Asthma is a disease of airway smooth muscle (ASM) dysfunction. ASM hypertrophy, hyper-reactivity, pathological remodeling and airway obstruction are well-established disease factors in asthma patients. A more recent concept is that ASM cells are immune effectors that contribute to inflammatory signaling in asthma. Calmodulin kinase II (CaMKII) is a hypertrophic and proinflammatory signal in myocardium. Although CaMKII is abundant in ASM, the potential for CaMKII to contribute to hypertrophy and inflammation in ASM are unknown. We hypothesize that CaMKII is a key determinant for these asthma-related ASM phenotypes. Collaborative work by our team has already established that CaMKII is a drugable target that contributes to hypertrophy, dysfunction and abnormal intracellular Ca2+ homeostasis in myocardium. Our exciting preliminary data suggest CaMKII contributes to bronchiolar hyper-reactivity in vivo and that CaMKII activates hypertrophic and proinflammatory gene programs in smooth muscle in vitro and in vivo. Despite the potential for CaMKII to play a central role in the pathophysiology of ASM, CaMKII has been neglected by the asthma field. Our studies will test the role of CaMKII in ASM:

Asthma is a severe disorder characterized by chronic inflammation of the airways and increased responsiveness to stimuli of bronchoconstriction. A number of complex inflammatory processes are involved in mediating the pathological manifestations of the disease. Both immune (e.g., T cells, dendritic cells) and non-immune inflammatory cells (e.g., macrophages and granulocytes), as well as mast cells play important roles in establishing inflammation in the lungs. In addition to cytokines, these cells secrete a number of proteases that act as mediators of the overall inflammatory process. While significant efforts have been made to understand the role of serine proteases such as tryptase, chymase and elastase and their roles in activation of protease activated receptors (PARs), very little is know about the roles of the cysteine cathepsins that are also secreted by immune/inflammatory cells at sites of inflammation in the lung. In this proposal we outline plans to assess the specific contributions of the cysteine cathepsins to disease pathology in asthma. Specifically we plan to 1) Use fluorescently labeled, activity-based probes to profile the in vivo repertoire of cysteine cathepsins in the lung in both acute and chronic mouse models of asthma and 2) Use small molecule inhibitors to assess the contribution of cysteine cathepsin activity to airway hyperreactivity (AHR), airway remodeling and inflammation. These studies will allow us to determine when, where and how cysteine cathepsins impact the overall process of inflammation in the lung and will help to validate this family of proteases as target for development of novel asthma drugs.

We have shown that ß2AR null mice mimic chronic nadolol treatment with regards to reducing mucous metaplasia and inflammatory cells in BALF. We also know that the ß2AR null mice have a greatly reduced airway resistance response to methacholine. To determine whether the critical role of ß2-adrenoceptor signaling in potentiation of allergic airway inflammation is in hematopoietic cells or lung parenchymal cells or both, we will perform the following experiments using ß2-adrenoceptor knockout (KO) mice and wild type (WT) controls. Based on preliminary studies our hypothesis is that ß2-adrenoceptors on airway epithelial cells are mediating mucous metaplasia. We will transplant bone marrow cells (for hematopoietic reconstitution) and splenocytes (for lymphoid reconstitution) harvested from ß2AR-null mice and littermate controls into lethally irradiated ß2AR-/- and ß2AR+/+ mice. We will then produce 4 groups of mice to study: (i) by transplanting inflammatory cells from ß2AR KO mice into irradiated WT mice, we will obtain mice with ß2AR in all tissues (including the lung) except in inflammatory cells (e.g. lymphocytes, dendritic cells, granulocytes, NK cells), (ii) by transplanting irradiated ß2AR KO mice with inflammatory cells harvested from WT mice we will obtain mice with ß2AR only in inflammatory cells, (iii) by transplanting inflammatory cells of WT mice into irradiated WT mice we will obtain mice with ß2AR in all tissues, and (iv) by transferring inflammatory cells from ß2AR KO mice into irradiated ß2AR KO mice we will obtain mice with no ß2AR in any tissue. A fifth group that will be an additional control will be WT mice that have had no treatment. We will then study each group in a model of allergic asthma as before (OVA sensitized and challenged) and determine which component (e.g. AHR, mucous metaplasia, eosinophilic airway inflammation, cytokine production) is altered by the absolute or selective absence on ß2AR signaling. We will measure mucous metaplasia with intracellular periodic acid fluorescent Schiff’s (PAFS) reagent in one lung, and from the second lung take BAL for cell counts and cytokine measurements. We will use the forced oscillation technique to measure changes in airway resistance in response to aerosolized methacholine or ATP. These experiments will allow us to determine whether the critical role of ß2-adrenoceptor signaling in potentiation of allergic airway inflammation is in hematopoietic cells or lung parenchymal cells or both, and thus help identify the target cell(s) of the anti-inflammatory effect of chronic ß-blockade.

