Deficiency of Innate Immunity against Pseudomonas aeruginosa Enhances Behavioral Avoidance via the HECW-1/NPR-1 Module in Caenorhabditis elegans.

To antagonize infection of pathogenic bacteria in soil and confer increased survival, Caenorhabditis elegans employs innate immunity and behavioral avoidance synchronously as the two main defensive strategies. Although both biological processes and their individual signaling pathways have been partially elucidated, knowledge of their interrelationship remains limited. The current study reveals that deficiency of innate immunity triggered by mutation of the classic immune gene pmk-1 promotes avoidance behavior in C. elegans and vice versa. Restoration of pmk-1 expression using the tissue-specific promoters suggested that the functional loss of both intestinal and neuronal pmk-1 is necessary for the enhanced avoidance. Additionally, PMK-1 colocalized with the E3 ubiquitin ligase HECW-1 in OLL neurons and regulated the expressional level of the latter, which consequently affected the production of NPR-1, a G-protein-coupled receptor (GPCR) homologous to the mammalian neuropeptide Y receptor, in RMG neurons in a non-cell-autonomous manner. Collectively, our study illustrates that once the innate immunity is impaired when C. elegans antagonizes bacterial infection, the other defensive strategy of behavioral avoidance can be enhanced accordingly via the HECW-1/NPR-1 module, suggesting that GPCRs in neural circuits may receive the inputs from the immune system and integrate those two systems for better adapting to the real-time status.

Strategic Aspects of NPY-Based Monoclonal Antibodies for Diagnosis and Treatment of Breast Cancer.

Immunotherapy emerges as a treatment strategy for breast cancer marker, diagnosis and treatment. In this review, monoclonal antibodies (mAbs)-based passive and peptide vaccines as active immunotherapy approaches like activation of B-cells and T-cells are studied. Passive immunotherapy is mAbs-based therapy effective against tumor cells, which acts by targeting HER2, IGF 1R, VEGF, BCSC and immune checkpoints. Neuropeptide Y (NPY) and GPCR are the areas of interest to target BC metastases for on-targeting therapeutic action. Neuropeptide S (NPS) or NPS receptor 1, acts as a biomarker for Neuroendocrine tumors (NET), mostly characterized by synaptophysin and chromogranin-A expression or Ki-67 proliferation index. The protein fusion technologies arise as a promising avenue in plant expression systems for increased recombinant Ab accumulation and cost-efficient purification. Recently, mAbs-based immunotherapy effectiveness is appreciated as a novel therapeutic combination of chemotherapy and immunotherapy to reduce the side effects and improve therapeutic responsiveness. Synthetic drug resistance will be overcome by mAbs-based therapy through several clinical trials and detection methods need to be optimized for accuracy and precision. Pharmacokinetic attributes need to be accessed for preferred receptor-agonist activity without ligand accumulation.

Non-neuronal neuropeptide Y and its receptors during acute rejection of rat pulmonary allografts.

This study tested the hypothesis that neuropeptide Y (NPY) and NPY receptors 1 (Y1) and 2 (Y2) participate in lung allograft rejection. Inflammation in grafts may include interaction between blood leukocytes and graft endothelial cells and marked accumulation of intravascular blood leukocytes. Fewer leukocytes accumulate in lung than in kidney allografts. Lung transplantion was performed in the Dark Agouti to Lewis rat strain combination. Intravascular and intraalveolar leukocytes were isolated from the grafts, and we evaluated the mRNA expression of NPY, Y1, and Y2 by real-time RT-PCR as well as the peptide expression of NPY by radioimmunoassay and immunohistochemistry. NPY and Y1 were expressed by pulmonary intravascular and intraalveolar leukocytes. Y1 was up-regulated by pulmonary intravascular and intraalveolar leukocytes during allograft rejection while Y2 could not be detected. Higher NPY expression levels in intravascular leukocytes were observed in lung compared to kidney allografts, which were investigated previously. Our findings suggest that an increased leukocytic expression of NPY in lung compared to kidney allografts results in a reduced accumulation of leukocytes in allograft vessels.

The homeostatic role of neuropeptide Y in immune function and its impact on mood and behaviour.

