Lee Limbird - US grants

The goal of the present studies is to determine the sequence of events involved in luteinizing hormone (LH) receptor synthesis, and regulation of these events by physiological agents that have been shown to alter the expression of LH receptor binding on gonadal tissue. Thus, the specific aims of the grant are 1) to prepare and 2) characterize monoclonal antibodies against the receptor as specific reagents for following the synthesis of the glycoprotein LH receptor in cultured cells that have been metabolically labeled using radiolabeled amino acids or sugars, 3) outline the translational and post-translational modifications of the LH receptor that occur prior to and following insertion into the surface membrane and expression of receptor functions, i.e. the binding of hormone and activation of adenylate cyclase and 4) evaluate changes in the sequelae determined in #3 that occur when receptor density is increased by estradiol or platelet-derived growth factor (PDGF) or attenuated by epidermal growth factor (EGF) or human chorionic gonadotropin (hCG). The LH-receptor plays an initial and crucial role in mediating LH-induced physiological effects in gonadal tissue. Consequently, these studies should outline the molecular basis for LH receptor expression in a target cell, and provide new insights into the biochemical events that result in a functional receptor molecule as well as changes in these events that are elicited by modulators of LH receptor density and function, e.g. estradiol, EGF, PGDF and the agonist hCG itself.

The goal of the research proposed is to further understand the molecular basis of activation and inhibition of adenylate cyclase. A large number of hormones and drugs act by binding to cell surface receptors coupled to activation of adenylate cyclase and generation of cAMP intracellularly. Very often, the biological effects of these activating agents are countered or attenuated by agents which-recently-have been realized to inhibit adenylate cyclase and dampen the increases in intracellular cAMP induced by activating agents. for example, epinephrine and other beta-adrenergic catecholamines increase the strength and rate of beating of the heart by elevating intracellular cAMP levels. Acetylcholine, acting through muscarinic cholinergic receptors, attenuates the actions of epinephrine and decreases cardiac inotropy and chronotropy. The purpose of the present studies is to learn whether separate signaling systems or shared molecular components confer both activating and inhibiting information to adenylate cyclase using receptor binding and other established biochemical probes. It is hoped that such insights will provide arational basis for the design of drug and other therapeutic interventions in disease states characterized by altered hormonal responsiveness.

The long-term goal of this proposal is to understand the molecular basis for physiological effects elicited by epinephrine via alpha-adrenergic receptors of the pharmacological alpha-2-subtype. We will address this long-term goal by focusing on the manner in which sodium ion (Na+) influences alpha-2-receptor-agonist interactions and alpha-2-receptor-induced function. The specific aims of the present proposal are to 1) purify the alpha-2-receptor to homogeneity, 2) evaluate whether the effects of Na+ on the purified alpha-2-receptor are conveyed via the betagamma heterodimer of the GTP-binding protein heterotrimer (Gi: alphabetagamma) which modulates alpha-2-receptor functions or, alternatively, via a Na+/H+ antiporter, 3) reconstitute the alpha-2-adrenergic receptor with the putative Na+-effector component in lipid vesicle preparations to test the conclusions drawn in #2 and 4) utilize permeabilized platelets to determine whether Gi represents the interface between the alpha-2-receptor and the Na+/H+ exchange mechanism and/or phospholipase A-2 activity postulated to be involved in epinephrine-provoked platelet secretion. Alpha-2-receptors are one of a number of hormone and neurotransmitter receptor populations which are linked to inhibition of adenylate cyclase activity. A number of lines of evidence suggest that the resultant decreases in cAMP levels are not sufficient to "signal" a physiological effect, but that other important biochemical changes must also be evoked by these receptor populations. Receptors which can inhibit cAMP accumulation are all known to be modulated by Na+. Our studies, which focus on the basis for this role of Na+, should provide fundamental new insights into how alpha-2-adrenergic receptors elicit their physiological effects, in particular, and what are the alternate signaling mechanisms utilized by receptors linked to inhibition of adenylate cyclase activity, in general. Our model system for assessment of alpha-2-receptor-provoked function, i.e. epinephrine-induced platelet aggregation and secretion, will be of particular significance in understanding normal and pathological hemostasis, since platelet plug formation is necessary to prevent excessive blood loss but, when occurring inappropriately, forms thrombi that can precipitate strokes or myocardial infarction.

The present studies are designed to determine the sequence of molecular events which link alpha2-adrenergic receptor occupancy to blockade of neurotransmitter secretion. Previous findings suggest that alpha2-adrenergic receptors provoke secretion from the human platelet by a pathway involving Na+/H+ exchange. Recently, we have observed alpha2-receptor activation of Na+/H+ exchange in a neuroblastoma x glioma hybrid cell line, NG108-15. Our proposed studies are intended to resolve the following questions: Do other receptor populations on NG108-15 cells that are linked to inhibition of adenylate cyclase, e.g. opiate and muscarinic receptors, also accelerate Na+/H+ exchange? Do GTP-binding proteins link receptor occupancy to activation of Na+/H+ exchange? Does differentation of NG108-15 cells to an electrically excitable state alter the properties of receptor-mediated Na+/H+ exchange? Does the alkalinization of NG108-15 cells that occurs as a consequence of Na+/H+ exchange cause an increase in outward K+ conductance, and associated hyperpolarization, thus providing a molecular basis for alpha2-receptor-mediated inhibition of neurotransmitter release? Does acceleration of Na+/H+ exchange activate a phospholipase A2 enzyme in NG108-15 cells, as it does in human platelets? If so, do arachidonic acid metabolites influence receptor-modulated neurotransmitter release? The present studies seek to provide novel insights into the sequence of events responsible for alpha2-adrenergic-receptor-induced changes in cellular responce by dissecting the pathway which leads from receptor occupancy to inhibition of neurotransmitter release. Perhaps more important is the probability that these studies will shed new light on the mechanism(s) by which all receptor populations linked to inhibition of adenylate cyclase modulate cellular response, since a large body of data suggests that decreases in cAMP levels, by themselves, are not sufficient to evoke changes in cell function.