We have identified a new potential therapeutic target for asthma: p21-activated kinase (Pak)-1. In the course of our studies of Pak1 - a protein kinase that is an effector for the small GTPases Rac and Cdc42 – we found that it has a profound positive signaling role in mast cells, which are a key element in the pathogenesis of asthma. Deletion of the pak1 gene in mice has no effect on their viability, longevity, or fertility, but such mice do show remarkably improved airflow in a model of chronic asthma. We have noted that mast cells derived from these animals do not efficiently degranulate or secrete inflammatory cytokines in response to activators. In addition, pak1-null mice are much less sensitive to IgE-mediated cutaneous anaphylaxis than normal mice, and mast cells from such animals do not migrate efficiently to sites of inflammation. As mast cell activity is an important contributor to airway inflammation, these findings suggest that inhibition of Pak1 could be beneficial in preventing or treating asthma. In addition to creating useful genetic models for Pak1 deficiency, we have developed the first specific small molecule Pak inhibitor. We propose the following specific aims. (1) Determine how Pak1 activity is regulated during mast cell activation. (2) Identify the substrates of Pak1 in mast cells that mediate its effects on degranulation and cytokine secretion. (3) Establish if loss of Pak1 function in mast cell affects airway responsiveness in mice. (4) Determine if chemical Pak1 inhibitors reduce mast cell function.

We propose to determine if thiol reductases, in particular Gamma Interferon-inducible Lysosomal Thiolreductase (GILT), secreted by activated alveolar macrophages prevent oxidative tissue damage and/or affect the redox status of membrane glycoproteins, such as integrins, on infiltrating T cells and lung epithelial and endothelial cells, modulating the localized inflammatory response in asthma. Aim 1. To identify the surface glycoproteins on mouse splenic T cells that have disulfide bonds reduced by low concentrations of a chemical reducing agent or GILT, and to use the well-established mouse ovalbumin (OVA)-induced asthma model to examine the redox status of these glycoproteins on splenic T cells and T cells from the lungs of control mice and mice sensitized by OVA inhalation. Aim 2. To compare lung tissues from wild type and GILT knock-out mice, sensitized or not by OVA inhalation, for glycoprotein redox status, levels of reactive oxygen species, evidence of inflammation and GILT content. Aim 3. To identify the surface glycoproteins on human T lymphocytes, lung epithelial cells, and endothelial cells with disulfide bonds that are reduced by low concentrations of a chemical reducing agent or GILT, and to determine the concentrations of GILT in lung fluids and plasma from asthmatic patients and control subjects. The OVA model is well established. However, we will also examine mice expressing inducible IL-13 and vascular endothelial growth factor (VEGF) in the lung that develop an asthma-like inflammatory response. These mice will be bred onto a GILT knockout background to evaluate the role of GILT in modulating inflammation.

P2X receptors are ATP-gated ion channel proteins expressed on the surface of a broad range of cell types, serving as high affinity sensors of extracellular ATP, and modulating a spectrum of cell properties that include smooth muscle contraction, dendritic cell activation and histamine release from mast cells. Despite the significance of P2X receptors in human physiology, including inflammation and asthma, there is a dearth of small molecule agonists, antagonists and allosteric modulators. To a great extent, the development of pharmacological agents directed toward P2X receptors has been hobbled by the fact that the receptors are oligomeric and glycosylated integral membrane proteins rich in disulfide bonds. Thus, even the basic task of receptor expression has proven challenging, and more detailed analysis, such as structural studies by x-ray crystallography, has thus far been without success. Here, I propose to use new technology developed in my laboratory to discover P2X receptors that are suitable for detailed biochemical and structural analysis, to solve high resolution crystal structures by x-ray diffraction, and to use these structures as starting points for development of new therapeutic agents.

