR, at doses antagonizing FEN's analgesia, enhanced pressing for brain stimulation. DIPR's enhancement of pressing was antagonized by naloxone (100 micrograms/kg). When FEN and DIPR were given concurrently, pressing for brain stimulation was not reduced and was greater than after FEN alone was given. These data support a conclusion that different types of receptors are associated with opioid analgesia and reinforcement.     

Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance

Extending axons in the developing nervous system are guided in part by repulsive cues. Genetic analysis in Drosophila, reported in a companion to this paper, identifies the Slit protein as a candidate ligand for the repulsive guidance receptor Roundabout (Robo). Here we describe the characterization of three mammalian Slit homologs and show that the Drosophila Slit protein and at least one of the mammalian Slit proteins, Slit2, are proteolytically processed and show specific, high-affinity binding to Robo proteins. Furthermore, recombinant Slit2 can repel embryonic spinal motor axons in cell culture. These results support the hypothesis that Slit proteins have an evolutionarily conserved role in axon guidance as repulsive ligands for Robo receptors.     

Olfactory functions are mediated by parallel and hierarchical processing

How the human brain processes the perception, discrimination, and recognition of odors has not been systematically explored. Cerebral activations were therefore studied with PET during five different olfactory tasks: monorhinal smelling of odorless air (AS), single odors (OS), discrimination of odor intensity (OD-i), discrimination of odor quality (OD-q), and odor recognition memory (OM). OS activated amygdala-piriform, orbitofrontal, insular, and cingulate cortices and right thalamus. OD-i and OD-q both engaged left insula and right cerebellum. OD-q also involved other areas, including right caudate and subiculum. OM did not activate the insula, but instead, the piriform cortex. With the exception of caudate and subiculum, it shared the remaining activations with the OD-q, and engaged, in addition, the temporal and parietal cortices. These findings indicate that olfactory functions are organized in a parallel and hierarchical manner.     

Flight initiations in Drosophila melanogaster are mediated by several distinct motor patterns

We have monitored the patterns of activation of five muscles during flight initiation of Drosophila melanogaster: the tergotrochanteral muscle (a mesothoracic leg extensor), dorsal longitudinal muscles #3, #4 and #6 (wing depressors), and dorsal ventral muscle #Ic (a wing elevator). Stimulation of a pair of large descending interneurons, the giant fibers, activates these muscles in a stereotypic pattern and is thought to evoke escape flight initiation. To investigate the role of the giant fibers in coordinating flight initiation, we have compared the patterns of muscle activation evoked by giant fiber stimulation with those during flight initiations executed voluntarily and evoked by visual and olfactory stimuli. Visually elicited flight initiations exhibit patterns of muscle activation indistinguishable from those evoked by giant fiber stimulation. Olfactory-induced flight initiations exhibit patterns of muscle activation similar to those during voluntary flight initiations. Yet only some benzaldehyde-induced and voluntary flight initiations exhibit patterns of muscle activation similar to those evoked by giant fiber stimulation. These results indicate that visually elicited flight initiations are coordinated by the giant fiber circuit. By contrast, the giant fiber circuit alone cannot account for the patterns of muscle activation observed during the majority of olfactory-induced and voluntary flight initiations.Abbreviations DLM/DLMn dorsal longitudinal muscle/motor neuron - DVM/DVMn dorsal ventral muscle/motor neuron - GF(s) giant fiber interneuron (s) - PSI peripherally synapsing interneuron - TTM/TTMn tergotrochanteral muscle/motor neuron     

Proliferative effect of acetylcholine on rat trachea epithelial cells is mediated by nicotinic receptors and muscarinic receptors of the M1-subtype

Acetylcholine (ACh), synthesized in mammalian non-neuronal cells such as epithelial cells of the airways, digestive tract and skin, is involved in the regulation of basic cell functions (so-called non-neuronal cholinergic system). In the present experiments rat trachea epithelial cells have been cultured to study the proliferative effect of applied ACh by [3H]thymidine incorporation. ACh (exposure time 24 h) caused a concentration-dependent increase in cell proliferation with a doubling of the [3H]thymidine incorporation at a concentration of 0.1 microM. This effect was partly reduced by 30 microM tubocurarine and completely abolished by the additional application of 1 microM atropine. The stimulatory effect of acetylcholine, remaining in the presence of tubocurarine, was prevented by 1 microM pirenzepine (preferentially acting at M1-receptors), but neither by 1 microM AFDX 116 (preferentially acting at M2-receptors) nor by 1 microM hexahydrosiladifenidol (preferentially acting at M3-receptors). The combination of tubocurarine and pirenzepine halved the basal [3H]thymidine incorporation. In conclusion, ACh produces a proliferative effect in rat trachea epithelial cells, the effect being mediated by both nicotinic receptors and muscarinic receptors of the M1-subtype.     