Neuropeptide Y (NPY), one of the most abundant peptides in the nervous system, exerts its effects via five receptor types, termed Y1, Y2, Y4, Y5 and Y6. NPY's pleiotropic functions comprise the regulation of brain activity, mood, stress coping, ingestion, digestion, metabolism, vascular and immune function. Nerve-derived NPY directly affects immune cells while NPY also acts as a paracrine and autocrine immune mediator, because immune cells themselves are capable of producing and releasing NPY. NPY is able to induce immune activation or suppression, depending on a myriad of factors such as the Y receptors activated and cell types involved. There is an intricate relationship between psychological stress, mood disorders and the immune system. While stress represents a risk factor for the development of mood disorders, it exhibits diverse actions on the immune system as well. Conversely, inflammation is regarded as an internal stressor and is increasingly recognized to contribute to the pathogenesis of mood and metabolic disorders. Intriguingly, the cerebral NPY system has been found to protect against distinct disturbances in response to immune challenge, attenuating the sickness response and preventing the development of depression. Thus, NPY plays an important homeostatic role in balancing disturbances of physiological systems caused by peripheral immune challenge. This implication is particularly evident in the brain in which NPY counteracts the negative impact of immune challenge on mood, emotional processing and stress resilience. NPY thus acts as a unique signalling molecule in the interaction of the immune system with the brain in health and disease.

Differential expression of neuropeptide Y Y1 receptors during pulpal inflammation.

AIM: To study the pattern of neuropeptide Y (NPY) Y1 receptor (Y1R) localization in the normal dental pulp and during different stages of pulpal inflammation. The hypothesis was that the expression of Y1R varies during different stages of pulpitis. METHODOLOGY: Pulp exposure injury was made on first molar teeth of Sprague-Dawley rats. Animals were killed 3, 7, 14, 21, 28 and 46 days after pulp exposure. Jaws were removed, decalcified and processed for immunohistochemistry for identification and localization of Y1R. Double labelling was performed using antibodies for laminin, CD43 and CD4 with Y1R. RT-PCR was performed to verify gene expression of Y1R in dental pulp and trigeminal ganglion. RESULTS: RT-PCR revealed the presence of NPY-Y1R in the dental pulp, trigeminal ganglion and gingiva. With immunohistochemistry, in control rats, Y1R was mainly located in capillaries and small vessels in the dental pulp as well as in the odontoblastic layer. No YIR was noted on immune cells in normal pulp. In pulpitis, immune cells such as CD43+ granulocytes and CD4+ lymphocytes expressed Y1R. As pulpitis progressed, there was a significant decrease in number of blood vessels expressing Y1R in the odontoblast layer when compared to control rat pulp. CONCLUSIONS: This study gives evidence that Y1R is a modulator of pulpal inflammation.

Modulation of granulocyte functions by peptide YY in the rat: age-related differences in Y receptors expression and plasma dipeptidyl peptidase 4 activity.

It has been acknowledged that aging exerts detrimental effects on cells of the innate immune system and that neuropeptides, including neuropeptide Y (NPY) and NPY-related peptides fine-tune the activity of these cells through a receptor specific mechanism. The present study investigated the age-dependent potential of peptide YY (PYY) to modulate different granulocyte functions. The PYY reduced the carrageenan-elicited granulocyte accumulation into the air-pouch of aged (24 months) rats, and markedly decreased the phagocytosis of zymosan, as well as the H(2)O(2) production, when applied in vivo (20 microg/air-pouch). The anti-inflammatory effect of PYY was less prominent in adult (8 months) and young (3 months) rats. However, the proportions of granulocytes expressing Y1, Y2 and Y5 receptor subtypes were significantly lower in both aged and young rats when compared to adult rats. Furthermore, the aging was found to be associated with the diminished dipeptidyl peptidase 4 (DP4, an enzyme converting the NPY and PYY to Y2/Y5 receptor selective agonists) activity in plasma. In conclusion, the diverse age-related anti-inflammatory effect of PYY in rats originates from different expression levels of Y1, Y2, and Y5 receptor subtypes in addition to different plasma DP4 activity.

Neural regulation of immunity: role of NPR-1 in pathogen avoidance and regulation of innate immunity.

