Cathleen R. Carlin - US grants

epidermal growth factor; gene expression; aging; protein tyrosine kinase; messenger RNA; fibroblasts; cell membrane; premature aging; cell cycle; embryo /fetus cell /tissue; gangliosides; tissue /cell culture; acylation;

We have found that group c human adenoviruses (Ad) down-regulate the receptor for epidermal growth factor (EGF-R); i.e. EGF-K is no longer on the cell surface. Using virus mutants, we mapped the effect to a putative 10,400 MW (10.4K) protein encoded in the E3 transcription unit. This represents an important discovery because the function of E3 is largely a mystery. Moreover, EGF-K is a key growth regulatory molecule, and inappropriate EGF signal transduction is often associated with malignancy. Using EGF-R antibodies, we are unable to detect mature EGF-K in 10.4K- expressing cells when EGF-R is metabolically labeled with (35S)Cys, labeled on the cell surface with 125I, or autophosphorylated in vitro. Also, 10.4K prevents binding of EGF to EGF-R. EGF induces a variety of effects in cells with specific receptors. EGF stimulates the intrinsic protein tyrosine kinase of EGF-R; the EGF/EGF-R complex then clusters in clathrin-coated pits, internalizes via endosomes, is transported to lysosomes, and degraded. This process transduces EGF signals which activate cellular metabolism, DNA synthesis, and mitosis. Although 10.4K differs in sequence from EGF and is presumably a membrane rather than a secreted protein, 10.4K may mimic some of the EGF effects on EGF-R. This hypothesis is based on our data above, as well as immunofluorescence, which suggest that 10.4K does not inhibit synthesis of EGF-R, but it does induce internalization and degradation of EGF-R. Activation of EGF-R should benefit Ad in vivo, since Ad multiplication is probably more efficient in stimulated versus quiescent cells. Our major goals are to determine the mechanism of action of 10.4K, and whether it stimulates or abrogates the cellular responses to EGF signal transduction. We will develop antibodies against 10.4K, characterize 10.4K biochemically, generate 10.4K mutants for structure/function studies, and express wild type and mutant forms of 10.4K in eukaryotic and prokaryotic expression vectors. We will determine {i) the sub-cellular localization of 10.4K and whether it binds to EGF-R, (ii) which domains in EGF-R and 10.4K are important for the 10.4K effect, (iii) whether 10.4K alone can stimulate the EGF-R kinase, nutrient uptake, transcription of specific genes, DNA synthesis, and/or mitosis, (iv) whether 10.4K acts on other receptors with protein tyrosine kinase activity, and (v) whether all six groups of human Ads and mouse Ad have a 10.4K- equivalent protein. This project is an ideal merging of the expertise of C. Carlin in EGF-R and cell biology, and of W. Wold in the molecular biology of Ad region E3.

Monolayers of polarized kidney tubule epithelial cells form a selective barrier fundamental to homeostasis, achieved in part by structural and functional polarity of the plasma membrane. Membrane proteins with specialized functions are targeted to apical or basolateral surfaces by intrinsic sorting signals. The overall goal of this proposal is to understand the biogenesis and polarized sorting of three related plasma membrane proteins that play important roles in renal genesis and tubular cell proliferation: the membrane-bound precursor of the polypeptide growth factor EGF (preproEGF), which is expressed on apical surfaces of epithelial cells in the thick ascending limb of Henle (TALH) and distal convoluted tubule; the membrane-bound precursor of a structurally related polypeptide, transforming growth factor alpha (proTGFalpha), which appears to be expressed on apical membranes of collecting tubule cells; and the cellular receptor for EGF and TGFalpha (EGFR), which is found on basolateral surfaces of most renal tubule segments. Asymmetric localization of ligand and receptor may help explain why renal epithelial cells are normally quiescent in vivo except during tubular damage. PreproEGF, TGFalpha, and EGFR surface polarity may also influence kidney organogenesis, since their expression is tightly regulated during development. It has recently been shown that surface polarity of some membrane proteins is altered in certain pathophysiological conditions. There are reports, for example, that EGFR polarity is partially reversed in cystic epithelia from patients with autosomal dominant polycystic kidney disease (ADPKD). If true, then EGF/TGFalpha signalling could be profoundly influenced by asymmetric expression of other cellular components in ADPKD epithelial cells, depending on where EGFR is expressed. The specific aims of this proposal will delineate intracellular trafficking of newly synthesized preproEGF, proTGFalpha, and EGFR in renal tubular epithelial cells; identify intrinsic sorting signals for preproEGF, proTGFalpha and EGFR in renal tubular epithelial cells; and determine the effect of regulated posttranslational Ser/Thr phosphorylation on EGFR trafficking in renal tubular epithelial cells, with an emphasis on post-endocytotic sorting pathways.