Anorectic actions of prolactin-releasing peptide are mediated by corticotropin-releasing hormone receptors

Prolactin-releasing peptide (PrRP) reduces food intake and body weight and modifies body temperature when administered centrally in rats, suggesting a role in energy homeostasis. However, the mediators of PrRP's actions are unknown. The present study, therefore, first examined the possible involvement of the anorectic neuropeptides corticotropin-releasing hormone (CRH) and the melanocortins (e.g., alpha-melanocyte-stimulating hormone) in PrRP's effects on food intake and core body temperature and, second, determined if PrRP affects energy expenditure by measuring oxygen consumption (Vo2). Intracerebroventricular injection of PrRP (4 nmol) to 24-h-fasted male Sprague-Dawley rats decreased food intake and modified body temperature. Blockade of central CRH receptors by intracerebroventricular coadministration of the CRH receptor antagonist astressin (20 microg) reversed the PrRP-induced reduction in feeding. However, astressin's effect on PrRP-induced changes in body temperature was complicated because the antagonist itself caused a slight rise in body temperature. In contrast, intracerebroventricular coadministration of the melanocortin receptor-3/4 antagonist SHU-9119 (0.1 nmol) had no effect on any of PrRP's actions. Finally, intracerebroventricular injection of PrRP (4 nmol) caused a significantly greater Vo2 over a 3-h test period compared with vehicle-treated rats. These results show that the anorectic actions of PrRP are mediated by central CRH receptors but not by melanocortin receptors-3/4 and that PrRP can modify Vo2.     

The targeting and functions of miRNA-383 are mediated by FMRP during spermatogenesis

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumors. However, the mechanisms underlying the targeting and functions of miR-383 during spermatogenesis remain unknown. In this study, we found that fragile X mental retardation protein (FMRP) was associated with 88 miRNAs in mouse testis including miR-383. Knockdown of FMRP in NTERA-2 (NT2) (testicular embryonal carcinoma) cells enhanced miR-383-induced suppression of cell proliferation by decreasing the interaction between FMRP and miR-383, and then affecting miR-383 binding to the 3′-untranslated region of its target genes, including interferon regulatory factor-1 (IRF1) and Cyclin D1 both in vivo and in vitro. On the other hand, FMRP levels were also downregulated by overexpression of miR-383 in NT2 cells and GC1 (spermatogonia germ cell line). miR-383 targeted to Cyclin D1 directly, and then inhibited its downstream effectors, including phosphorylated pRb and E2F1, which ultimately resulted in decreased FMRP expression. Reduced miR-383 expression, dysregulated cyclin-dependent kinase 4 expression (one of the downstream genes of miR-383) and increased DNA damage were also observed in the testes of Fmr1 knockout mice and of MA patients with a downregulation of FMRP. A potential feedback loop between FMRP and miR-383 during spermatogenesis is proposed, and FMRP acts as a negative regulator of miR-383 functions. Our data also indicate that dysregulation of the FMRP–miR-383 pathway may partially contribute to human spermatogenic failure with MA.     

PSA-NCAM: Synaptic functions mediated by its interactions with proteoglycans and glutamate receptors

Dynamic regulation of glycosylation of the neural cell adhesion molecule (NCAM) by an unusual large negatively charged polysialic acid (PSA) is the major prerequisite for correct formation of brain circuitries during development and for normal synaptic plasticity, learning and memory in the adult. Traditionally, PSA is viewed as a de-adhesive highly hydrated molecule, which interferes with cell adhesion and promotes cellular/synaptic dynamics by steric hindrance. Analysis of synaptic functions of PSA-NCAM highlighted additional features of this molecule. First, PSA promotes interaction of NCAM with heparan sulfate proteoglycans and thus stimulates synaptogenesis. Second, PSA-NCAM modulates glutamate receptors: it restrains activity of extrasynaptic GluN2B-containing NMDA receptors and facilitates activity of a subset of AMPA receptors. Perturbation in polysialylation and/or NCAM expression in mouse models recapitulates many symptoms of human brain disorders such as schizophrenia, depression, anxiety and Alzheimer's disease.     

