Wylie Vale - US grants

Neuroendogastrointestinal peptides act at various sites to modulate central nervous system (CNS), endocrine and gastrointestinal functions. The presence in or access to each of these tissues by such peptides is suggestive of their playing physiologic roles as members of a diffuse extracelluar messenger system. We have three major goals: 1. The isolation, chemical characterization and synthesis of new regulatory peptides including growth hormone releasing factor (GRF), corticotropin/beta-endorphin/beta-LPH releasing factor (CRF), mammalian bombesin-like peptide, big somatostatin and adrenal hyperglycemic factor. 2. The investigation of the physiologic and pathophysiologic roles of selected neuroendogastrointestinal peptides particularly as they modulate the functions of the anterior pituitary gland, pancreas and brain control of behavior, the sympathetic nervous system, and glucose metabolism. 3. The development of peptide analogs of these key regulatory peptides which can provide important investigational and clinical tools. Potent, long acting, orally effective, selective agonists and antagonists might be applied to the study, diagnosis or even therapeutic management of conditions such as diabetes mellitus, abnormal growth and development, cardiovascular disease, endocrine neoplasia, and behavioral disorders.

This Program Project of 25 years brings together five Projects and three Cores from 2 institutions and 4 laboratories to explore the physiologic and pathophysiologic roles and signaling mechanisms of neuroendocrine peptides. Over the next period, we will continue our focus on receptors and ligands related to corticotropin releasing factor (CRF), a peptide we chemically identified at the inception of the Program. CRF is the principal neuroregulator of the hypothalamic-pituitary-adrenal (HPA) axis and acts within the brain to integrate endocrine, autonomic and behavioral responses to stress. Many human disorders including anorexia nervosa, anxiety, obesity, drug addiction and depression are associated with perturbations of the HPA axis and changes in sensitivity to CRF. In previous grant periods, we have chemically and biologically characterized multiple components of this system including two receptor genes, a soluble CRF binding and inactivating protein and the neuropeptide, urocortin (Ucn), which has high affinity for both receptor types. Over the past grant period we have identified two new neuropeptides, Ucn 2 and Ucn 3, both of which are highly selective for the second CRF receptor and both exert CRFR2-mediated effects on the endocrine, central nervous, gastrointestinal and cardiovascular systems. We have now developed mutants that are deficient in urocortins 1, 2 and 3. The brain and peripheral distribution and regulation of the two new urocortins are being studied along with their potential physiologic and pathophysiologic roles. The 3-dimensional structure of a major binding domain of CRFR2 has been solved by NMR and a native soluble protein encompassing that domain identified. New potent and selective peptide antagonists of CRFR1 and CRFR2 have been developed and are being used in acute studies to probe the physiologic significance and the pharmacologic promise of these important signaling systems. All of the Projects in this Program are testing hypotheses relating to CRF and Ucn, their regulation and physiologic importance and are taking advantage of recently characterized ligands and receptors, knockout mice, antibodies and improved and selective CRF receptor agonists and antagonists developed by various components within the Program, which are as follows: W. Vale, PD: CRF and urocortins and their receptors; J. Rivier, PD: Pharmacology of Neuroendocrine peptides; R. Riek, PD: Structural studies of the interaction between CRF G-protein coupled receptors and their ligands; G. Koob, PD: Behavioral significance of neuroendocrine peptides; P. Sawchenko, PD: Anatomy of neuroendocrine peptide pathways in the brain; Core A, W. Vale, CD: Administrative; Core B, K-F. Lee and W. Vale, CD's: Biology; Core C, J. Rivier and W. Fischer, CD's: Chemistry.

Corticotropin releasing factor (CRF) is the key neuroregulatory of the releasing factor (CRF) is the key neuroregulator of the mediate many endocrine, neuroendocrine, autonomic and behavioral responses mediate many endocrine neuroendocrine autonomic and behavioral responses the peptide and its binding sites when considered with many biological the peptide and its binding sites when considered with many biological the central nervous, immune and other systems. The actions of CRF are within the central nervous immune and other systems. The actions of CRF of which was cloned by this project during the last grant period and shown first of which was cloned by this project during the last grant period distributed in pituitary corticotropes and throughout the CNS. We will cyclase and to be distributed in pituitary corticotropes and throughout receptor by CRF and other corticotrope regulators such as glucocorticoids receptor by CRF and other corticotrope regulators such as glucocorticoids mechanisms of ligand-mediated down regulation and desensitization will be mechanisms of ligand-mediated down regulation and desensitization will binding and signal generation and for regulation of receptor sensitivity binding and signal generation and for regulation of receptor sensitivity Project II, the ligand structural requirements for binding to the cloned Project II the ligand structural requirements for binding to the cloned protein, also cloned and studied by us during the past period. An protein also cloned and studied by us during the past period. An we will screen for cDNA's encoding this gene and will complete its analysis we will screen for cDNA s encoding this gene and will complete its will be identified by molecular biological techniques or will be purified related ligands will be identified by molecular biological techniques or will be purified and chemically characterized