Monolayers of polarized kidney tubule epithelial cells form a selective barrier fundamental to homeostasis, achieved in part by structural and functional polarity of the plasma membrane. Membrane proteins with specialized functions are targeted to apical or basolateral surfaces by intrinsic sorting signals. The overall goal of this proposal is to understand the biogenesis and polarized sorting of three related plasma membrane proteins that play important roles in renal genesis and tubular cell proliferation: the membrane-bound precursor of the polypeptide growth factor EGF (preproEGF), which is expressed on apical surfaces of epithelial cells in the thick ascending limb of Henle (TALH) and distal convoluted tubule; the membrane-bound precursor of a structurally related polypeptide, transforming growth factor alpha (proTGFalpha), which appears to be expressed on apical membranes of collecting tubule cells; and the cellular receptor for EGF and TGFalpha (EGFR), which is found on basolateral surfaces of most renal tubule segments. Asymmetric localization of ligand and receptor may help explain why renal epithelial cells are normally quiescent in vivo except during tubular damage. PreproEGF, TGFalpha, and EGFR surface polarity may also influence kidney organogenesis, since their expression is tightly regulated during development. It has recently been shown that surface polarity of some membrane proteins is altered in certain pathophysiological conditions. There are reports, for example, that EGFR polarity is partially reversed in cystic epithelia from patients with autosomal dominant polycystic kidney disease (ADPKD). If true, then EGF/TGFalpha signalling could be profoundly influenced by asymmetric expression of other cellular components in ADPKD epithelial cells, depending on where EGFR is expressed. The specific aims of this proposal will delineate intracellular trafficking of newly synthesized preproEGF, proTGFalpha, and EGFR in renal tubular epithelial cells; identify intrinsic sorting signals for preproEGF, proTGFalpha and EGFR in renal tubular epithelial cells; and determine the effect of regulated posttranslational Ser/Thr phosphorylation on EGFR trafficking in renal tubular epithelial cells, with an emphasis on post-endocytotic sorting pathways.

Protein transport and sorting are essential processes for many cellular functions. Understanding molecular interactions that regulate intracellular protein transport is therefore a fundamental question in cell biology. The present proposal, in its fifth year of funding, seeks continued support for investigating EGF receptor (EGFR) degradation induced by a novel human group C adenovirus (Ad) protein called E3- 13.7/11.3 kDa. This application has been revised since it was originally reviewed in October, 1993, in response to the comments of the study section and our additional preliminary data. Our major accomplishments in the last funding period have been: a) The discovery that E3-13.7/11.3 kDa is translated in Ad-infected cells, and is an integral membrane protein with two potential membrane-spanning alpha-helices, an extracellular loop domain connecting the helices, and a cytosolic tail region essential for function. b) The discovery that the EGFR cytosolic juxtamembrane domain is required for E3-13.7/11.3 kDa-induced degradation. c) The discovery that the insulin receptor, IGF1 receptor and pp185(c-neu) are also down- regulated by E3-13.7/11.3 kDa, but the PDGF receptor and aFGF receptor are not. d) The discovery that E3-13.7/11.3 kDa increases trafficking of constitutively internalized EGFRs to multivesicular bodies (MVBs) where they are sorted to lysosomes, but does not increase the rate of EGFR internalization or induce intrinsic EGFR tyrosine kinase activity. These findings have led us to hypothesize that E3-13.7/11.3 kDa regulates EGFR plasma membrane (PM) to lysosome (Ly) sorting. If this supposition is correct, this is the first viral protein identified that uses this mechanism, and as such will be a valuable model system for characterizing host cell proteins that regulate membrane protein traffick. Towards that end, we have recently identified cellular proteins that bind specifically to a bacterial fusion protein expressing the E3-13.7/11.3 kDa cytosolic tail in vitro. In order to understand the interaction between EGFR and E3- 13.7/11.3 kDa in proper physiological context, we have also demonstrated the feasibility of studying this process in filter-grown polarized epithelial cells where EGFRs are located predominantly in the basolateral (BL) PM. We have recently shown that E3-13.7/11.3 kDa selectively sorts to the BL PM and also induces EGFR degradation in polarized cells. Surprisingly, a significant fraction of EGFRs missort to the apical (Ap) PM in cells infected with an Ad E3-13.7/11.3 kDa deletion mutant. This suggests that E3-13.7 kDa mediated EGFR degradation may counteract EGFR missorting induced by a separate event during early Ad infection. The new specific aims are: 1. To learn whether E3-13.7/11.3 kDa affects recycling of endocytosed EGFRs to PM, or transport of newly synthesized EGFRs from trans Golgi network to PM. 2. To learn whether E3-13.7/11.3 kDa is an organelle-associated protein that regulates EGFR protein transport. This will be achieved by determining the steady-state subcellular distribution of E3-13.7/11.3 kDa; which amino acids in the cytosolic tail of 13.7/11.3 kDa are important for proper expression and function; and whether E3- 13.7/11.3 kDa interacts with host cell proteins. 3. To precisely define the minimum receptor tyrosine kinase sequence required for E3-13.7/11.3 kDa-induced degradation in EGFR, the insulin receptor, and pp185(c-neu). 4. To establish a physiological model for studying Ad-induced effects on EGFR trafficking, by studying this effect in polarized epithelial cells.