The present studies are aimed at examining, using a molecular biological approach, the structural basis for the diverse functional properties of alpha2-adrenergic receptors. We plan to clone the porcine gene that codes for the alpha2-adrenergic receptor that we have purified to homogeneity. We will exploit insights gained from domain mapping and biochemical analysis of the purified receptor to delineate those areas of the receptor that we can analyze further by deletion and site-directed mutagenesis to learn what structural components are involved in ligand recognition, allosteric effects on adrenergic binding by Na+, H+ and 5-amino-substituted analogs of amiloride, receptor-GTP binding protein interactions, receptor-accelerated Na+/H+ exchange and receptor mediated inhibition of adenylate cyclase. Furthermore, we plan to examine what role different functional domains of the receptor play in the overall physiological functions influenced by alpha2-adrenergic receptors, such as suppression of neurotransmitter release, by expressing mutated versus wild type receptors in appropriate target cells to determine what effect discrete deletion or site mutations have on particular alpha2-receptor functions. We anticipate that the proposed studies will provide new insights into the structural basis for many of the functional properties of alpha2-receptors and perhaps suggest novel loci for drug development in mimicking, or blocking, the effects of alpha2-adrenergic agents in particular physiological processes.

The long-term goal of this proposal is to understand the molecular basis for physiological effects elicited by epinephrine via alpha-adrenergic receptors of the pharmacological alpha-2-subtype. We will address this long-term goal by focusing on the manner in which sodium ion (Na+) influences alpha-2-receptor-agonist interactions and alpha-2-receptor-induced function. The specific aims of the present proposal are to 1) purify the alpha-2-receptor to homogeneity, 2) evaluate whether the effects of Na+ on the purified alpha-2-receptor are conveyed via the betagamma heterodimer of the GTP-binding protein heterotrimer (Gi: alphabetagamma) which modulates alpha-2-receptor functions or, alternatively, via a Na+/H+ antiporter, 3) reconstitute the alpha-2-adrenergic receptor with the putative Na+-effector component in lipid vesicle preparations to test the conclusions drawn in #2 and 4) utilize permeabilized platelets to determine whether Gi represents the interface between the alpha-2-receptor and the Na+/H+ exchange mechanism and/or phospholipase A-2 activity postulated to be involved in epinephrine-provoked platelet secretion. Alpha-2-receptors are one of a number of hormone and neurotransmitter receptor populations which are linked to inhibition of adenylate cyclase activity. A number of lines of evidence suggest that the resultant decreases in cAMP levels are not sufficient to "signal" a physiological effect, but that other important biochemical changes must also be evoked by these receptor populations. Receptors which can inhibit cAMP accumulation are all known to be modulated by Na+. Our studies, which focus on the basis for this role of Na+, should provide fundamental new insights into how alpha-2-adrenergic receptors elicit their physiological effects, in particular, and what are the alternate signaling mechanisms utilized by receptors linked to inhibition of adenylate cyclase activity, in general. Our model system for assessment of alpha-2-receptor-provoked function, i.e. epinephrine-induced platelet aggregation and secretion, will be of particular significance in understanding normal and pathological hemostasis, since platelet plug formation is necessary to prevent excessive blood loss but, when occurring inappropriately, forms thrombi that can precipitate strokes or myocardial infarction.

The present studies propose to examine what structural domain(s) within the alpha2-adrenergic receptor (alpha2AR) are responsible for targeting and/or retention of this receptor on the basolateral membrane of renal epithelial cells. The Madin-Darby canine kidney (MDCKII) cell line will be the primary model system for these studies. Site and deletion mutagenesis strategies will be employed to explore whether N-terminal glycosylation, secondary structure within the large third cytoplasmic loop, endocytosis signals located at the base of predicted transmembrane helix 7, endofacial aromatic residues and/or acylation of the alpha2AR play a critical role in alpha2AR polarization in MDCKII cells. Permanent transformants of MDCKII cells expressing genes coding for wild-type and mutant alpha2AR will be cloned and characterized for alpha2AR expression. Polarization of the MDCKII cells will be achieved by growth on permeable supports (Transwell culture wells) and alpha2AR distribution monitored using three independent strategies, including 1) biotinylation/extraction/streptavidin fractionation, 2) morphological localization and 3) cell surface ELISA techniques. The studies proposed will provide novel insights into the molecular basis for targeting of alpha2AR in renal epithelia that likely will reflect structural features exploited by all GTP-binding protein-coupled receptors for localization to specialized cellular domains. We will extend our findings in renal epithelial cells by examining whether or not similar structural domains target the alpha2AR to the basolateral domain of intestinal epithelial polarized in culture. Finally, future studies informed by the proposed experiments hopefully will reveal whether or not basolateral targeting/retention signals within varying alpha2AR subtypes determine receptor delivery to discrete regions within neurons, e.g., the somatodendritic versus synaptic terminal membranes.