The nervous and immune systems consist of complex networks that have been known to be closely interrelated. However, given the complexity of the nervous and immune systems of mammals, including humans, the precise mechanisms by which the two systems influence each other remain understudied. To cut through this complexity, we used the nematode Caenorhabditis elegans as a simple system to study the relationship between the immune and nervous systems using sophisticated genetic manipulations. We found that C. elegans mutants in G-protein coupled receptors (GPCRs) expressed in the nervous system exhibit aberrant responses to pathogen infection. The use of different pathogens, different modes of infection and genome-wide microarrays highlighted the importance of the GPCR NPR-1 in avoidance to certain pathogens and in the regulation of innate immunity. The regulation of innate immunity was found to take place at least in part through a mitogen-activated protein kinase signaling pathway similar to the mammalian p38 MAPK pathway. Here, the results that support the different roles of the NPR-1 neural circuit in the regulation of C. elegans responses to pathogen infection are discussed.

Control of delayed-type hypersensitivity by ocular- induced CD8+ regulatory t cells.

The immunoregulatory pathway from the eye to the peri - pheral immune system is comprised of the iris, ciliary body, circulation, thymus and spleen, and is influenced by the sympathetic nervous system. At the splenic end of this pathway are antigen-specific CD8+ regulatory T cells (Tregs) that mediate directly the suppression of T cells that effect delayedtype hypersensitivity (DTH). Here we review investigations that demonstrate: (i) the injection of antigen into the anterior chamber (AC) attracts circulating monocytic cells to the iris/ciliary body that recirculate to the thymus and spleen. In the thymus, ocular-influenced monocytic cells activate natural killer T (NKT) cells that migrate to the spleen where, in concert with the ocular-influenced monocytic emigrants, they (ii) activate CD4+ and CD8+ immunoregulatory T cells. (iii) The generation of the CD8+ Tregs is dependent on NKT cells in the thymus and the periphery that are influenced by the sympathetic nervous system. (iv) The suppression of DTH by the AC-induced CD8+ Tregs is dependent on the cytokines transforming growth factor-Beta and interferon-gamma and is restricted by the expression of major histocompatibility complex-associated Qa-1b antigens. In aggregate, this oculothymic- splenic pathway is a well-controlled response to ocular injury that utilizes a systemic response to antigen that may protect ocular tissue and systemic tissue.

NPY and receptors in immune and inflammatory diseases.

Growing evidence suggests that neuropeptide Y (NPY) plays an important role in the immune system. NPY is produced by the central and peripheral nervous system but also by immune cells in response to activation. NPY has pleiotropic effects on both the innate and adaptive arms of the immune system, with effects ranging from the modulation of cell migration to macrophage, T helper (Th) cell cytokine release, and antibody production. Subsequent studies have confirmed the importance of this system in immunity in particular via the demonstration that Y1, a receptor for NPY, plays a fundamental role in autoimmunity and inflammation using Y1-deficient animals. Furthermore, clinical studies have suggested a role for NPY in other immune disorders such as asthma and arthritis. This review provides the latest information on the role of NPY and Y1 in the immune system, and discusses the potential new opportunities of this work for the development of a new generation of immuno-modulatory treatments of autoimmune and inflammatory diseases.

Src tyrosine kinases mediate activations of NF-kappaB and integrin signal during lipopolysaccharide-induced acute lung injury.

Src tyrosine kinases (TKs) are signaling proteins involved in cell signaling pathways toward cytoskeletal, membrane and nuclear targets. In the present study, using a selective Src TK inhibitor, PP1, we investigated the roles of Src TKs in the key pulmonary responses, NF-kappaB activation, and integrin signaling during acute lung injury in BALB/C mice intratracheally treated with LPS. LPS resulted in c-Src phosphorylation in lung tissue and the phospho-c-Src was predominantly localized in recruited neutrophils and alveolar macrophages. PP1 inhibited LPS-induced increases in total protein content in bronchoalveolar lavage fluid, neutrophil recruitment, and increases in the production or activity of TNF-alpha and matrix metalloproteinase-9. PP1 also blocked LPS-induced NF-kappaB activation, and phosphorylation and degradation of IkappaB-alpha. The inhibition of NF-kappaB activation by PP1 correlated with a depression of LPS-induced integrin signaling, which included increases in the phosphorylations of integrin beta(3), and of the focal adhesion kinase (FAK) family members, FAK and Pyk2, in lung tissue, and reductions in the fibrinogen-binding activity of alveolar macrophages. Moreover, treatment with anti-alpha(v), anti-beta(3), or Arg-Gly-Asp-Ser (RGDS), inhibited LPS-induced NF-kappaB activation. Taken together, our findings suggest that Src TKs play a critical role in LPS-induced activations of NF-kappaB and integrin (alpha(v)beta(3)) signaling during acute lung injury. Therefore, Src TK inhibition may provide a potential means of ameliorating inflammatory cascade-associated lung injury.

