Philip D. Stahl - US grants

Macrophages express a cell surface receptor that binds and internalizes glycoproteins and glycoconjugates terminating in mannose and fucose. The receptor also mediates phagocytosis of mannose-coated organisms (e.g., yeast). The receptor is a 175 KD glycoprotein that has been isolated from rabbit macrophages and human placenta. The placental receptor is found in trophoblasts. Within placenta, the receptor has an apical distribution being found associated with the brush border facing maternal blood. The macrophage receptor recycles constitutively from the cell surface to an acid intracellular compartment where receptor ligand dissociation occurs. Before an analysis of receptor function can be undertaken, the structure of the receptor must be elucidated. We propose to isolate cDNA encoding for the mannose receptor using lambda gtll placental and macrophage libraries. The libraries will be screened with oligonucleotide probes, derived from receptor sequence and antibodies, prepared aganist human receptor. Using information from cDNA's encoding for the receptor and other peptide sequence, the orientation of the receptor in the membrane will be established. The localization of N-linked and O-linked oligosaccharides within the receptor will be determined as well as other post-translational modifications. In parallel experiments, the biosynthesis, intracellular transport and turnover of the receptor in monocyte- derived macrophages will be determined. Of particular interest is whether the receptor is assembled as a monomer or an oligomer in the plasma membrane. Glucocorticoids elevated mannose receptor levels while gamma-interferon lowers receptor levels. The biochemcial basis for these responses will be investgated using 35 S-methionine pulse-chase experiments followed by immunoprecipitation and Northern blots to quantitate messenger RNA levels. The intracellular localization of receptor will be determined in macrophages and placenta using frozen-thin section immuno-gold cytochemistry at the EM level. Transfection experiments will be carried out to express the mannose receptor cDNA, and constructs with various deletions, in polarized and non-polarized cells. The questions to be adddressed include whether the same receptor mediates phagocytosis and pinocytosis, what structural information accounts for the polarized distribution of the receptor in placental trophoblasts, and for receptor-ligand recycling. The mannose receptor may be a good model for the study of receptor mediated pinocytosis and phagocytosis and its regulation in macrophages and placenta.

The extracellular levels of lysosomal enzymes have been implicated in the development of uncontrolled cellular growth, tumor invasiveness, and the inflammatory response. Previous work in our laboratory has uncovered a mannosyl/glucosyl-specific receptor system associated with macrophage plasma membranes which mediates adsorptive pinocytosis (uptake) of lysosomal glycosidases and glycoproteins having mannose in the terminal position. The receptor also recognizes glucose and N-acetylglycosamine but not galactose. We are studying the mode and mechanism of uptake of lysosomal enzymes by macrophages. To accomplish this we have developed methods to fractionate macrophages by Percoll gradient sedimentation and to follow the uptake of ligands by electron microscopy. We have identified an intracellular compartment in macrophages which appears to mediate separation of receptor-ligand complexes and recycling of free receptors to the cell surface. The regulation of the receptor will be studied by following ligand binding and by preparing receptor-specific antibody. The interrelationships between ligand and internalization and lysosomal enzyme biosynthesis and turnover are being investigated. (E)

This proposal requests funds to purchase a preparative ultracentrifuge. The instrument will facilitate research on protein involved in neuronal growth, to understand the molecular basis of the spatial and temporal control of actin filament assembly and to determine the role of actin assembly in nonmuscle cell motility, on the structure, function, and regulation of Na,KATPase, on the mechanism of endocytosis and receptor- recycling and the cell biology of the macrophage mannose receptor, and the molecular basis of intracellular motility. The senior investigators have good track records and are engaged in high-quality research. Support is recommended.

Abnormalities of urinary acidification are frequent in chronic renal failure and other kidney diseases. The cellular mechanisms for these disorders are not understood, but likely entail changes in biosynthesis, targetting, and degradation of the kidney H+ ATPase. The object of this proposal is to examine the steps in biosynthesis, and pathways of targetting and degradation of the kidney proton pump in the LLC-PK1 cell line. Cell proteins will be labeled with 35S-methionine under pulse-chase conditions, and immunoprecipitation of pump subunits, using antisera both to the whole enzyme and to individual subunits, will be performed. The analysis will include immunoprecipitation from cytosol, from isolated vacuolar organelle fractions, and from plasma membrane. These studies should allow the precise sequence of assembly of the proton pump and its subsequent intracellular itinerary to be established. These findings will contribute greatly toward elucidating the potential steps from which abnormalities or urinary acidification may arise.