The 14th Gordon Research Conference on Molecular Pharmacology will focus on "Chemical and Electrical Messages for Signal Transduction". Recent advances in molecular genetics have combined with biochemical, morphological and electrophysiological measurements at the level of single molecules/cells to allow the study of complex signal transducing systems. The conference is designed to promote the exchange of information and ideas among participants who are studying a broad range of problems in these areas. The speakers have been chosen from universities and industrial research laboratories to represent diverse approaches to the problem of signal transduction. Excellent young scientists and more established investigators have been chosen in all of the fields represented. About 120 participants will be chosen from an expected large number of applicants based on three criteria: (1) broad representation of disciplines, approaches and institution; (2) selection of young scientists; students and fellows and scientists under - represented in the profession, including women and minorities; (3) probability of significant contributions to discussion. A change in the conference over previous years is that it will be held in the winter, March 1-5, 1993, at the Casa Sirena Resort in Oxnard, California. This to avoid the scientific overlap that has occurred with other summer Gordon Conferences and offer a scientifically intensive and stimulating conference on this topic in the winter months. Biological signal transduction increasingly appears to be mediated by relatively few general mechanisms: receptor-regulated G proteins, cellular second messengers, ligand-and voltagegated channels, and protein phosphorylation/dephosphorylation. These mechanisms form the basis of signaling in the nervous system, control of cardiovascular function, regulation of cellular differentiation in normal cells and following malignant cellular proliferation, control of inflammatory and immune responses, and the molecular bases underlying normal development. The topics and approaches are thus relevant to NIGMS, NINDS, NHLBI, NIDDK, NCI, NIAID and NCHHD.

The present studies are aimed at examining, using a molecular biological approach, the structural basis for the diverse functional properties of alpha2-adrenergic receptors. We plan to clone the porcine gene that codes for the alpha2-adrenergic receptor that we have purified to homogeneity. We will exploit insights gained from domain mapping and biochemical analysis of the purified receptor to delineate those areas of the receptor that we can analyze further by deletion and site-directed mutagenesis to learn what structural components are involved in ligand recognition, allosteric effects on adrenergic binding by Na+, H+ and 5-amino-substituted analogs of amiloride, receptor-GTP binding protein interactions, receptor-accelerated Na+/H+ exchange and receptor mediated inhibition of adenylate cyclase. Furthermore, we plan to examine what role different functional domains of the receptor play in the overall physiological functions influenced by alpha2-adrenergic receptors, such as suppression of neurotransmitter release, by expressing mutated versus wild type receptors in appropriate target cells to determine what effect discrete deletion or site mutations have on particular alpha2-receptor functions. We anticipate that the proposed studies will provide new insights into the structural basis for many of the functional properties of alpha2-receptors and perhaps suggest novel loci for drug development in mimicking, or blocking, the effects of alpha2-adrenergic agents in particular physiological processes.

The long-term goal of this proposal is to understand the molecular basis for physiological effects elicited by epinephrine via alpha-adrenergic receptors of the pharmacological alpha-2-subtype. We will address this long-term goal by focusing on the manner in which sodium ion (Na+) influences alpha-2-receptor-agonist interactions and alpha-2-receptor-induced function. The specific aims of the present proposal are to 1) purify the alpha-2-receptor to homogeneity, 2) evaluate whether the effects of Na+ on the purified alpha-2-receptor are conveyed via the betagamma heterodimer of the GTP-binding protein heterotrimer (Gi: alphabetagamma) which modulates alpha-2-receptor functions or, alternatively, via a Na+/H+ antiporter, 3) reconstitute the alpha-2-adrenergic receptor with the putative Na+-effector component in lipid vesicle preparations to test the conclusions drawn in #2 and 4) utilize permeabilized platelets to determine whether Gi represents the interface between the alpha-2-receptor and the Na+/H+ exchange mechanism and/or phospholipase A-2 activity postulated to be involved in epinephrine-provoked platelet secretion. Alpha-2-receptors are one of a number of hormone and neurotransmitter receptor populations which are linked to inhibition of adenylate cyclase activity. A number of lines of evidence suggest that the resultant decreases in cAMP levels are not sufficient to "signal" a physiological effect, but that other important biochemical changes must also be evoked by these receptor populations. Receptors which can inhibit cAMP accumulation are all known to be modulated by Na+. Our studies, which focus on the basis for this role of Na+, should provide fundamental new insights into how alpha-2-adrenergic receptors elicit their physiological effects, in particular, and what are the alternate signaling mechanisms utilized by receptors linked to inhibition of adenylate cyclase activity, in general. Our model system for assessment of alpha-2-receptor-provoked function, i.e. epinephrine-induced platelet aggregation and secretion, will be of particular significance in understanding normal and pathological hemostasis, since platelet plug formation is necessary to prevent excessive blood loss but, when occurring inappropriately, forms thrombi that can precipitate strokes or myocardial infarction.

The long-term goal of this proposal is to understand the molecular basis for physiological effects elicited by epinephrine via alpha-adrenergic receptors of the pharmacological alpha-2-subtype. We will address this long-term goal by focusing on the manner in which sodium ion (Na+) influences alpha-2-receptor-agonist interactions and alpha-2-receptor-induced function. The specific aims of the present proposal are to 1) purify the alpha-2-receptor to homogeneity, 2) evaluate whether the effects of Na+ on the purified alpha-2-receptor are conveyed via the betagamma heterodimer of the GTP-binding protein heterotrimer (Gi: alphabetagamma) which modulates alpha-2-receptor functions or, alternatively, via a Na+/H+ antiporter, 3) reconstitute the alpha-2-adrenergic receptor with the putative Na+-effector component in lipid vesicle preparations to test the conclusions drawn in #2 and 4) utilize permeabilized platelets to determine whether Gi represents the interface between the alpha-2-receptor and the Na+/H+ exchange mechanism and/or phospholipase A-2 activity postulated to be involved in epinephrine-provoked platelet secretion. Alpha-2-receptors are one of a number of hormone and neurotransmitter receptor populations which are linked to inhibition of adenylate cyclase activity. A number of lines of evidence suggest that the resultant decreases in cAMP levels are not sufficient to "signal" a physiological effect, but that other important biochemical changes must also be evoked by these receptor populations. Receptors which can inhibit cAMP accumulation are all known to be modulated by Na+. Our studies, which focus on the basis for this role of Na+, should provide fundamental new insights into how alpha-2-adrenergic receptors elicit their physiological effects, in particular, and what are the alternate signaling mechanisms utilized by receptors linked to inhibition of adenylate cyclase activity, in general. Our model system for assessment of alpha-2-receptor-provoked function, i.e. epinephrine-induced platelet aggregation and secretion, will be of particular significance in understanding normal and pathological hemostasis, since platelet plug formation is necessary to prevent excessive blood loss but, when occurring inappropriately, forms thrombi that can precipitate strokes or myocardial infarction.