Neuropeptide Y Y1 receptors mediate morphine-induced reductions of natural killer cell activity.

Morphine suppresses a number of immune parameters, such as natural killer (NK) cell activity and lymphocyte proliferation, by acting through mu-opioid receptors in the central nervous system. Prior studies have implicated the sympathetic nervous system in mediating the immunomodulatory effects of acute morphine treatment. However, the peripheral mechanism whereby morphine inhibits NK cell activity is not fully understood. The aim of the present study was to investigate the role of the sympathetic transmitter neuropeptide Y (NPY) in mediating morphine-induced immune alterations. The results showed that administration of the selective NPY Y1 receptor antagonist BIBP3226 blocked morphine's effect on splenic NK activity but did not attenuate the suppression splenocyte proliferative responses to Con-A or LPS. Furthermore, intravenous NPY administration produced a dose-dependent inhibition of splenic NK activity but did not suppress lymphocyte proliferation. Recent studies from our laboratory have demonstrated that morphine modulates NK activity through a central mechanism that requires the activation of dopamine D1 receptors in the nucleus accumbens. Results from the present study showed that microinjection of the D1 receptor agonist SKF-38393 into the nucleus accumbens shell induced a suppression of NK activity that was reversed by BIBP3226. Collectively, these findings demonstrate that NPY Y1 receptors mediate morphine's suppressive effect on NK activity and further suggest that opioid-induced increases in nucleus accumbens D1 receptor activation inhibit splenic NK activity via NPY released from the sympathetic nervous system.

A fundamental bimodal role for neuropeptide Y1 receptor in the immune system.

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY) in the central nervous system is a major regulator of numerous physiological functions, including stress. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five Y receptors (Y1, Y2, Y4, Y5, and y(6)). Using Y1-deficient (Y1(-/-)) mice, we showed that Y1(-/-) T cells are hyperresponsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signaling through Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, Y1(-/-) mice were resistant to T helper type 1 (Th1) cell-mediated inflammatory responses and showed reduced levels of the Th1 cell-promoting cytokine interleukin 12 and reduced interferon gamma production. This defect was due to functionally impaired antigen-presenting cells (APCs), and consequently, Y1(-/-) mice had reduced numbers of effector T cells. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. Our findings uncover a sophisticated molecular mechanism regulating immune cell functions that can lead to stress-induced immunosuppression.

Neuropeptide Y: a new mediator linking sympathetic nerves, blood vessels and immune system?

Neuropeptide Y (NPY(1-36)), a sympathetic cotransmitter and neurohormone, has pleiotropic activities ranging from the control of obesity to anxiolysis and cardiovascular function. Its actions are mediated by multiple Gi/o-coupled receptors (Y1-Y5) and modulated by dipeptidyl peptidase IV (DPPIV/cd26), which inactivates NPY's Y1-agonistic activity but generates the Y2 and Y5-agonist, NPY(3-36). Released by sympathetic activity, NPY is a major mediator of stress, responsible for prolonged vasoconstriction via Y1 receptors. Y1 receptors also mediate NPY's potent vascular growth-promoting activity leading in vivo in rodents to neointima formation. This and the association of a polymorphism of the NPY signal peptide with increased lipidemia and carotid artery thickening in humans strongly suggest NPY's role in atherosclerosis. NPY and DPPIV/cd26 are also coexpressed in the endothelium, where the peptide activates angiogenesis. A similar system exists in immune cells, where NPY and DPPIV/cd26 are coactivated and involved in the modulation of cytokine release and immune cell functions. Thus, NPY, both a messenger and a modulator for all three systems, is poised to play an important regulatory role facilitating interactions among sympathetic, vascular and immune systems in diverse pathophysiological conditions such as hypertension, atherosclerosis and stress-related alterations of immunity.

Localization of neuropeptide Y1 receptor immunoreactivity in the rat retina and the synaptic connectivity of Y1 immunoreactive cells.