Asthma varies with circadian rhythm and possibly to light exposure. It grows worse at night, and lack of sunlight may increase its incidence and severity. Current therapies, either incidentally or by intent, influence circadian and photic factors. For example, corticosteroids, in addition to anti-inflammatory effects, can regulate clock gene expression and override normal daily cycles of the hypothalamic-pituitary-adrenal axis, and phototherapy is useful for asthma-related conditions, such as atopic eczema. Treatments targeting circadian and photic triggers of asthma could therefore prove valuable. Eosinophils are responsible for inflammation in asthma and might contribute to asthma’s circadian variance, because their numbers also vary with circadian rhythm. Older studies comparing sighted to blinded animals indicate that eosinophil counts synchronize with light exposure, but that, surprisingly, non-visual perception of light, in addition to ocular detection of light, harmonizes eosinophils’ circadian periodicity. In plants, as well as animals, extra-visual photoreception is frequently mediated by “flavoproteins”, in which the fluorescent cofactor flavin adenine dinucleotide (FAD) entrains light to circadian cycles. One light-sensing flavoprotein, cryptochrome, is particularly important, because it is also a molecular component of circadian clocks. Eosinophils are “autofluorescent”—a remarkable property sometimes evolutionarily coupled to photodetection. The pigment responsible for fluorescence is FAD. Although the corresponding FAD-containing protein remains undefined, eosinophils intriguingly harbor extraordinarily high levels of the transcript encoding cryptochrome. We thus hypothesize that eosinophils contain a light-sensing flavoprotein (possibly cryptochrome), conceivably linking photic and circadian variation of eosinophils to asthma and that might point the way to new therapeutic targets. We propose to identify the FAD-containing protein associated with eosinophil autofluorescence, determine if light can trigger signaling pathways in eosinophils, and evaluate circadian and light-dependent asthma-related functions of eosinophils.

Asthma patients suffer from bronchoconstriction in response to allergens or irritants, airway hyper-responsiveness, as well as airway edema and mucus hypersecretion potentially causing mucus plugging—a feature of fatal asthma in both adults and children. Whereas current asthma therapy with corticosteroid plus bronchodilators aims primarily at controlling airway inflammation and bronchoconstriction, many thousands of patients with chronic asthma still suffer daily from severe symptoms. Despite the knowledge that CaCC channel blockers could reduce a wide range of asthma symptoms as well as the underlying airway hyper-responsiveness and mucous hyperplasia, development of more specific CaCC blockers is difficult without molecular characterization of CaCC channels that are expressed in the lung and many other cell types including exocrine glands. Our recent cloning of CaCC with expected channel properties and novel molecular nature enables us to do the following: Aim 1. Characterizing the airway expression patterns of mRNAs for CaCC and its family members. Aim 2. Raising antibodies for CaCC and family members for immunocytochemical studies of their localization in the airway and submucosal glands. Aim 3. Electrophysiological recording from cells heterologously expressing CaCC and relatives found in the airway and submucosal glands. Aim 4. Establishing stable cell lines expressing CaCC and its relatives present in the airway and submucosal glands for screening small molecular CaCC modulators. Aim 5. Generating floxed CaCC alleles for conditional knockout for future studies of the physiological functions of these chloride channels in different cell types.

In the last 25 years, asthma and autoimmune diseases have increased in the industrialized world. The “hygiene hypothesis” relates this increase to a failure of proper microbial stimulation of the immune system due to increased sanitary conditions early in life. Scarlet Fever, caused by the Streptococcus pyogenes bacterium, is inversely correlated with asthma. S.pyogenes induces large amounts of antibody to cell wall-associated N-acetyl glucosamine (GlcNAc), which is also the major component of chitin, a component of many allergen-bearing organisms. We will study the link between innate and adaptive immunity and asthma induced by environmental allergens containing GlcNAc and other conserved carbohydrates. We will quantitate the specificity, clonality, airway association and longevity of the antibody response to S.pyogenes, chitin and Aspergillus fumigatus. We will determine the role of B cells and antibody in modifying innate cell functions and activation of CD4 T cells involved in the chronic inflammatory process and airway obstruction of asthma. Apart from the established paradigm involving the shift on TH1/Th2 functionality and the important but not definitive role of IgE in asthma, B cell and antibody functions in the disease have not been extensively studied. By focusing on the role of B cells and antibodies, we may shed light on the mechanisms involved in the developmental induction of allergic asthma and treatment of established disease.