DESCRIPTION: The long range goal of studies in the principal investigator's laboratory is to understand, in as precise molecular detail as possible, the mechanism(s) by which alpha2 adrenergic receptors couple to distinct effector systems (such as inhibition of adenylate cyclase, suppression of calcium currents, activation of potassium currents) to elicit their diverse regulatory effects on excitation-secretion coupling. This next phase of the research is based on the discovery that a mutation in a highly conserved residue in the receptor, Asp79Asn, resulted in a loss of allosteric regulation of the receptor to sodium and, more significantly, a selective loss in coupling of the receptor to potassium currents in AtT20 cells. The selective loss of response by the mutated receptors offers an excellent opportunity to explore the roles of hormonal responsive currents in suppression of peptide hormone release in these cells and to gain insight into basic coupling mechanisms of the receptor. Specific aims include: 1) Determination of the relative abilities of wildtype and various mutant receptors to suppress peptide hormone secretion via measurement of ACTH release form AtT20 cells. 2) Evaluation of a series of mutants in the Asp79 position of the alpha2 receptor with regard to allosteric modulation by cations and coupling to inhibition of adenylate cyclase, ion channels, and secretion. 3) Clarification of the molecular basis for perturbation of coupling of the D79N mutation to activation of potassium currents in the AtT20 cells as a means to reveal the G protein involved in coupling this and other similar receptors to potassium currents. 4) Identification of alpha2A-adrenergic receptor associated effector complexes involved in receptor mediated signal transduction by selective immuno-isolation of effector proteins with wildtype versus functionally modified mutant receptors. Overall, the PI believes that these studies will provide new molecular insights regarding alpha2- adrenergic coupling to diverse signal transduction mechanisms as a first step toward developing novel therapeutic agents that are targeted downstream of receptor occupancy, and may provide a degree of specificity for function not attainable by simultaneous modulation of activity at all alpha2 receptors of a given pharmacological type.

DESCRIPTION: The goal of this proposal is to understand the molecular mechanism for GPCR localization in polarized target cells. Using MDCK cells as a model system, the PI has shown that the alpha 2A and 2C adrenergic receptors (ARs) are directly delivered to and retained on the basolateral surface while A1-ARs are predominantly localized (65-85%) on the apical surface and the alpha 2B-AR achieves basolateral localization by random delivery and preferential retention on that surface. The PI has also been able to eliminate several possible motifs such as the TM7 NPVIYTIF sequence, glycosylation, the i3 loop (delta 241-360) and fatty acid acylation as well as the requirement for functional coupling to G proteins as potential localization signals for the alpha 2A-AR, although the deletion of most of the i3 loop resulted in increased rate of loss of the alpha 2A-AR from the basolateral surface. There are three specific aims. The first is to use chimeric alpha 2A-AR/A1-AdR or alpha 2B-ARs to determine subdomains that are critical for direct basolateral, as opposed to apical, localization. The second aim utilizes several approaches including the yeast two-hybrid system, lamba-zap expression libraries, gel overlays, co-immuno-isolation and/or reversible cross-linking, and co-purification with loop i3-GST fusion proteins to identify proteins that interact with cytosolic domains of the alpha 2A, 2B and 2C ARs and may play a role in their localization and/or signaling. The final aim is to use permeabilized MDCK cells expressing epitope-tagged alpha 2A or A1-AdR, targeting-competent cell domains or targeting-defective chimeras, to identify interacting proteins in the trans golgi network (TGN) or its budded-off vesicles by cross-linking and immunoprecipitation that play a role in selective targetting. An alternative approach is to first isolate the TGN of MDCK cells and use epitope-tagged alpha 2-AR subtype and hexa-his tagged A1-AdR to co-purify associated proteins or associated proteins trapped with reversible cross-linkers. Taken together, these results should yield information that increases our knowledge of the components and mechanisms involved in GPCR localization.

The overall goal of the research in our laboratories is to understand the mechanisms of alpha-2 adrenergic receptor (alpha2AR) signaling in enough detail to be able to intervene with ingenuity in a variety of pathophysiological states. The present proposal seeks to establish the in vivo functional relevance of several partial reactions that follow agonist occupancy: receptor phosphorylation, receptor binding to arrestin, and receptor endocytosis. In addition, we wish to understand the functional relevance of differing trafficking itineraries for the three alpha2AR subtypes and of alpha2AR interactions with 14-3-3 proteins and with spinophilin in vivo. These linked goals ultimately will be addressed by introducing alpha2AR structures with modified trafficking properties or altered partial reactions into the alpha2AR locus of the mouse, using Cre-loxP based homologous recombination strategies. However, to prioritize which mouse lines expressing mutant alpha2AR should be developed as well as to gain unprecedented insights concerning the structure-function relationships of alpha2AR in the context of native target cells, we will utilize stereotactic procedures to deliver adenoviral constructs encoding these various alpha2AR structures into the fourth ventricle of the brain of alpha2AAR "knockout" mice. We will then evaluate the trafficking properties and the cellular functions elicited by these receptor structures in the locus ceruleus. For subsequently-developed homozygous mouse cell lines, we will evaluate alpha2AR suppression of Ca2+ currents and activation of K+ currents in the locus ceruleus and in superior cervical ganglion neurons. We will also evaluate a number of physiological parameters (including sedation, analgesia, lowering of blood pressure and suppression of epileptogenesis) and behavioral parameters, including measures of the efficacy of anti-depressant agents and indices for pre-existing "depressive states". These proposed studies, representing a collaboration between the laboratories of Lee Limbird and Brian Kobilka, co- investigators in this study, represent the first effort to explore, in the context of native target cells and in vivo, the impact of partial reactions of alpha2 receptor signaling and of alpha2 receptor trafficking. We anticipate that the insights we obtain will inform the development of novel therapeutic strategies for a number of cardiovascular, neurological and behavioral disorders regulated by alpha2AR.