Neuropeptide Y (NPY), an inhibitory neuropeptide expressed by a moderately dense population of wide-field amacrine cells in the rat retina, acts through multiple (Y1-y6) G-protein-coupled receptors. This study determined the cellular localization of Y1 receptors and the synaptic connectivity of Y1 processes in the inner plexiform layer (IPL) of the rat retina. Specific Y1 immunoreactivity was localized to horizontal cell bodies in the distal inner nuclear layer and their processes in the outer plexiform layer. Immunoreactivity was also prominent in cell processes located in strata 2 and 4, and puncta in strata 4 and 5 of the IPL. Double-label immunohistochemical experiments with calbindin, a horizontal cell marker, confirmed Y1 immunostaining in all horizontal cells. Double-label immunohistochemical experiments, using antibodies to choline acetyltransferase and vesicular acetylcholine transporter to label cholinergic amacrine cell processes, demonstrated that Y1 immunoreactivity in strata 2 and 4 of the IPL was localized to cholinergic amacrine cell processes. Electron microscopic studies of the inner retina showed that Y1-immunostained amacrine cell processes and puncta received synaptic inputs from unlabeled amacrine cell processes (65.2%) and bipolar cell axon terminals (34.8%). Y1-immunoreactive amacrine cell processes most frequently formed synaptic outputs onto unlabeled amacrine cell processes (34.0%) and ganglion cell dendrites (54.1%). NPY immunoreactivity in the rat retina is distributed primarily to strata 1 and 5 of the IPL, and the present findings, thus, suggest that NPY acts in a paracrine manner on Y1 receptors to influence both horizontal and amacrine cells.

Effect of neuropeptide Y on inflammatory paw edema in the rat: involvement of peripheral NPY Y1 and Y5 receptors and interaction with dipeptidyl-peptidase IV (CD26).

Several lines of evidence suggest that neuropeptide Y (NPY) may exert regulatory effects in local inflammatory responses. Here, we show that intraplantarly (i.pl.) applied NPY, peptide YY (PYY), and an NPY Y5 receptor-selective agonist dose-dependently potentiate concanavalin A (Con A)-induced paw edema in the rat. The NPY Y1 receptor antagonist BIBO 3304 abolishes the pro-inflammatory action of both NPY and PYY while the dipeptidyl-peptidase IV (CD26) inhibitor Ile-thiazolidide exerted synergistic and potentiating effects in vivo. Taken together, the present data reveal an NPY Y1/Y5 receptor interplay and an involvement of CD26 in the NPY-induced potentiation of paw edema in the rat.

Association of neuropeptide Y Y1 receptors with glutamate-positive and NPY-positive neurons in rat hippocampal cultures.

The hippocampus is particularly enriched with neuropeptide tyrosine (NPY) and NPY receptors including the Y1, Y2 and Y5 subtypes. We have previously reported on the enrichment of cultured rat hippocampal neurons in specific [125I][Leu31, Pro34]PYY/BIBP3226-sensitive (Y1) binding sites and Y1 receptor mRNAs [St-Pierre et al. (1998) Br. J. Pharmacol., 123, p183]. We have now identified which cell types express the Y1 receptor. The majority of Y1 receptors, visualized using either the radiolabeled probe [125I][Leu31,Pro34]PYY or two antibodies directed against distinct domains of the Y1 receptor, was expressed in neurons as revealed by neuron-specific enolase (NSE) immunostaining. One antibody was directed against the second extracelllular loop of the Y1 receptor (amino acids 185-203) whereas the second was directed against the intracellular C-terminal loop (amino acids 355-382). The labelling was evident over both perikarya and processes. Neurons labelled by the various Y1 receptor probes were mostly glutamate-positive as revealed by double immunostaining. Most interestingly, a number of NPY-positive cultured hippocampal neurons were also enriched with the Y1 receptor, suggesting that this subtype may act as an autoreceptor to regulate NPY release in the hippocampus. These results thus provide an anatomical basis for the modulation of glutamate and NPY release by the Y1 receptor in the hippocampus.

Neuropeptide Y innervation and neuropeptide-Y-Y1-receptor-expressing neurons in the paraventricular hypothalamic nucleus of the mouse.