The extracellular levels of lysosomal enzymes have been implicated in the development of uncontrolled cellular growth, tumor invasiveness, and the inflammatory response. Previous work in our laboratory has uncovered a mannosyl/glucosyl-specific receptor system associated with macrophage plasma membranes which mediates adsorptive pinocytosis (uptake) of lysosomal glycosidases and glycoproteins having mannose in the terminal position. The receptor also recognizes glucose and N-acetylglycosamine but not galactose. We are studying the mode and mechanism of uptake of lysosomal enzymes by macrophages. To accomplish this we have developed methods to fractionate macrophages by Percoll gradient sedimentation and to follow the uptake of ligands by electron microscopy. We have identified an intracellular compartment in macrophages which appears to mediate separation of receptor-ligand complexes and recycling of free receptors to the cell surface. The regulation of the receptor will be studied by following ligand binding and by preparing receptor-specific antibody. The interrelationships between ligand and internalization and lysosomal enzyme biosynthesis and turnover are being investigated. (E)

The Gordon Conference on Lysosomes will provide an informal forum for discussion of current advances and new directions in the cell and molecular biology of intracellular organelles. Special attention will be paid to pathophysiologic processes which are based on aberrations in subcellular structure and function. Sessions will be devoted to the mechanisms by which cells sort newly synthesized proteins; vesicular transport and the motor- proteins that drive vesicle movement in cells; organelle and lysosome biogenesis and new directions in intracellular targeting' receptor-mediated endocytosis and the reconstitution of fusion events along the endocytic pathway; antigen and protein processing by immune cells and the mechanisms by which certain microbes evade immune destruction; selection protein turnover by lysosomal and cytoplasmic proteases and the movement of proteins between these two compartments. Finally, a session will be organized on the progress in our understanding of lysosomal storage disease with special emphasis on the mechanisms involved and the use of transgenic animals for studies of enzyme replacement therapy. We will make special effort to include young investigators on the program and to encourage such individuals to participate in one of two poster sessions planned for the afternoons. ADditionally, young investigators will be selected from the poster session to present their work to the assembled group in each of the sessions. This should allow for the maximum dissemination of new data and should allow young workers to make important contacts with those in complementary disciplines. We request that this application be reviewed by the Cell Biology Study Section, a group with special expertise int his area. We also would like this proposal considered for partial funding by several NIH institutes in addition to NIGMS. The involvement of lysosomes and other intracellular compartments involving secretion, processing and endocytosis in the pathophysiology of a variety of disease states will be a significant overall part of the meeting. The diversity of the program presented should allow for wide participation of individuals with diverse research interests.

The American Society for Biochemistry and Molecular Biology sponsors an annual fall meeting series focusing on emerging fields of biochemistry and molecular biology. This fall three ASBMB Symposia are planned. The first of these symposia is entitled "GTP-binding proteins and vesicular transport in eukaryotic cells," and will be held September 25-28, 1992 in Keystone, Colorado. The organizers are Philip D. Stahl (Washington University Medical School), William Balch (Scripps), and Peter Novick (Yale University Medical School). This proposal requests support for travel for post-docs and students to attend this symposium. The development of the concept of a role for GTP-binding proteins in intracellular transport has occurred only over the past 5 years. Intracellular transport in general and the role of GTP-binding protein in particular, cuts across the entire field of eucaryotic cell and molecular biology. A meeting of this kind will bring together individuals who primarily work on lower organisms as well as individuals who work predominantly on complicated multicellular organisms. The approaches vary, but the problems that each faces in determining and delineating the role of GTP-binding proteins in transport have a common theme. Often the techniques available and employed by one investigator are foreign to another. One of the major problems to be clarified in this meeting is the precise relationship between and among the various GTP-binding proteins that orchestrate intracellular transport and the factors that regulate and modulate such GTP-binding proteins. One of the goals of this meeting is to being together individuals with diverse backgrounds and interests, along with a significant percentage of students and post-docs to provide opportunity for unique interactions which may lead to long-term consequences.