DESCRIPTION (Verbatim from the Applicant's Abstract): These studies address the mechanisms by which G protein-coupled receptors (GPCR) achieve their discrete localization in target cells. Localization is determined by targeting mechanisms that govern receptor delivery to the surface and tethering mechanisms that govern receptor stability at its final surface "destination." Studies funded by this grant, focusing on alpha2AR subtypes and the Al adenosine receptor as models of GPCR, have revealed that targeting to the apical (A1AdoR) or basolateral (alpha2AR subtypes) surface is driven by multiple, independent and hierarchical sequences in or near the bilayer. In contrast, tethering to the basolateral surface involves the large third intracellular (3i) loop of alpha2AR subtypes. We identified two interacting proteins for the alpha2AR subtype 3i loops: 14-3-3 proteins and spinophilin, both multi-domain proteins implicated as scaffolds in various signaling pathways. The aims of this proposal are to 1) determine if spinophilin or 14-3-3 proteins tether the alpha2AR subtypes to the basolateral surface of MDCK II cells, a model system for polarized renal epithelia, 2) determine the trafficking itinerary of wildtype V2 vasopressin receptors in MDCK II cells and the point(s) at which varying alleles of the V2R that contribute to X-Iinked nephrogenic diabetes insipidus (NDI) are interrupted in their direct delivery to the basolateral surface, and the cell surface "rescue" of these mutant V2R by temperature shifts or by chemical chaperones, and 3) use molecular cloning strategies to identify cDNAs encoding molecules that rescue cell surface expression of mutant V2R. We will also explore whether these cDNAs can enrich the cell surface expression of the alpha2cAR subtype, which exists predominantly in an intracellular precursor pool in a variety of cell backgrounds. There are two reasons for including another GPCR, i.e. the V2R, as a model system in our studies to understand the mechanisms underlying GPCR localization. First, our finding that multiple, non-contiguous membrane-embedded sequences dictate the delivery of GPCR to polarized surfaces in MDCKII cells means that a single sequence region cannot serve as a "ligand" to identify the targeting machinery for these receptors. Second, many diseases result from intracellular accumulation of molecules that must achieve surface expression to regulate cell function; cDNAs that rescue cell surface expression of intracellularly trapped receptors identified in the proposed studies likely encode targets for therapeutic intervention not only in NDI, but also in retinitis pigmentosa, cystic fibrosis, and other inherited diseases.

DESCRIPTION (from applicant's abstract): Support is requested to partially underwrite the costs of a conference entitled "Neurogenomics: Building a Better Brain" to be held May 20 to May 23, 2001 at the conference center on Peabody campus of Vanderbilt University. The general purpose of this conference is to assemble the top biomedical researchers in this field along with promising young investigators, trainees and more senior faculty, in order to educate the audience as to possibilities afforded by the "roll-out" of the human genome project. It is felt that attention has to be focused on senior faculty members as well, as they adapt their laboratories to meet the exciting new challenges and opportunities presented by having available to all, this new genomic information base. A series of workshops are also planned. The workshops will provide practical advice in terms of the pros/cons of available technologies and methodological approaches, hands-on experience with select technologies, available statistical analysis, and even the legal and ethical problems faced while performing genetic research. The conference will be publicized in scientific journals, on the Internet, and announcements will be mailed to members of neuroscience societies, as well as to individuals known to be active in the field. Electronic publicity will be accomplished by a Vanderbilt-linked conference web site with details of the program and on-line registration, and by listing it on established web sites providing meeting announcements. A concerted effort will be made to attract young investigators by the inclusion of travel awards, including those considered underrepresented in science fields, such as women and minorities.

DESCRIPTION (provided by applicant): The proposed construction project will make possible the build out of shell space for an expanded animal facility in Medical Center North (MCN) at Vanderbilt University. Currently, the University is building a three-floor mouse facility on the rooftop of MCN (referred to as MCNII throughout this application); floors six (staff) and eight [mice; Biosafety Level 3 containment suite; rodent quantitative suites; cage washing] will be completed in spring 2002. Unfortunately, limited University resources have required that floor seven (11,000 cage holding capacity) be shell space. Yet this space is absolutely essential to accommodate funded, pending and planned peer-reviewed research programs of existing faculty under recruitment. Several of these programs are multidisciplinary and involve creating and screening mouse models. The University aggressively manages colony size by providing investigators with core functions for mouse embryo cryo-preservation and breeding and colony monitoring. Despite these efforts, there is desperate need of mouse holding space. This application outlines the overall strategic animal space planning at Vanderbilt University. The growth in mouse research projects required the current investment in and building of additional mouse facilities, and a perspective on the scientific research. Funds are requested to build-out what otherwise would be shell space to increase mouse housing capacity by 40 percent.

DESCRIPTION (provided by applicant): The proposed construction project will make possible the build out of shell space for an expanded animal facility in Medical Center North (MCN) at Vanderbilt University. Currently, the University is building a three-floor mouse facility on the rooftop of MCN (referred to as MCNII throughout this application); floors six (staff) and eight [mice; Biosafety Level 3 containment suite; rodent quantitative suites; cage washing] will be completed in spring 2002. Unfortunately, limited University resources have required that floor seven (11,000 cage holding capacity) be shell space. Yet this space is absolutely essential to accommodate funded, pending and planned peer-reviewed research programs of existing faculty under recruitment. Several of these programs are multidisciplinary and involve creating and screening mouse models. The University aggressively manages colony size by providing investigators with core functions for mouse embryo cryo-preservation and breeding and colony monitoring. Despite these efforts, there is desperate need of mouse holding space. This application outlines the overall strategic animal space planning at Vanderbilt University. The growth in mouse research projects required the current investment in and building of additional mouse facilities, and a perspective on the scientific research. Funds are requested to build-out what otherwise would be shell space to increase mouse housing capacity by 40 percent.