Inhibins and activins were initially recognized as gonadal protein hormones which reciprocally modulate follicle stimulating hormone production by the anterior pituitary. This Project played a key roll in the initial characterization of these dimeric proteins which share a common subunit. Activins are now recognized to be produced by and exert important endocrine, paracrine and autocrine actions in reproductive and other tissues to regulate cell proliferation, development and differentiated functions. Inhibin (alphabeta) opposes some but not all actions of activin (betabeta) and both are structural and functional members of the transforming growth factor beta (TGFbeta) superfamily of growth and differentiation factors. Similar to TGFbeta, activins signal through two classes of membrane receptors. During the last grant period we cloned and characterized the Type II receptor for activin (ActRII), and thereby discovered the first vertebrate receptor serine kinase (RSK) as well as the first receptor for any member of the TGFbeta superfamily. The field has now identified over a dozen RSK family members including a second Type II activin receptor, the Type II activin receptor, the Type II TGFbeta receptor and Type I receptors for both activin and TGFbeta. The first aim of this Project is to examine the nature of the interactions between the Type I and Type II activin receptors and to examine the roles played by the dimeric ligand in both complex (involving activin and both the Type I and II receptors) formation, cis and trans receptor phosphorylation and signalling. The second aim proposes to clone and characterize the components of the inhibin receptor and then to explore several hypotheses concerning the mechanisms by which inhibin blocks activin. The importance of this interaction has been convincingly demonstrated by reports of the appearance of gonadal and other tumors in 100% of mice whose ability to produce inhibin had been abolished by deletion of the inhibin alpha gene. During the past grant period we proposed that activin produced locally in the pituitary was a key modulator of gonadotrope functions and uncovered multiple regulatory loops involving locally produced activin and follistatin, an activin binding protein. Aim 3 will examine the regulation of Type I and Type II activin receptors in the anterior pituitary. The board spectrum of critical biological actions of these hormones/growth factors and their possible applications to the treatment of reproductive, bone, hematopoietic and central nervous system disorders forms a compelling rationale for the exploration of their receptors, signalling mechanisms and local regulatory roles.

Activins and inhibins are members of the TGFbeta family of growth and differentiation that were discovered by virtue of their reciprocal effects on the production of FSH. These pleiotropic proteins play a wide variety of endocrine, paracrine and autocrine roles within normal and neoplastic reproductive and other tissues. The binding of activin to its specific type II receptor serine kinase kinase (RSK) leads to the recruitment and trans- phosphorylation of its cognate type I RSK (Activin Like Kinase-4) and the subsequent phosphorylation of downstream mediators, the pathway- specific Smads. Under Aim I, we will examine the nature of the interactions between the components of activin signaling. Based on the elucidation of the structure of the ligand binding domain of the type II activin receptor by Project III, we are expressing a series of mutant receptors in mammalian cells and analyzing their affinities for activin, inhibin and the type I receptor (ALK4). These efforts are highly complementary to Choe (Project III), who is working to solve the structure of the ActRII/activin/ALK4 complex and to Fischer (Project IV) who is mutating activin itself.. The second Aim involves the study of mechanisms that serve to limit the biological effects of activin itself. We have observed cellular desensitization of the transcriptional response to activin and will explore mechanisms, including the possibility that this is mediated by the inhibitory Smad, Smad7. We will continue to explore the mode of action of inhibin; this will include a continued effort to clone an inhibin specific binding component. If we are successful, a series of biochemical and cellular experiments will ensure to determine the importance of this protein. If warranted, we will generate and analyze mice deficient in the inhibin receptor. Under Aim III we will continue to explore paracrine and autocrine mechanisms within the pituitary, examining the interesting possibility that some cytokine effects may be mediated through increased production of follistatin. The roles and regulation of Smad7 in the pituitary and gonadotropes, will be explored by a variety of approaches, including the generation of Smad 7 null mice, in which, Smad7 is conditionally knocked out in gonadotropes. This intra-pituitary modulatory network provides a mechanism for the integration of central and peripheral inputs and may be critical for the differential production of FSH and LH under defined physiological circumstances. Drugs and other approaches targeting components of the extracellular and intracellular activin signaling system may prove to be useful for the management of human fertility and reproductive disorders.