Expression of the blistered/DSRF gene is controlled by different morphogens during Drosophila trachea and wing development

The Drosophila serum response factor (DSRF) is expressed in the precursors of the terminal tracheal cells and in the future intervein territories of the third instar wing imaginal disc. Dissection of the DSRF regulatory region reveals that a single enhancer element, which is under the control of the fibroblast growth factor (FGF)-receptor signalling pathway, is sufficient to induce DSRF expression in the terminal tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is active in the central intervein sector and is induced by the Hedgehog signalling pathway. The other element is under the control of Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector. Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and the localized expression of a gene required for the final differentiation of this territory.     

Two thermosensors in Drosophila have different behavioral functions

Insects inhabit extreme temperature environments and have evolved mechanisms to survive there. Small insects are especially susceptible to rapid changes in body temperature. Therefore, the rapid detection of environment and body temperature is important for their survival. Little, however, is known about the thermosensors that detect those temperatures. Using rapid thermosensitivity assays with temperature step gradients and a spatial learning paradigm (the heat-box) in which elevated temperature serves as the negative reinforcer, two thermosensors were identified and their behavioral functions assessed. A low-temperature thermosensor is located on the antenna, detects relatively low temperatures, and can detect spatial temperature gradients directly. Thus, the antennae can be used by Drosophila to quickly orient with respect to temperature cues. A high-temperature thermosensor of unknown location appears to have a roughly similar sensitivity to temperature differences as the low-temperature thermosensor (< or = 3 degrees C) and is both necessary and sufficient for memory formation in the heat-box spatial learning paradigm. Therefore, the high-temperature thermosensor is important for remembering spatial positions in which dangerously high temperatures were encountered.     

Novel mechanisms of tube-size regulation revealed by the Drosophila trachea

The size of various tubes within tubular organs such as the lung, vascular system and kidney must be finely tuned for the optimal delivery of gases, nutrients, waste and cells within the entire organism. Aberrant tube sizes lead to devastating human illnesses, such as polycystic kidney disease, fibrocystic breast disease, pancreatic cystic neoplasm and thyroid nodules. However, the underlying mechanisms that are responsible for tube-size regulation have yet to be fully understood. Therefore, no effective treatments are available for disorders caused by tube-size defects. Recently, the Drosophila tracheal system has emerged as an excellent in vivo model to explore the fundamental mechanisms of tube-size regulation. Here, we discuss the role of the apical luminal matrix, cell polarity and signaling pathways in regulating tube size in Drosophila trachea. Previous studies of the Drosophila tracheal system have provided general insights into epithelial tube morphogenesis. Mechanisms that regulate tube size in Drosophila trachea could be well conserved in mammalian tubular organs. This knowledge should greatly aid our understanding of tubular organogenesis in vertebrates and potentially lead to new avenues for the treatment of human disease caused by tube-size defects.     

Slit and Robo control the development of dendrites in Drosophila CNS

The molecular mechanisms that generate dendrites in the CNS are poorly understood. The diffusible signal molecule Slit and the neuronally expressed receptor Robo mediate growth cone collapse in vivo. However, in cultured neurons, these molecules promote dendritic development. Here we examine the aCC motoneuron, one of the first CNS neurons to generate dendrites in Drosophila. Slit displays a dynamic concentration topography that prefigures aCC dendrogenesis. Genetic deletion of Slit leads to complete loss of aCC dendrites. Robo is cell-autonomously required in aCC motoneurons to develop dendrites. Our results demonstrate that Slit and Robo control the development of dendrites in the embryonic CNS.     