DESCRIPTION (provided by applicant): The proposed construction project will make possible the build out of shell space for an expanded animal facility in Medical Center North (MCN) at Vanderbilt University. Currently, the University is building a three-floor mouse facility on the rooftop of MCN (referred to as MCNII throughout this application); floors six (staff) and eight [mice; Biosafety Level 3 containment suite; rodent quantitative suites; cage washing] will be completed in spring 2002. Unfortunately, limited University resources have required that floor seven (11,000 cage holding capacity) be shell space. Yet this space is absolutely essential to accommodate funded, pending and planned peer-reviewed research programs of existing faculty under recruitment. Several of these programs are multidisciplinary and involve creating and screening mouse models. The University aggressively manages colony size by providing investigators with core functions for mouse embryo cryo-preservation and breeding and colony monitoring. Despite these efforts, there is desperate need of mouse holding space. This application outlines the overall strategic animal space planning at Vanderbilt University. The growth in mouse research projects required the current investment in and building of additional mouse facilities, and a perspective on the scientific research. Funds are requested to build-out what otherwise would be shell space to increase mouse housing capacity by 40 percent.

DESCRIPTION (provided by applicant): The proposed construction project will make possible the build out of shell space for an expanded animal facility in Medical Center North (MCN) at Vanderbilt University. Currently, the University is building a three-floor mouse facility on the rooftop of MCN (referred to as MCNII throughout this application); floors six (staff) and eight [mice; Biosafety Level 3 containment suite; rodent quantitative suites; cage washing] will be completed in spring 2002. Unfortunately, limited University resources have required that floor seven (11,000 cage holding capacity) be shell space. Yet this space is absolutely essential to accommodate funded, pending and planned peer-reviewed research programs of existing faculty under recruitment. Several of these programs are multidisciplinary and involve creating and screening mouse models. The University aggressively manages colony size by providing investigators with core functions for mouse embryo cryo-preservation and breeding and colony monitoring. Despite these efforts, there is desperate need of mouse holding space. This application outlines the overall strategic animal space planning at Vanderbilt University. The growth in mouse research projects required the current investment in and building of additional mouse facilities, and a perspective on the scientific research. Funds are requested to build-out what otherwise would be shell space to increase mouse housing capacity by 40 percent.

DESCRIPTION (provided by applicant): The proposed construction project will make possible the build out of shell space for an expanded animal facility in Medical Center North (MCN) at Vanderbilt University. Currently, the University is building a three-floor mouse facility on the rooftop of MCN (referred to as MCNII throughout this application); floors six (staff) and eight [mice; Biosafety Level 3 containment suite; rodent quantitative suites; cage washing] will be completed in spring 2002. Unfortunately, limited University resources have required that floor seven (11,000 cage holding capacity) be shell space. Yet this space is absolutely essential to accommodate funded, pending and planned peer-reviewed research programs of existing faculty under recruitment. Several of these programs are multidisciplinary and involve creating and screening mouse models. The University aggressively manages colony size by providing investigators with core functions for mouse embryo cryo-preservation and breeding and colony monitoring. Despite these efforts, there is desperate need of mouse holding space. This application outlines the overall strategic animal space planning at Vanderbilt University. The growth in mouse research projects required the current investment in and building of additional mouse facilities, and a perspective on the scientific research. Funds are requested to build-out what otherwise would be shell space to increase mouse housing capacity by 40 percent.

The Fisk University Planning Grant will identify sustainable best-practices to increase student retention, learning success and career interest in STEM at the institution. The specific efforts to achieve this includes undertaking a self-study of it STEM undergraduate programs and research infrastructure using a Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis, studying national models for undergraduate research, utilizing evidence-based results to develop and institutional plan. A STEM Knowledge Map will be developed to digital capture the content and objectives of all of the STEM courses and depict the relationships to each other. This tool will help in determining what alignments and modifications will be needed in the STEM programs following the self-study. The planning process will include a synthesis of current knowledge about virtual mentoring and plans will include creation of a virtual mentoring community for FISK STEM students. This self evaluation approach can serve as a model for similar other institutions to utilize in gaining understanding of their STEM programs strengths, weaknesses, and opportunities and how to integrate undergraduate research experiences throughout the curriculum..

DESCRIPTION (provided by applicant): The overarching goal of our proposed Fisk University MARC U*STAR Program is to develop URM undergraduate students for successful application to and graduation from highly selective Ph.D. biomedical training programs, aligning fully with Fisk's mission is to produce graduates from diverse backgrounds with the integrity and intellect required for substantive contributions to society. Fisk University requests support for three juniors and 3 seniors in its first year, and four juniors and seniors/year in subsequent years, ultimately growing to a steady state of 8 MARC Scholars/year. The Specific Goals of our MARC Program are to 1) Increase interest in biomedical research careers via multiple, complementary strategies, including a) development of a Fisk Community of Biomedical Scholars that hosts: i) monthly informational and skill-building sessions, ii) a Career Roundtable, and iii) the monthly university-wide COOL SCIENCE CAF¿; and also b) engaging in a 6 wk. intensive summer 'Interdisciplinary Approaches to Biomedical Discovery' course after the Freshman year, with an Introduction to Responsible Conduct of Research. 2) Increase application, acceptance and completion of PhD- programs in the biomedical and behavioral sciences by MARC Scholars by engaging in a) summer biomedical research experiences at Research I institutions; b) MayMester I and II 3-week intensive courses preceding summer research experiences; c) the Professional Skills for Graduate Studies Success course with 1st year Fisk-Vanderbilt Masters-to-PhD students in our URM Bridge program; and d) Academic year research opportunities. 3) Enhance implementation of inquiry- and problem-based instruction in STEM courses and their associated laboratories via multiple faculty development strategies led by consultant P. Marstellar, PhD, Emory University. Measurable outcomes, tracked in collaboration with G Pion, PhD, Vanderbilt University, evaluator, include an increase in STEM retention from 72% to 85%, and an on-time graduation rate from 59% to 75%; with > 75% of our MARC U*STAR Scholars entering and completing a biomedical sciences PhD program within 7 years of follow- up, yielding an overall 50 % increase in biomedical PhDs emerging from Fisk compared to our baseline. Together, our integrated and inter-dependent evidence-based strategies proposed, our ~1:14 faculty-to-student ratio permitting individualized advising and mentoring, and the prevailing faculty commitment to what is best for our students affirm that Fisk University can successfully implement and achieve the goals of this proposed MARC U*STAR program .