Formation of epithelial cell polarity is a fundamental process in embryonic development and organogenesis. Polarized epithelia in adult animals form a physical barrier between the host and external environment essential for homeostasis. Polarized epithelia maintain distinct apical (Ap) and basolateral (BL) membrane domains, by actively regulating membrane distribution of lipids and proteins as well as the sub- membranous cytoskeleton unique to each surface. Many questions remain about how membrane polarity is achieved, chief among them how Ap and BL proteins are packaged in distinct transport vesicles at the trans-Golgi- network (TGN). Sorting of BL proteins is mediated in part through recognition of distinct cytoplasmic sorting signals that also appear to regulate polarized sorting in endosomes. Although many BL sorting signals have common features, consensus motifs have not emerged, making it unclear whether all BL signals mediate transport by the same or different pathways. We propose to address this question by studying a novel autonomous Bl signal which we have identified in the EGF receptor (EGFR). This signal is located between residues K652 to A674 in the EGFR juxta- membrane domain, and mediates BL transport of cytoplasmically truncated EGFRs and protein chimeras containing a luminal. This BL signal critical tyrosine and leucine residues and do not overlap any of the known EGFR endocytic signals, features that distinguish it from many other well- characterized BL sorting signals. Computer modeling suggests a propensity for this region to form an amphipathic helix, in contrast to other BL signals whose critical structure suggests a propensity for this region to form a amphipathic helix, in contrast to other BL signals whose critical structure in a beta-turn. This region also induce T654, a known substrate for protein kinase C, raising the possibility that its activity is regulated by phosphorylation. Immediate goals are to understand the mechanism by which this signal establishes and maintains EGFR' polarity. A long-term goal is to understand how genes which cause polycystic kidney disease alter this process, since non-polar EGFR expression is a common finding that renal cysts both in humans and animals disease models. The specific aims will: test the hypothesis that BL sorting of cytoplasmically truncated EGFRs is critically dependent on particular amino acids in the BL signal; test the hypothesis that residues K652 to A674 regulate BL transport of full-length EGFRs; test the hypothesis that residues K652 to A674 regulate polarized sorting in endosomes; and characterize elements of the EGFR sorting machinery by identifying proteins that interact with EGFR residues K652 to A674.