The mannose receptor (MR), discovered and characterized in alveolar macrophages, has only recently been recognized as the prototype of a new family of endocytically-and phagocytically-active membrane receptors. The MR appears to play a role in innate immunity and in first-line host defense, especially in the lung and at the interfaces between the immune system and the extracellular environment. Recent work has suggested the presence of a signal transduction pathway associated with ligand binding to the MR. These new data raise fundamental questions about the role of the MR in host defense. Moreover, they raise the generic question of whether scavenger receptors, normally associated with the uptake and disposal of microorganisms and extracellular debris, can initiated signaling pathways leading to cytokine production. The long-range goal of this project is to identify and characterize the MR signaling pathway as a means of delineating the role of the MR, and other family members, in host defense and innate immunity. The proposed work will be carried out as three specific aims. The first specific aim is to follow several leads to identify a MR-activated signal transduction pathway. Three complementary approaches will be used. We will characterize several tyrosine phosphorylated proteins which are phosphorylated in response to MR activation. The response to MR activation is wortmannin sensitive linking MR to PI 3-kinase. Protein kinase B/akt and phosphatidylinositol (4) 5-kinase, kinases linked to PI 3-kinase and to link receptors to the cytoskeleton and to activation of gene expression, will be explored as components of the MR signaling pathway. IL-6 amd IL-12 secretin will be used as readouts. Since expression of the MR is closely regulated by IFN-gamma and IL-4, the effects of these cytokines on MR signal transduction will be monitored. The second approach is based on preliminary data indicating that the MR can be isolated with one or more associated proteins. The goal of this part of the proposed is to purify and characterize MR-associated proteins and to determine their role in MR signal transduction. The third approach is to compare the MR with other members of the MR family to determine the structural requirements for signal transduction. By deleting or swapping MR domains with those of other MR family members, we hope to identify regions of the MR required for signaling. The second specific aim is to identify motifs present on the cytoplasmic tail of the MR required for endocytosis and to determine whether the same motifs are required for phagocytosis and/or signaling. Moreover, we will determine whether internalization motifs present in the cytoplasmic tail mediate activation of Rab5, the GTPase which is rate-limiting for endocytosis and required for phagocytosis. The last specific aim is based on preliminary work suggesting the presence of a phenyl-FITC binding site on the MR. We plan to characterize the phenyl binding site using fluorescence spectroscopy and to map its location on the MR using truncation constructs. The overall goal of this project is to identify the molecular components of the MR signaling pathway as a means of delineating the role of the MR, and other family members, in host defense and innate immunity.

9310342 Stahl This U.S.-Argentina Program award will support the collaboration of Philip Stahl of Washington University and Luis Mayorga of the University of Cuyo in Mendoza, Argentina. The project aims to advance knowledge of phagocytosis, a receptor-mediated process carried out by eucaryotic cell types specializing in host defense. The project will explore mechanisms whereby phagosomes, containing newly internalized materials, fuse with the appropriate intracellular vesicles (endosomes) to achieve targeting of the endocytosed materials. This is an important process for eukaryotic cells, with broad implications for such diverse processes as food uptake in unicellular organisms, vertebrate immune functions, the function of retinal photoreceptors in vision, and general homeostasis in multicellular organisms. The U.S. group will focus on the reconstitution of fusion events in permeabilized cell preparations and in identifying protein factors. The Argentine group will use magnetic latex spheres to purify phagosomes and to determine the role of receptors in intracellular fusion events. ***