DESCRIPTION (provided by applicant): Fisk University proposes to plan a novel accelerated Fisk undergraduate STEM (3 yr.) -Computer Science Master's (2yr) Bridge to a Vanderbilt PhD in Biomedical Informatics program in parallel with a real-time mentoring tracking system that adapts extant medical informatics tools to support reciprocal mentor-mentee interactions. Our BUILD Planning Grant addresses three key initiatives of the NIH: 1) Enhancing Diversity in the NIH-Funded Workforce, 2) nurturing individuals from diverse backgrounds across the lifespan of a research career, and 3) preparing an interdisciplinary workforce capable of fully exploiting the interrogation of Big Data for biomedical discovery. Our experience at Fisk, an historically black ( > 95 percent URM ) primarily undergraduate liberal arts college, affirms that programs linking 3 years of undergraduate education to accelerated entry into engineering or professional training programs attracts our most talented and highly motivated students. Findings that minorities disproportionately obtain Master's degrees en route to the PhD in natural sciences led to the 2005 launch of the Fisk-Vanderbilt Masters-to-PhD program, with > 92 percent of its entrants retained through PhD to postdoctoral or research positions. The BUILD planning grant will exploit the strengths of these programs and collaboration with the internationally recognized Vanderbilt Department of Biomedical Informatics (DBMI) to: Aim 1: Craft a Fisk STEM undergraduate (3 yr.)-Masters in Computer Science (2yr) Bridge to a Vanderbilt PhD in Biomedical Informatics. Academic and mentoring plan; Aim 2: Develop a plan to adapt DBMI-created tools for individualized health care and linkages to national clinical trials for seamless tracking of academic progress and local/national mentoring interactions in behalf of our Fisk-Vanderbilt trainees from undergraduate admission through the PhD, postdoctoral experience and early academic career, and Aim 3: Develop, implement, and analyze, in collaboration with the Institute for Broadening Participation, assessments that determine pre- College and early undergraduate awareness of the impact of Big Data on biomedical discovery and health care outcomes, and interest in careers in careers in these areas.

The project at Fisk University, a Historically Black University, builds on lessons learned from a planning grant. The overall goal of the project is to enhance undergraduate student interest, retention, learning success, and pursuit of post-graduation training or careers in science, technology, engineering and mathematics (STEM) areas. Three main strategies were selected to overcome barriers to student success: innovation in developmental and early mathematics courses to achieve accelerated acquisition of STEM pre-requisite skills; introduction of Supplementary Instruction in all Gatekeeper STEM courses to achieve deeper learning and student retention; and introduction of course-embedded research in two required courses in each Natural Science discipline, and in one mathematics and computer science course, to enhance STEM interest, critical thinking and concept mastery.

The project will collect data concerning the suitability of evidence-based practices successful in research-based universities for implementation in small liberal arts colleges focused on increasing minority student access to STEM careers. Additionally, accelerated mathematics confidence and a deeper understanding of fundamental concepts for application in novel settings will facilitate academic success of students at Fisk University as STEM majors and in seeking STEM careers.

This project has the potential for becoming a model for STEM education at small liberal arts institutions. The project is likely to have an impact on STEM education, student learning, and faculty practice.

Vanderbilt University, Fisk University, and Wake Forest University will collaborate to develop, study and refine a model to recruit, retain and advance historically underrepresented minority (URM) women from doctoral degree attainment to postdoctoral fellowship to tenured track positions in STEM. This alliance was created in response to the NSF's Alliances for Graduate Education and the Professoriate (AGEP) program solicitation (NSF 16-552) The AGEP program seeks to advance knowledge about models to improve pathways to the professoriate and success of URM graduate students, postdoctoral fellows and faculty in specific STEM disciplines and/or STEM education research fields. AGEP Transformation Alliances develop, replicate or reproduce; implement and study, via integrated educational and social science research, models to transform the dissertator phase of doctoral education, postdoctoral training and/or faculty advancement, and the transitions within and across the pathway levels, of URMs in STEM and/or STEM education research careers.

As our nation is confronted with a STEM achievement gap between URM and non-URM undergraduate and graduate students, our universities and colleges struggle to recruit, retain and promote URM STEM faculty who serve as role models and academic leaders for URM students to learn from, to work with and to emulate. Recent NSF reports indicate that URM STEM associate and full professors occupy 8% of these senior faculty positions at all 4-year colleges and universities and about 6% of these positions at the nation's most research-intensive institutions. URM women hold smaller shares of these academic STEM positions and an increase in their representation is essential since female URM undergraduate students, enrolled in STEM majors, outnumber their male peers. The current AGEP project has potential to advance a model to improve the representation of URM women in STEM faculty positions, eventually providing URM STEM role models to a STEM undergraduate and graduate students at postsecondary academic institutions.