High and low frequency electroacupuncture analgesia are mediated by different types of opioid receptors at spinal level: a cross tolerance study

Previous studies have shown that rats subjected to low or high frequency electroacupuncture (EA) stimulation release enkephalins or dynorphins respectively to produce analgesia. This conclusion was tested in the present study by using cross tolerance technique for further analysing their receptor mechanisms. The main results were as follows: (1) In rats subjected to 2 Hz EA for 6 h, there was a gradual decrease in the analgesic effect, leading to a state of tolerance to 2 Hz EA analgesia. These rats, however, still responded to 100 Hz EA. Likewise, rats made tolerant to 100 Hz EA were still effective to 2 Hz EA stimulation, showing not significant cross tolerance between 2 Hz and 100 Hz EA analgesia. (2) Rats made-tolerant to 100 Hz EA analgesia showed a diminished response to intrathecal dynorphin A (1-13), a kappa agonist, whereas the analgesic effect of the delta agonist [D-Pen2, D-pen5] enkephalin (DPDPE) remained intact. (3) Rats made tolerant to 2 Hz EA analgesia showed a cross tolerance to DPDPE, but not to dynorphin A (1-13). Results obtained from aforementioned cross tolerance studies suggest that 2 Hz and 100 Hz EA analgesia are mediated by delta and kappa opioid receptors, respectively, at the spinal cord of the rat.     

Induction and repression of the Drosophila Sgs-3 glue gene are mediated by distinct sequences in the proximal promoter.

The normal developmental expression of the Drosophila salivary gland secretion protein gene Sgs-3 requires the interaction of a distal and proximal regulatory element. A deletion/replacement analysis of the proximal promoter in stably transformed lines shows that induction of an Sgs-3/Adh fusion gene is normal if sequences from +10 to -50 are replaced by those of the hsp70 gene. Sequences between -98 and -50 are necessary for this expression but there is internal redundancy within this region as two distinct upstream sequences of 18 and 22 bp respectively are sufficient for stage- and tissue-specific expression, albeit at reduced levels. A point mutation at -53 eliminates the ecdysone-mediated repression of the Sgs-3 promoter at pupariation. We report mosaicisms of expression within the salivary gland for a number of stably transformed lines.     

Single Cl- channels activated by Ca2+ in Drosophila S2 cells are mediated by bestrophins

Mutations in human bestrophin-1 (VMD2) are genetically linked to several forms of retinal degeneration but the underlying mechanisms are unknown. Bestrophin-1 (hBest1) has been proposed to be a Cl(-) channel involved in ion and fluid transport by the retinal pigment epithelium (RPE). To date, however, bestrophin currents have only been described in overexpression systems and not in any native cells. To test whether bestrophins function as Ca(2+)-activated Cl(-) (CaC) channels physiologically, we used interfering RNA (RNAi) in the Drosophila S2 cell line. S2 cells express four bestrophins (dbest1-4) and have an endogenous CaC current. The CaC current is abolished by several RNAi constructs to dbest1 and dbest2, but not dbest3 or dbest4. The endogenous CaC current was mimicked by expression of dbest1 in HEK cells, and the rectification and relative permeability of the current were altered by replacing F81 with cysteine. Single channel analysis of the S2 bestrophin currents revealed an approximately 2-pS single channel with fast gating kinetics and linear current-voltage relationship. A similar channel was observed in CHO cells transfected with dbest1, but no such channel was seen in S2 cells treated with RNAi to dbest1. This provides definitive evidence that bestrophins are components of native CaC channels at the plasma membrane.     

DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context.

The homeo box gene even-skipped (eve) encodes a 376-amino-acid protein that binds with high affinity to sequences located near the 5' termini of the eve and en genes. The 5' en sites are A + T rich and contain copies of the 10-base-pair (bp) consensus sequence T-C-A-A-T-T-A-A-A-T. In contrast, the 5' eve sites are G + C rich and contain the 9-bp sequence T-C-A-G-C-A-C-C-G. Among the five different homeo box proteins that have been tested for binding, eve is unique in that it shows virtually equal preference for the A + T-rich 5' en binding sites and the G + C-rich 5' eve sites. Most of the other proteins bind with a relatively higher affinity to the en sites than to the eve sites. In an effort to identify the regions of the eve protein that are responsible for its efficient binding to both classes of recognition sequences, we analyzed the DNA-binding properties of various mutant eve proteins. These studies suggest that the homeo domain of the eve protein is responsible for both binding activities. However, mutations in distant regions of the protein influenced the binding behavior of the eve homeo domain and caused a reduction in binding to the G + C class of recognition sites. We propose that the protein context of the homeo domain can influence its DNA-binding properties.