Asthma is rooted in childhood when the immune system is prone to Th2 polarization. Since chronic exposure to in- and out-door allergens promotes and provokes asthma, repeated exposure of the innate immune system may polarize T-cells to allergen. In studying fungi, we have found that chitin coats their cell surface; others have recently reported that chitin strongly induces innate allergic inflammation. These findings raise two questions whose answers will clarify asthma pathogenesis and prompt new therapies: (i) What is the principal innate immune receptor for chitin? (ii) How does its activity shape T-cell behavior? We postulate that the principal chitin receptor remains unknown; its engagement triggers IL-4 producing cells and downstream products that dictate Th2 polarization, promoting the development of asthma. To test this hypothesis, we will: (1) Identify novel macrophage receptors that sense chitin, using gene silencing of identified candidates, and biochemical and expression cloning methods. (2) Define the influence of chitin receptors in allergic inflammation and chitin-induced asthma by silencing chitin receptors, and testing responses to chitin in reporter mice for IL-4+ cells and alternatively activated macrophages, and in murine models of fungal chitin induced asthma. (3) Reveal the role of chitin receptors on Th2 differentiation and effector function in asthma, using fungal chitin related asthma models such as due to Aspergillus, together with Aspergillus transgenic mice and T-cells to monitor T-cell activity. By revealing the receptor that links innate and adaptive immunity upon chitin exposure, we will clarify how chitin promotes asthma and facilitate new therapeutic strategies.

Our recent research has identified the hypoxia-inducible transcription factor-1α (HIF1α) as a master regulator of the inflammatory and bactericidal capacity of phagocytes. Using conditional deletion or constitutive expression of HIF1α in the myeloid lineage (neutrophils and macrophages), we have observed post-transcriptional stabilization of HIF1α to control expression of antimicrobial peptides, cytokines and iNOS. HIF1α responds to bacterial and microbial products even at normoxia, and supports innate defense function of phagocytic cells in vitro and in vivo. Additional data reveals that HIF1α is regulated at the transcriptional level by IKK/NFκB activation, further supporting the interdependence of the hypoxic response and innate immunity. In this proposal, we will explore the role of HIF1α in regulation of inflammatory and innate immune responses that may underpin asthma, in the following Specific Aims. (1) Determine the role of HIF1α, vHL and VEGF deletion on macrophage and neutrophil production of cytokines and molecular effectors implicated in asthma; (2) Determine the roles of HIF1α, vHL and VEGF on lung function and inflammation in murine asthma models; (3) Determine the influence of bacterial infection and smoke in HIF1α mediated asthma responses; (4) Determine the effect of pharmacologic modulation of HIF1α on lung function and inflammation during asthma. It is anticipated this research will provide several unique and novel insights into the fundamental pathogenesis of asthma and reveal novel targets for therapeutic intervention.

T lymphocytes play a critical role in asthma development and severity. We have recently shown that regulation of glucose uptake is a central feature of T cell function. Upon stimulation, T cell glucose uptake increases by fifty-fold, leading to a highly glycolytic phenotype that is necessary for proliferation and proper cytokine production. Upregulation of glucose uptake directly contributes to T cell responses as transgenic expression the Glucose Transporter 1 (Glut1) in T cells is sufficient to augment T cell activation, inhibit cell death, and promote autoimmunity in aged mice. In contrast, treatment with PPAR agonists, which promote oxidative rather than glycolytic metabolism, has been reported to suppress inflammation and asthma, although the mechanism is unclear. We hypothesize that a glycolytic phenotype in stimulated lymphocytes contributes to asthma severity. Furthermore, manipulation of lymphocyte metabolism may provide an effective strategy in asthma management. We propose to (1) determine how manipulation of lymphocyte bioenergetics affect T cell activation and function using in vitro biochemical, metabolic, and immunologic approaches. (2) Analyze the metabolic phenotype of T cells at various stages in an asthmatic response compared to splenic T cells using a murine asthma model. (3) Determine how transgenic expression of Glut1 to enhance glycolysis or treatment with PPAR agonists to limit glycolysis affect T cell metabolism and function as well as the severity of asthma in vivo. Together, these aims will establish how T cell metabolism impacts asthma and may reveal a novel therapeutic avenue for immunosuppression and asthma treatment.