The project includes activities to transition postdoctoral fellows into faculty positions, or a postdoc-to-faculty bridge program, to provide junior faculty with mentoring and to assist junior faculty in developing strong scholarly identities. The integrated research will include cross-sectional surveys, three-year longitudinal surveys and small-group interviews to gain a better understanding of the processes facilitating the choices women and URMs make in their STEM careers. Variables to study include gender and race differences, social relationship influences, the academic-professional culture and the institutional context. Vanderbilt and Fisk Universities will institutionalize the key model interventions, stage the model components for implementation at Wake Forest University, and disseminate the model to the network of 40 institutions represented in the Collaborative to Advance Equity through Research. The National Academy of Science's Ford Foundation Diversity Fellows program will work with the alliance to identify and recruit promising postdoctoral associates for project participation. The Anna Julia Cooper Center at Wake Forest will conduct scale up and dissemination activities for the alliance. Formative and summative evaluation work will be performed by an external evaluation team, via a subaward from Vanderbilt to the Institute for Broadening Participation. An external advisory board will provide advice to the project team through annual consultation.

Implementation projects provide support to Historically Black Colleges and Universities (HBCUs) to design, implement, and assess strategies that can lead to comprehensive institutional efforts to increase the number of students receiving undergraduate degrees in science, technology, engineering and mathematics (STEM) and enhance the quality of their preparation by strengthening STEM education and research. The project at Fisk University seeks to establish the foundation for building STEM student success. The goal of the project is to prepare students to contribute in an enduring way to new knowledge as a result of more competitive preparation for graduate school and careers.

Activities that are part of this project are: increasing students' deeper learning and successful transition to advanced courses by supplementary instruction into General Biology and College Algebra courses; increasing student confidence and competence in quantitative and computational approaches related to the biological sciences; and increasing enriched faculty-mentored undergraduate research opportunities across the STEM disciplines assured by intentional quantitative and computational integration into courses and faculty research collaborations. The activities and strategies are evidence-based and will inform further efforts by the institution to strengthen STEM undergraduate education.

The overall goal of the Fisk-Vanderbilt R25 Bridge to the Biomedical PhD Program (R25-BMP) is to provide research experiences, coursework and professional development training to assure at least 70% of trainees transition to PhD granting programs at Vanderbilt or other institutions nationally, and more than 80% of those who transition to the PhD complete the degree. This proposal adds a number of innovative strategies to the original R25 programming, based on lessons learned, including: 1) direct linkage of the Biomedical Track in the Fisk Master?s phase to Vanderbilt?s Interdisciplinary Graduate Program, specifically the embedded Vanderbilt Initiative for Maximizing Student Development (IMSD) Program, linked to 16 possible bio-medically relevant PhD-granting programs; 2) embedding incoming Master?s trainees in IMSD student led data/journal clubs the Fall of their first year; 3) participation in the Bio-regulation course, required of all VU biomedical PhD programs, during year two of the Master?s for PhD-level credit for students who earn a B or higher; 4) National Networking Liaisons at research focused institutions where our Biomedical Bridge trainees have transitioned previously and/or whose research areas align with the Master?s phase research, and 5) IMSD peer mentors connected to Master?s phase trainees from the start of their first year.

PROJECT SUMMARY Racial and ethnic minorities and women have been historically under-represented in quantitative sciences. Even within biology, diversity in quantitative sub-branches is much lower than that in experimental counterparts, with the historical data clearly showing that the more mathematical and computational skills a discipline requires, the fewer the enrollment of these under-represented students. The proposed training program seeks to ameliorate these especially pronounced disparities with the biomedical sciences by establishing a streamlined bridge between Master?s programs at Fisk University and doctoral programs at the University of Illinois, Urbana- Champaign (UIUC). Our bridge program designed to nurture a diverse future generation of active minds specifically in the areas of biomedical data science and quantitative biology is named FUTURE-MINDS-QB (Fisk- UIUC Training of Under-represented Minds in Data Science and Quantitative Biology), where quantitative biology encompasses bioinformatics, computational biology, genomic biology, and biophysics. This training program will significantly contribute to diversifying the pool of Ph.D. researchers to include those currently under- represented in biomedical discovery and leadership To achieve our goal, we will accomplish the following short-term and medium-term objectives: (a) establish pathways for transitioning 20 Fisk M.S. students to UIUC Ph.D. programs over five years by providing ample opportunities to strengthen their background in relevant fields and acquire core computational and mathematical skill sets; (b) ensure the trainees? timely Ph.D. attainment within 5 years after Master?s degree; (c) accelerate the admission to and completion of Ph.D. programs by creating a new 4+1 M.S. track at Fisk, rigorously preparing undergraduates for a shortened 1-year M.S degree at Fisk and successful completion of a Ph.D. degree at UIUC; (d) create an inclusive and diverse inter-institutional environment by training both students and faculty in equity- focused teaching, mentoring, peer interactions, rigor, reproducibility, and the responsible conduct of research; (e) devise effective career development plans and opportunities; (f) implement a longitudinal survey of the development of individual trainees, and disseminate an open network of current trainees, graduates, and faculty; and, (g) make FUTURE-MINDS-QB a dynamic entity that continually improves by integrating feedback from trainees, faculty, oversight committees, and independent evaluators. As outcomes of our training, we expect that our seamless infrastructure and appealing inclusive environment will significantly increase the recruitment of under-represented students to quantitative biomedical sciences and that the reinforced academic and psychological preparation will increase the completion of doctoral degrees by under-represented students and ultimately improve their long-term retention in biomedical sciences. We thus expect FUTURE-MINDS-QB to establish an exemplary foundation for training under-represented graduate students and have a long-lasting scientific and socioeconomic impact stemming from their persistence and leadership in their careers.