DESCRIPTION: Receptor-mediated endocytosis is a fundamental process carried out by all cells and is characterized by the internalization of plasma membrane-derived coated vesicles and a series of ordered and specific fusion events among coated vesicles, endosomes, and lysosomes. During the last grant period, in vitro assays were developed to reconstituted early fusion events. Heterotrimeric G proteins and a variety of other factors emerged as important regulators of endocytic vesicle fusion. These assays will now be used to identify key GTP binding proteins and other novel regulators such as phospholipase A2 and characterize their mechanisms of action. Dr. Stahl plans four major specific aims. (1) To use density shift and/or immunoisolation techniques to prepare enriched preparations of endosomes for biochemical and morphological analysis. These fractions will be used to identify cytosolic and membrane proteins associated with endosomes that have been "primed" for recognition and fusion. Ultrastructural characterization of putative fusion pores will be continued. Finally, monoclonal antibodies will be prepared that inhibit fusion. (2) To elucidate the role of heterotrimeric G proteins and ADPribosylation factor (ARF) in fusion, G proteins present in the enriched vesicle fraction will be identified by western blot and mutant or wild type G protein subunits and different ARF family members overexpressed in intact cells using the Sindbis virus expression system. Effects on in vitro fusion or endocytosis in intact cells will be sought. (3) Since G proteins are likely to be activated by upstream effectors, such effectors will be sought. Given that clathrin adaptor proteins have an effect on the in vitro fusion assay, the possibility that adaptins themselves serve this effector function will be evaluated. The role of downstream effectors, such as rab5, NSF and phospholipase A2 will also be evaluated. (4) Endosome-lysosome fusion will be reconstituted in permeabilized cell systems or by using Xenopus oocytes injected with mRNA encoding molecules of interest.

Mycobacterium avium has emerged as a major opportunistic pathogen in AIDS patients. M.avium normally resides within the vacuolar compartment of the macrophage where it restricts access to the digestive and processing capabilities of the host cell. Access appears to be restricted by impaired membrane protein trafficking into and out of the M.avium and M.tuberculosis phagosome. Because of this and the similarity of intracellular M.avium to M.tuberculosis, M.avium may provide an ideal organism to investigate fundamental mechanism of protein traffic in mononuclear phagocytes. Newly formed phagosomes fuse and exchange membrane with multiple intracellular compartments including lysosomes and that the exchange of such proteins is critical to intracellular killing. We have developed methods to reconstitute phagosome fusion events in vitro and we have begun to identify the factors that are required for phagosome membrane traffic. Among the proteins that are likely to play structural and regulatory roles in phagosome membrane trafficking are the GTP binding proteins (Heterotrimeric G proteins, Rab GTP binding proteins and ADP- ribosylation factors-ARFs ) and the newly described SNARE docking proteins (i.e., receptors for the NEM-sensitive fusion protein ). Our initial experiments have successfully reconstituted the in vitro fusion of early phagosomes with endosomes. We propose to exploit the in vitro assay to determine the biochemical requirements for fusion of endosomes with phagosomes isolated from normal macrophages and from macrophages infected with M.avium. Our initial effort is to identify key GTP-binding proteins and SNARE docking proteins that mediate protein trafficking into and out of normal and M.avium phagosomes. Using membranes and cytosol from M.avium infected cells, we will investigate possible inhibitors produced by the organism. In our initial experiments we have begun to catalogue the biochemical changes occurring in phagosomal membranes during phagosome maturation. We propose to develop in vitro assays to reconstitute fusion of phagosomes with vesicles derived from the trans-Golgi Network and with lysosomes and to use these assays to determine whether products of M.avium intracellular infection impair membrane transport. Lastly, we will utilize in vitro assays using Mycobacterium avium to determine the influence of the live organism on the fusion of phagosomes with endosomes, vesicles derived from the trans Golgi Network and lysosomes.