The paraventricular hypothalamic nucleus (PVH) serves as integrator and link between the neuroendocrine and autonomic nervous systems. Neuropeptide-Y (NPY)-producing neurons in the arcuate nucleus project to the PVH, where neurons expressing NPY Y1 receptor (Y1R) have been demonstrated. This projection has been suggested to be involved in the regulation of parameters related to energy metabolism, e.g. food intake and thermoregulation. The present study aimed at characterizing this pathway and chemically defining Y1R-expressing neurons by means of immunohistochemistry. The densely distributed NPY-immunoreactive (ir) terminals in the PVH co-stained for agouti gene-related protein (AGRP) mainly in the medial parvocellular regions, indicating an origin in the arcuate nucleus. This was in contrast to noradrenergic/adrenergic terminals in the PVH, which were less frequently seen to contain NPY-like immunoreactivity. Furthermore, AGRP-ir terminals were seen forming abundant close appositions on Y1R-ir cell bodies. Double staining revealed co-existence of Y1R-like immunoreactivity and immunoreactivities for thyrotropin-releasing hormone (TRH) and, to a minor extent, cocaine- and amphetamine-regulated transcript peptide in parvocellular neurons. No Y1R-like immunoreactivity was noted in parvocellular neurons expressing corticotropin-releasing hormone or in magnocellular neurons expressing vasopressin or oxytocin. The present results suggest that the arcuatoparaventricular NPY projection targets the TRH neurons preferentially via the Y1R, whereas the NPYergic regulation of corticotropinergic and magnocellular neurons may be relayed through other subtypes of NPY receptors. This study further defines the link between NPY-induced feeding and the hypothalamus-pituitary-thyroid axis.

Comparison of antibodies directed against receptor segments of NPY-receptors.

The Y1-, Y2-, Y4- and Y5-receptor, which belong to the rhodopsin-like G-protein coupled, 7 transmembrane helix spanning receptors, bind the 36-mer neuromodulator NPY (neuropeptide Y) with nanomolar affinity. Synthetic fragments of the second (E2) and third (E3) extracellular loop were used to generate subtype selective anti-receptor antibodies against the Y-receptors. As investigated on intact receptors by ELISA and on solubilized receptors by SDS-PAGE and subsequent Western blotting, subtype selectivity was only partly achieved. Nevertheless, selectivity can be obtained by using several antisera in combination. These antibodies represent tools for molecular mass determination, receptor purification by affinity chromatography with antibody-columns and receptor localization studies.

Diazepam and buspirone alter neuropeptide Y-like immunoreactivity in rat brain.

Neuropeptide Y-like immunoreactivity (NPY-LI) was investigated in naIve Sprague-Dawley rats subjected to acute, subchronic (7 days) or chronic (21 days) intraperitoneal treatment with diazepam (1 or 3 mg/kg once daily) or buspirone (1.5 or 5 mg/kg twice daily). NPY-LI was determined by radioimmunoassay in the amygdala, nucleus accumbens, hypothalamus and frontal cortex 24 h after the last dose of the drugs. Amygdala NPY-LI decreased after acute diazepam (3 mg/kg) or buspirone (1.5 mg/kg) and increased after subchronic treatment with both doses of diazepam and after chronic buspirone (1.5 mg/kg) treatment. Both diazepam and buspirone given in subchronic and chronic doses decreased NPY-LI levels in the nucleus accumbens. Hypothalamic NPY-LI changed only after chronic treatment: it decreased after diazepam and increased after buspirone (5 mg/kg). NPY-LI content in the frontal cortex decreased after subchronic diazepam (3 mg/kg) treatment and slightly increased after buspirone. The study has shown that both diazepam and buspirone affect NPY-LI levels in rats. These results suggest that the NPY system in the amygdala and nucleus accumbens is implicated in the anxiolytic effects of the drugs studied.

Probing of the neuropeptide Y-Y1-receptors interaction with anti-receptor antibodies.

The Y1 receptor, which belongs to the family of rhodopsin-like GTP-binding protein-coupled, seven-transmembrane helix-spanning receptors, binds the 36-mer neuromodulator neuropeptide Y (NPY) with nanomolar affinity. Synthetic fragments of the N-terminus, extracellular loops and C-terminus of the Y1 receptor were used to generate 18 anti-receptor antibodies; ten of them recognize the receptor expressed on intact cells as well as on membranes that have been prepared (with the exception of one antibody raised against the intracellular C-terminus) as investigated by ELISA. SDS/PAGE of solubilized membranes, subsequent Western blotting and staining with the antibodies revealed two proteins of 73 kDa and 51 kDa for both, the rat and the human receptor. Competition with neuropeptide Y showed that the binding of seven antibodies is strongly inhibited in the presence of the native ligand. Using photoactivatible analogues, it could be demonstrated that the competition efficiency strongly depends on the position of the crosslinker within the ligand. Based on these studies, a model for the ligand-receptor interaction is suggested. These antibodies represent novel tools for the structural characterization of the Y1 receptor and its interaction with NPY and antagonists as well as for localization studies.