Airway epithelial cells, which come in contact with the environment, are thought to undergo rapid turnover (via apoptosis). Yet, relatively little is known about how these apoptotic epithelial cells are engulfed, and whether defects in clearance could contribute to the development of asthma. It is now beginning to be accepted that non-professional phagocytes such as epithelial cells may do much of the engulfment during normal cellular turnover within tissues. Since alveolar epithelial cells can engulf apoptotic targets in vitro, an intriguing possibility is that these cells engulf neighboring apoptotic epithelial cells in the lung. Importantly, phagocyte recognition of cells at the earliest stages of apoptosis is actively anti-inflammatory through section of cytokines such as TGF- and IL-10 (both linked to asthma), and the generation and maintenance of tolerance to antigens. In contrast, late-stage apoptotic corpses or necrotic cells induce an inflammatory response and are linked to break in tolerance to self-antigens. We propose that in healthy individuals such normal turnover/engulfment may create an environment where immunological responses to common allergens are dampened or suppressed. In contrast, failure in apoptotic cell clearance or overwhelming of the engulfment machinery could predispose such individuals to developing asthma. Our laboratory has cloned several proteins linked to phosphatidylserine dependent recognition of apoptotic cells and has generated mice with conditionally targeted Elmo1 or Gulp1 loci. Using Cre mice that can efficiently delete floxed loci within the airway epithelium, this provides a great opportunity to directly address the role of engulfment mediated by epithelial cells in the context of normal lung physiology, and how alterations in engulfment influence symptoms in mouse models of asthma. We will further validate some of these observations using human patient samples.

When cells undergo necrosis in vivo they release danger signals that stimulate inflammation. While this response can be beneficial (e.g. in infections) it also damages adjacent healthy tissue and can cause disease. In asthma, cells in the airways are damaged and undergo necrosis. We hypothesize that danger signals from these dying cells amplify inflammation and is so doing contributes to acute airway dysfunction and chronic irreversible airway changes in this disease. We have two specific Aims to test this hypothesis. In Aim 1, we will investigate the role of Danger signals in the inflammatory response and collateral damage in the lung to necrosis and asthma. This aim is based on our recent discoveries that there are two major danger signals that initiate the inflammatory response to necrosis. One is monosodium urate (MSU), which works though a TLR-independent pathway, while a second distinct danger signal(s) works through TLR2+4. We propose to use novel mouse models that are genetically-deficient in MSU (or MSU-signaling pathways) and/or TLR2+4 to define the role of these danger signals in asthma. In Aim 2, we will test the hypothesis that cellular danger signals orchestrate the inflammatory response in asthma through the IL-1-IL-1R pathway. The rationale for this hypothesis is our recent finding in other tissues that danger signals induce IL-1 and that the IL-1-IL-1R pathway is essential for the inflammatory response and collateral damage to necrosis. We propose to test our hypothesis using mouse models that are genetically deficient in the IL-1-IL-1R response and biologics that block this pathway.

The goal of our research is to uncover the nature of a novel putative target for anti-viral therapy against human rhinovirus, a positive-strand RNA virus belonging to the picornavirus family. Infections by human rhinoviruses are major exacerbating factors in disease morbidity for individuals suffering from asthma. Anti-viral therapies against human rhinovirus are particularly desirable as they would lessen both the severity and duration of upper respiratory distress in asthmatic individuals. However, the available anti-viral treatments targeting specific steps in the human rhinovirus replication cycle have not been shown to be efficacious. Thus, there is a critical need to develop new targets for anti-rhinovirus chemotherapy. One such target is the cellular activity (termed “unlinkase”) that removes a small viral peptide (VPg) from the 5’ end of human rhinovirus genomic RNA by cleaving a protein-nucleotidyl bond prior to the onset of viral-specific protein synthesis. This proposal aims to identify the protein(s) responsible for unlinkase activity and to determine the consequences for viral replication when unlinkase activity is down-regulated during a rhinovirus infection of human cells. Aim 1. Identification and purification of rhinovirus VPg unlinkase activity. Aim 2. Characterization of unlinkase activity in uninfected and human rhinovirus-infected cells. Aim 3. Determining the functional consequences of down regulation of VPg unlinkase activity. Results from our proposed studies will allow the design of small molecule inhibitors of VPg unlinkase activity whose chemotherapeutic potential can then be evaluated for the inhibition of rhinovirus replication and possible treatment of infected individuals suffering from asthma.