95005083 Stahl This U.S.-Argentina Workshop award supports travel expenses of the P.I., Dr. P. Stahl, Washington Univ. , Saint Louis, MO., five other senior scientists, and three post-doctoral fellows to the workshop which will be held at the Universidad de Cuyo, Mendoza, Argentina, in October, 1995. Travel expenses will also be provided for five South- American Fellows, from countries that are not covered under bilateral agreements with NSF. Some expendable supplies and services related to the workshop will also be covered. The Argentine co- organizer is Dr. Luis Mayorga of the Universidad de Cuyo, Mendoza; other Argentinean scientists involved in the workshop are Drs. Lopez and Sosa, also from the Univ. of Cuyo, and Dr. Maccioni, from Cordoba University. Over the past decade significant progress has been made in the understanding of the cellular and molecular basis of intracellular transport. Fundamental processes, such as secretion, endocytosis and mitosis, processes requiring selective and specific membrane fusion events, are increasingly open to study. Applications of new techniques to a variety of cellular systems, both in plants and animals provides unique opportunities to further expand the level of understanding of vesicular transport. The workshop will provide a unique opportunity to explore these developments, by gathering a small group of scientists from several laboratories from North and South America to exchange ideas about their work on the cell and molecular biology of intracellular transport. The workshop will substantially enhance and extend a very productive international collaboration between U.S. and Argentinean scientists, as well as scientists from other Latin American countries. The workshop will also provide an opportunity to engender long term interactions among faculty and students. This is particularly attractive, since the students will be, for the most part, in the formative years of their training, and wi ll be in an optimal position to benefit from potential international exchange. ***

A wide variety of human pathogens including Listeria monocytogenes (LM) take up residence and thrive within host cells by interfering with membrane trafficking events. LM is internalized into phagosomes where it actively inhibits maturation of the phagosome. Virulent LM escapes to the cytoplasm. Listeria mutants (LMhly-) that lack listeriolysin fail to access the cytoplasm but retain the ability block phagosome maturation. Newly formed phagosomes mature by dynamic remodeling via a series of sequential membrane fusion events followed by phagosome-lysosome fusion. Each fusion event appears to be regulated by a RabGTPase. Rab5a is required for phagosome- endosome fusion. Live LMhly- blocks phagosome maturation by interfering with Rab5a function. Thus, analysis of Rab5a provides an attractive opportunity to examine the regulation of phagosome maturation and the mechanism by which LM interferes with the process. Our central hypothesis is that the GTP/GDP cycle of Rab5a is tightly coupled to phagosome maturation and the activation of downstream GTPases required for efficient phagosome-lysosome fusion. Interferon gamma enhances intracellular killing of LM by selectively inducing Rab5a synthesis. Our goal is to determine how LM and Rab5a function in phagosome maturation, to define the role of interferon gamma in facilitating the process and to delineate the role of GTPases operating down-stream including Rab7 and Rab11. The Specific Aims include identifying the signal transduction mechanisms that control the guanine nucleotide status Rab5a during phagocytosis of LM. We will also delineate the role of protein kinase B/akt, a known regulator of Rab5. The second specific aim focuses on the mechanism by which IFNgamma stimulates phagosome maturation and killing. Interferon gamma selectively induces Rab5a synthesis and processing. We will investigate the mechanism by which IFNgamma elevates the prenylation of Rab5a. We will use phagosome-lysosome fusion assays to determine whether IFNgamma treatment enhances coupling of Rab5a to downstream Rab GTPases, Rab7 and Rab11. We will use knock out mice lacking the IFNgamma receptor to confirm the role played by this receptor. Since IFNgamma treatment selectively induces Rab5a but not Rab5b or Rab5c and since live LM causes Rab5a to accumulate on phagosomes, we will explore the possib ility that the endocytic apparatus is composed of sub-compartments marked by different rab5 isoforms. Rab5a may specifically connect the endocytic apparatus to the developing phagosomes whereas other Rab5 isoforms may have different functions. Using epitope tagged Rabs coupled with both light and electron microscopy and using GFP-Rab5 isoforms in living cells, we will identify a subset of endosomes that function in phagosome-endosome fusion. We will use GFP-Rab5 to observe in real time the docking and fusion of GFP-Rab5 isoform-marked endosomes to newly formed phagosomes harboring live or dead Listeria monocytogenes. We will determine the effects of IFNgamma treatment on vesicular traffic into and out of LM phagosomes using GFP-Rab5, GFP-Rab7 and GFP-Rab11.