[unreadable] DESCRIPTION (provided by applicant): The endosomal apparatus is a major site of membrane sorting in both endocytic and exocytic pathways. For ligand activated receptors it also provides a mechanism for achieving the proper balance of cell signaling from different membrane compartments. A main objective of this proposal is to understand how endocytosis controls cellular responses to the EGF receptor (EGFR), the prototype for the ErbB receptor family. The complexity of endocytic trafficking predicts that EGFRs use a multitude of distinct sorting signals at different locations in the cell. Characterization of these signals provides a framework for understanding regulation of EGFR transit in the endosomal apparatus. We have identified three non-overlapping sorting signals that control critical benchmarks in EGFR trafficking: 679-LL, which controls sorting in multivesicular endosome-to-lysosome transport intermediates or MVEs; 954-YLVI, which controls lysosomal sorting at a site intersecting the exocytic pathway; and 662-RxxxxPLTP, which controls sorting from endosomes to the plasma membrane. Completion of the proposed studies will provide new insights on the coordinated action of these signals during ligand-regulated EGFR trafficking. Since related ErbB receptors have divergent sorting signals compared to EGFR, we will also gain insight to how ErbB signaling in general is controlled by intracellular trafficking. This is particularly important for ErbB2, whose ligand-dependent activation by EGFR requires EGFR sequences that regulate post-endocytic trafficking and not intrinsic tyrosine kinase activity. The availability of dominant-inhibitory mutations that disrupt EGFR trafficking at defined steps also provides a unique set of reagents with which to study the temporal and spatial organization of signaling from endosomes. The following hypotheses will be tested: 1. Ligand-induced EGFR post-endocytic sorting to lysosomes is mediated by the concerted action of multiple sorting signals acting in a step-wise fashion. 2. The 679-LL MVE sorting signal is recognized as part of a larger motif whose physiological function is dependent on protein-protein interactions. 3. The 662-RxxxxPLTP sorting signal controls EGFR recycling to the plasma membrane, and is regulated by MAP kinase-mediated phosphorylation of Thr669. 4. The 679-LL sorting signal regulates EGFR signaling by controlling the balance of growth stimulatory pathways elicited during endocytosis. This hypothesis is based on new data suggesting that EGFRs with an inactivating L679A,L680A mutation selectively activate survival pathways compared to wild-type, and will be tested using both in vitro and in vivo experimental models.

OVERALL DESCRIPTION (Taken directly from the application) The overall objective for the development of an Interdisciplinary Center for Polycystic Kidney Disease Research at Case Western Reserve University (CWRU) is to attract a partnership of interdisciplinary research among investigators who will use complementary and integrated approaches to study the molecular and cellular pathophysiology of autosomal recessive polycystic kidney disease (ARPKD). ARPKD has an incidence of 1: 10,000 to 1: 40,000, has a mortality of 40-65% in the newborn period, and accounts for approximately 5% of all end-stage renal disease in children. Other than end-stage renal disease therapy and palliative measures designed to treat the complications of progressive portal hypertension, there is no known therapy for progressive renal cyst formation and enlargement or progressive biliary ectasia and fibrosis in ARPKD. Therefore the overall goal of the Center is to support scientific investigation directed at delineating the fundamental aspects of the disease process which will translate into the design of preventative and/or curative strategies for ARPKD. An ancillary objective of the Center is to attract new scientific expertise to the study of polycystic kidney disease. To achieve the stated objectives of the Center, the program involves a interdisciplinary research team drawn from the Departments of Pediatrics, Genetics, and Physiology & Biophysics at CWRU. The three Projects, three Cores, and two Pilot and Feasibility studies are scientifically integrated into an overall scheme which follows directly from current understanding of the molecular and cellular pathophysiology of ARPKD. Project 1, "Epithelial Growth Factor Mislocalization in ARPKD" focuses on a key process mediating abnormal epithelial cell proliferation. Project 2, "Altered Collecting Tubule Ion Transport in ARPKD" focuses on abnormalities in key ion transport processes which mediate altered tubular fluid secretion. Project 3, "Pharmacological and Genetic Therapy of ARPKD" is a translational project to develop therapeutic strategies which target key processes operative in the development and progression of disease. To support the scientific program, an Administrative Core will coordinate Center activities and specifically focus on maximizing scientific interactions of Center Investigators while monitoring and critically evaluating scientific process and encouraging new research in polycystic kidney disease-related areas. A Transgenic & Animal Resource Core will facilitate whole animal experimental approaches for all projects of the Center using state-of-the-art molecular genetic technology. A Cell Culture Core will provide and maintain primary cells and cell lines from human and murine control and cystic kidneys for the proposed studies. Though largely focused on the molecular and cellular pathophysiology of ARPKD, much of the basic knowledge and many of the treatment strategies developed by the Center will also have relevance to the study and treatment of autosomal dominant polycystic kidney disease.