Asthma, a chronic inflammatory disease of unknown aetiology, is a major complication worldwide with limited preventive options. It may result from direct involvement of the inflammation in airway tract and lung tissues. Inflammation is exemplar of cytotoxicity caused by the formation of NF-κB -dependent inflammatory cytokines and chemokines and their autocrine and paracrine effects. However, the mechanisms through which the increased inflammatory markers cause airway inflammation are not well understood. Our recent studies have shown that aldose reductase (AR), thought to be a glucose reducing enzyme, efficiently catalyzes the reduction of lipid peroxidation-generated lipid derived aldehydes and their glutathione conjugates (Km 10-30 μM) and is an obligatory mediator of cytokine-, chemokine-, growth factor- and endotoxin–induced activation of NF-κB and AP1 via PLC/PKC/IKK/MAPK. Also we have shown that AR inhibition prevents LPS-induced production of inflammatory markers such as nitric oxide, TNF-α, IL-6, IFN-γ, IL-1, MCP-1, iNOS and Cox-2 in macrophages. To investigate the feasibility of effectiveness of AR inhibitors in the treatment of asthma pathogenesis, we performed preliminary studies using ragweed pollen extract (RW)- and ovalbumin (OVA) -induced asthma models. We observed that AR inhibition significantly prevented inflammatory cell infiltration, expression of cytokines such as IL-5, IL-4, and MCP-1 in BAL, mucin production by goblet cells and airway hyperresponsiveness in both the mice models as well as activation of PKC/MAPK/NF-κB in human small airway epithelial cells (SAEC). Based on these results, our long-term goal is to understand the mechanisms through which AR contributes to airway inflammation, and to develop AR inhibitors to prevent asthma pathogenesis. We propose to systematically examine our hypothesis “that AR catalytical activity plays a pivotal role in the transduction of airway inflammatory response leading to asthma” by investigating the (1) effect of AR inhibitors in OVA- and RW-induced airway inflammation in mice asthma models, (2) OVA- and RW-induced airway inflammation in AR null mice and (3) mechanism(s) of AR mediation in bacterial endotoxin (LPS)-, RW-, TNF-α- and 4-hydroxynonenal (HNE)-induced signals in SAEC. Completion of these studies will reveal how AR mediates inflammatory signals by regulating the activation of NF-κB dependent airway inflammatory response and provide a novel therapeutic approach for preventing asthma and its associated respiratory complications by AR inhibition.

The cytokine IL-13 is strongly implicated in the pathogenesis of asthma. There exists tremendous interest in developing potent, selective, long-lasting and non-immunogenic inhibitors of IL-13 signaling. Here we propose to convert IL-13 itself from an agonist into an antagonist having the aforementioned properties through site-specific introduction of a synthetic element, an α-helix staple. Helix stapling has been shown to improve greatly the pharmacologic properties of peptides, and we hypothesize that similar enhancements will be observed when a staple is incorporated into the IL-13 protein. If successful, this work will pave the way not only for a new therapeutic avenue in asthma, but will also pave the way for the development of stapled cytokines and other proteins to treat a wide variety of diseases. Aim 1. Develop a practical semi-synthetic route to IL-13 analogs bearing a hydrocarbon staple on Helix-A. Aim 2. Produce a series of stapled IL-13 proteins differing in the location of the staple, its chemical composition and stereochemical configuration. Aim 3. Characterize, in collaboration with the laboratory of K. Chris Garcia (Stanford University), the binding of the stapled IL-13 analogs to IL-13Rα 1 and IL-4Rα, and determine structures of the relevant bound complexes. Aim 4. Test the ability of the stapled IL-13 analogs to induce or antagonize IL-13 signaling in cells. Aim 5. With a select set of stapled IL-13 antagonists, obtain measurements of the pharmacokinetics in rats.

Recently, the neuropeptide S receptor (NPSR), a G protein-coupled receptor (GPCR) also known as GPR154 and GPRA, has been implicated in asthma pathogenesis. Importantly, several single nucleotide polymorphisms within NPSR have been found to associated with asthma, some of which produce changes in the protein sequence. Following activation by agonist, signal transduction from GPCRs is extensively regulated by numerous processes. Of these regulatory mechanisms, rapid endocytosis is of particular interest because it can be differentially regulated by different ligands. For the vast majority of GPCRs, including NPSR, the effects of endogenous ligand and exogenous drugs on the endocytic trafficking of the receptor, as well as the influence of naturally-occurring receptor mutations on receptor trafficking, remains unknown. My laboratory has been instrumental in demonstrating the importance of receptor endocytosis and post-endocytic sorting in mediating drug responsiveness through several classes of GPCR important in addiction. Indeed, our initial interest in NPSR has been with regard to its role in behaviors related to addiction. However, clearly this receptor is important for asthma as well. The basic questions of 1) whether the NPSR undergoes regulated endocytosis and 2) where it sorts post-endocytically, have not been addressed. In addition, the effects of receptor mutations associated with an increased risk for asthma on the endocytosis and post-endocytic sorting of the NPSR remain unexplored. Importantly, an understanding of the trafficking properties of this receptor could help inform the development of new therapeutics to this target, any of which might be expected to influence receptor trafficking.