DESCRIPTION (provided by applicant): Type Ibeta phosphatidylinositol-4-phosphate-kinase (PIP5Ka) was initially identified as a partner of the M-CSF receptor. Subsequently, PIP5Kbeta was shown to be recruited to the M-CSF receptor and to the EGF receptor. Failure to recruit PIP5Kbeta prevented receptor internalization and altered signal transduction pathways. Our long term goal is to address three fundamental questions: (i) how M-CSF activation of the M-CSF receptor is coupled the activation of the type I PIP5K family of lipid modifying kinases, (ii) how M-CSFR interaction with PIP5K integrates signaling and endocytic trafficking of the activated receptor in macrophages and osteoclasts, (iii) what is the effect of PIP5K and M-CSF on macrophage and osteoclast endosome structure and composition. M-CSF (c-Fms) is one of the most important cytokines regulating monocyte/macrophage survival, proliferation and differentiation. Moreover, cells of the monocyte/macrophage lineage are precursors of the osteoclast, multinucleated cells essential for bone remodeling. Understanding the relationship between M-CSF receptor signaling and trafficking could reveal new therapeutic targets and have a profound effect on our understanding of a number of M-CSF dependent pathophysiological processes including osteoporosis. The goal of this proposal is to examine the interaction of PIP5Kbeta with the M-CSF receptor and to determine the role it plays in M-CSF signaling in osteoclasts, the major site of M-CSF activity and in macrophage/osteoclasts endosome structure and function. (1) We will study the interaction of the PIP5K with the MCSF receptor expressed in bone marrow macrophages and macrophages transfected with an Epo/c-fms chimeric receptor. We will take advantage of the many point and truncation mutants available in the Epo/c-fms chimeric receptor. (2) Preliminary data indicate that PIP5Ks are important early determinants of RTK signal transduction, both via the MAP kinase pathway and the PKB/akt pathway. We will establish the role of the PIP5Kbeta as a mediator of MCSF signal transduction using M-CSFR and Epo/c-fms expressing bone marrow macrophages. Our hypothesis is that PIP5Kbeta initiates receptor internalization which may favor one or more signal transduction pathways. (3) We will characterize the effect of PIP5K and M-CSF on macrophage and osteoclast endosome structure and composition. These studies will lay the groundwork for an osteoclast "endosome proteome" project. Osteoclast endosomes are predicted to be important organelles both in terms of regulating and modulating cell surface composition and function (e.g., bone resorption) and in generating signals from M-CSF and other osteoclastogenic growth factors. Membrane rafts may also play key roles in receptor signaling. Using established gradient sedimentation and fractionation procedures with cultured osteoclasts, we propose to explore the effect of PIP5K, following M-CSF and RANKL stimulation, on the structure and composition of the osteoclast endosomes and rafts using mass spectrometry and proteomics methodologies.

[unreadable] DESCRIPTION (provided by applicant): Critical to continued progress in imaging sciences is the integration of traditionally discrete lines of research. For example, advances in observing organelles and macromolecular complexes in living cells need to be integrated with new methodological approaches to imaging live animals; advances in biochemistry that produce novel imaging agents need to be integrated with new approaches to selective cellular delivery and employ transgenic animal models simulating human diseases. To realize the potential that evolving paradigms of molecular, cellular, live animal and human imaging offer, a cadre of 21st-century scientists must be trained-and a revolutionary synthesis of chemistry, biology, engineering, physics and mathematics must be achieved. The imaging science community at Washington University, because of its breadth, diversity and depth of funding, is in an excellent position to foster the development of a new approach by attracting and training next-generation imaging scientists. We propose to devise curricula and develop opportunities that reach out to students at the most formative stages of their academic careers-in their undergraduate and graduate years. Early access to students is imperative to develop a broadly-based academic foundation in both the physical and life sciences, and in engineering. An Undergraduate Imaging Sciences Pathway will be developed. Students in their junior and senior years will have the opportunity to take courses in chemistry, physics, computer science, engineering and molecular cell biology as they relate to imaging sciences, and to gain in-depth research experience in laboratories of imaging sciences faculty. For predoctoral students, we propose to create a Graduate Imaging Sciences Pathway that will be available to students in the biomedical sciences, physical and quantitative sciences, psychology, and engineering. We anticipate that the Graduate Pathway program will mature into a formal graduate program in 2008, when we have optimized the curriculum and developed the administrative mechanisms for interdisciplinary training of students having mentors in the Schools of Arts and Sciences, Medicine, and Engineering and Applied Sciences. We envision that a graduate student who has completed training in the Imaging Sciences Pathway or graduate program will be poised to broaden their horizons and open new opportunities for research in the new age of interdisciplinary science. [Relevant Institutes: NIBIB, NIGMS, NCI, NINDS, NHLBl [unreadable] [unreadable] [unreadable]