DESCRIPTION (provided by applicant): Endocytosis serves many important functions ranging from acquisition of extracellular nutrients to regulation of cell surface receptor expression and signal transduction, maintenance of cell polarity, and antigen presentation. Many intracellular pathogens hijack endocytic pathways in order to invade cells, proliferate, and ensure pathogen survival. Understanding the consequences of pathogenic infection has enabled greater understanding of the endocytic trafficking machinery. Isolated pathogenic genes have also been used to curb pathological processes associated with their cellular targets in human disease models. The focus of this proposal is to understand the molecular and cellular mechanisms employed by an integral membrane protein encoded by the early region 3 of human adenoviruses called RID?, which was originally identified because of its ability to divert constitutively recycling EGF receptors to lysosomes. We have recently discovered that RID? interacts with RILP and ORP1L, two known effectors for the Rab7 GTPase that governs transport from early to late endosomes and then to lysosomes. Importantly, RID? compensates for Rab7 loss-of-function, suggesting it modifies endosome membrane dynamics by coordinating recruitment of Rab7 effectors to compartments that would ordinarily be perceived as early sorting endosomes. To our knowledge this is the first protein encoded by a DNA virus that functions by Rab7 mimicry. Similar to other small GTPases, Rab7 acts by cycling between an active GTP- bound state and an inactive GDP-bound state. In contrast RID? is a non-enzymatic intrinsic membrane protein that lacks any sequence homology to Rab7, providing a remarkable example of convergent evolution. Four specific aims will test these hypotheses. 1) RID? controls microtubule-dependent vesicle transport by recruiting RILP and ORP1L which then activate minus end-directed dynein-dynactin motors. 2). RID?- RILP facilitates ESCRT-II-dependent EGF receptor sorting independent of receptor tyrosine kinase or ubiquitin status. 3) RID?-ORP1L regulates cholesterol efflux from endosomes necessary to maintain the proper lipid balance in cells. 4) RID? compensates for Rab7 loss-of-function during a productive adenovirus infection, and blunts adenovirus-induced inflammatory disease by interfering with a TNF?-EGF receptor signaling cascade that regulates IL-8 production. Altogether these studies will provide novel insights to the cell biology of membrane protein trafficking, and also the molecular basis for adenovirus-induced inflammatory disease. PUBLIC HEALTH RELEVANCE The proposed studies are relevant to public health because they will provide novel insights to the molecular pathogenesis of adenovirus pneumonia. They will generate new strategies for design of non-toxic adenovirus-based gene therapy vectors, and benefit patients with chronic obstructive pulmonary disease (COPD) and other chronic respiratory diseases where it is thought persistent adenovirus infections are an important disease risk factor. These studies will also provide the foundation for developing new treatments for patients with Niemann-Pick C disease, a progressive neurological disease that is always fatal.

ABSTRACT The apical surface of polarized epithelium constitutes one of the first points of contact between the host and pathogens such as human adenoviruses (HAdVs) that invade the lumen of the respiratory tract. In recent years, it has become clear that respiratory epithelial cells not only serve as functional and physical barriers, but actively contribute to the innate immune system providing initial protection against external pathogens. The EGF receptor (EGFR), which typically exhibits basolateral polarity, has emerged as a key player in the innate immunity of respiratory epithelium to a variety of infectious and noninfectious noxious stimuli. In contrast to the canonical ligand-stimulated EGFR pathway, a number of cellular stresses including HAdV infection (our studies) trigger an alternative mode of EGFR trafficking associated with sustained EGFR activity in non- degradative endosomes. However, molecular mechanisms regulating this pathway remain poorly understood. In addition, despite significant progress in understanding the pathological and therapeutic stresses that activate it, relatively little is known about EGFR function in the context of cellular stress. We have recently found that stress-induced EGFR signaling is involved in innate immune responses triggered by HAdV cell entry, as well as following exposure to the pro-inflammatory cytokine TNF-?, in respiratory epithelial cells. Moreover, HAdV encodes an early gene product that suppresses this pathway by promoting EGFR degradation, and which could provide new insights to potential targets for future anti-viral therapies. Interestingly, our preliminary studies have revealed that EGFR stress responses were tightly regulated by epithelial cell polarity, with robust stress-induced EGFR responses only observed when receptors were mistargeted to apical membranes. In addition, HAdV co-opted cellular pathways contributing to innate immune signaling by enabling dynamic EGFR membrane remodeling associated with enhanced stress-induced EGFR signaling from apical membranes. The newly described EGFR innate immune system is likely to have a central role in protecting the lung from infection with HAdVs and perhaps other pathogens. Conversely, failure to curb this signaling network may lead to tissue damage, respiratory compromise, and potentially systemic HAdV infections. Our research plan will identify novel mechanisms regulating dynamic EGFR membrane remodeling in polarized epithelial cells (Aim 1), and stress-induced EGFR innate immune signaling from endosomes (Aim 2); and analyze EGFR innate immune signaling pathways that are activated as a consequence of HAdV infection in primary human respiratory epithelial cells and new physiological cells models with enhanced apical EGFR expression (Aim 3). Although our studies will be carried out in the context of HAdVs, successful completion of this project will have a broad impact on a variety of respiratory conditions in need of new therapies.