Animals; Binding; Binding (Molecular Function); CRISP; Computer Retrieval of Information on Scientific Projects Database; Funding; GDI-2 protein, mouse; GDP; GDP dissociation inhibitor 2, mouse; GTP; Gdi2 protein, mouse; Grant; Guanosine 5'-(tetrahydrogen triphosphate); Guanosine 5'-(trihydrogen diphosphate); Guanosine 5'-Diphosphate; Guanosine Diphosphate; Guanosine Triphosphate; Institution; Investigators; Isoforms; Knockout Mice; Mice, Knock-out; Mice, Knockout; Molecular Interaction; NIH; National Institutes of Health; National Institutes of Health (U.S.); Null Mouse; Protein Isoforms; Proteins; Rab GDI-2 protein, mouse; Research; Research Personnel; Research Resources; Researchers; Resources; Sequence Homology; Signaling Protein; Source; Structure; United States National Institutes of Health; gene product; guanosine diphosphate dissociation inhibitor protein, mouse; homology (molecular); mouse Gdi2 protein

DESCRIPTION (provided by applicant): The molecular cell biology of human- and hominoid-specific genes is virtually a terra incognita in basic research. Understanding the molecular cell biology of human-specific genes will open a new vista of research of wide import to understanding human biology and disease. TBC1D3 is a widely expressed hominoid-specific gene, and recent work indicates that TBC1D3 is a powerful stimulant of the EGF and IGF-1 receptor pathways. Thus, expression of TBC1D3 promotes cellular proliferation and enhances the activation of Ras in response to growth factors. TBC1D3 contains a TBC domain that interacts with the small GTPase Rab5, a key regulator of endocytosis. We speculate that TBC1D3 may be both a growth factor receptor regulator and a Rab5 effector. This proposal focuses upon defining the biochemical mechanisms that are set in motion by TBC1D3 and result in cell proliferation. In Specific Aim 1, we will carry out a structure-function study to determine domains in TBC1D3 involved in cell localization and in Ras activation. To understand the linkage between TBC1D3 and the Ras pathway, we will identify serine/threonine and tyrosine-phosphorylation sites on TBC1D3 and validate the role TBC1D3-interacting proteins, Grb2 and 14-3-3, in Ras activation. In Specific Aim 2, we will quantify the interaction between TBC1D3 and the Rab5 subfamily of GTPases. By modeling the TBC domain of TBC1D3 using 3-dimensional structures of known TBC domains, we will identify key residues at the TBC1D3:Rab5 interface and test their role in Rab5 binding. To confirm and extend these initial structural studies, the structure of the TBC domain will be solved. In Specific Aim 3, we hypothesize that TBC1D3 stimulates cell signaling/proliferation by modulating IRS1 phosphorylation and via the interaction between TBC1D3 and the E3 ubiquitin ligase Cul7. We will characterize the effect of TBC1D3 on IRS1 phosphorylation and turnover. To understand the role of Cul7 in TBC1D3 function, we will determine the domains that mediate TBC1D3-Cul7 interaction and using deletion mutants, determine the function of the TBC1D3-Cul7 complex. The cell and molecular biology of TBC1D3 may represent a model to understand the evolution of human-specific genes that function to modulate and regulate evolutionarily conserved cell signaling pathways. PUBLIC HEALTH RELEVANCE: TBC1D3 is a hominoid-specific gene that regulates growth factor receptor signaling. TBC1D3 has been shown to be an oncogene and to be over-expressed in certain proliferative disorders. Human specific genes have not been extensively studied in spite of their relevance to the human condition and their potential role in human specific diseases. This work proposes to elucidate the mechanism of action of TBC1D3 at the cellular and molecular level. TBC1D3 may serve as a model for developing an understanding of the function of human-specific genes in regulating complex signaling pathways.