This Program Project, comprised of 3 Projects and 2 Cores, explores the signaling mechanisms of activins, BMPs, inhibins and their receptors and binding proteins as they modulate the reproductive and other systems. Project I (Vale) recently identified the proteoglycan, betaglycan, as a putative inhibin co-receptor. This project will investigate the mechanisms by which inhibin interacts with betaglycan and activin receptors to antagonize activin and selected BMPs. Furthermore, the physiologic importance of betaglycan will be tested by immunoneutralization or disruption of its expression in vitro and in vivo. Project I will characterize betaglycan variants and seek additional inhibin co-receptors. Project II (Choe) has solved the X-ray crystallographic structure of the type II receptor extracellular ligand-binding domain (ActRII- ECD) both alone and in a complex with the activin paralog, BMP-7. The structure of BMP-7 with its inhibitory binding protein, noggin, has also been solved. Project II proposes to elucidate additional structures, including triple complexes, comprising activins or BMPs bound to their type II and/or type I ECDs as well as inhibin bound to ActRII and/or betaglycan ECDs. As additional structural information is provided by Project II it will be used by Projects I and Ill to further characterize the structure/function relationships between inhibins, activins, BMPs and their receptors. Project III (Fischer) uses computer modeling and X-ray structure data, obtained in close collaboration with Project II, to design mutant ligands and receptors to be tested for their effects on ligand:receptor binding interactions. Activin mutants will be used to test hypotheses regarding interactions with type II and type I activin receptors and follistatin. Core A (Vale) provides administrative and computer support, evaluation of progress and coordinates interactions with the Advisory Panel and NICHD staff. Core B (Fischer, Rivier, Choe & Vale) provides technical support and reagents, including antisera towards activin/inhibin signaling molecules, and conducts RIAs for pituitary and gonadal hormones. This Core provides synthetic peptides and expresses and purifies recombinant activin and inhibin and various other proteins including receptor components. The Core characterizes native and recombinant proteins by automated sequencing and mass spectrometry. This Program brings together a diversity of approaches to address fundamental issues surrounding activin and BMP signaling and their counter-regulation by inhibin and binding proteins. Because of the physiologic and pathophysiologic significance of activins, BMPs, and inhibins, these studies should serve to illuminate potential means for the control of human fertility and management of medical disorders.

DESCRIPTION (provided by applicant): TGF-B and activin are functionally related members of the TGF-Beta superfamily of growth and differentiation factors. TGF-Beta and activin activate Smad2 and Smad3 and they both regulate tissue homeostasis by potently inhibiting proliferation of multiple cell types, including epithelial cells. Disruption of the antiproliferative response to activin and/or TGF-Beta is associated with dysregulated cellular proliferation, oncogenesis and progression toward the malignant phenotype. Cripto is a developmental oncoprotein that is overexpressed in multiple human tumors including approximately 80% of human breast tumors. Cripto overexpression transforms mammary epithelial ceils and activates the growth promoting Erk/MAPK and PI3K signaling pathways. Cripto and related EGF-CFC proteins also play essential signaling roles during embryonic development and Cripto is required for mesoderm induction and cardiogenesis. It has been shown that Cripto acts as a coreceptor to facilitate receptor assembly and signaling via activin type II/I receptors by the TGF-Beta superfamily members nodal, Vgl and GDF1. This led us to test whether Cripto may regulate the ability of activin and possibly other TGF-Beta ligands to assemble functional receptor complexes and we showed that Cripto binds activin in the presence of type II activin receptors, competes with ALK4 for binding to activin and antagonizes activin signaling. We now have evidence showing that Cripto also binds TGF-Beta in the presence of TBRII, competes with ALK5 for TGF-Beta binding and antagonizes TGF-Beta signaling; we propose, therefore, that Cripto may be generally capable of blocking antiproliferative Smad2/3 signals. Under Aim I of this proposal we will identify the molecular determinants on Cripto that mediate Cripto binding to activin/TFG-Beta and antagonism of activin/TGF-Beta signaling. We also propose to develop reagents including anti-Cripto neutralizing antibodies designed to prevent the ability of Cripto to antagonize activin and TGF-Beta signaling. Aim II proposes to characterize the ability of Cripto to block type I receptor recruitment to ligand-type II receptor complexes and block type I receptor phosphorylation in breast cancer cells in vitro in response to activin/TGF-Beta and the ability of Cripto to block phosphorylation of Smad2/3 following activin/TGF-beta treatment of breast cancer cells in vitro. Under Aim III we will characterize the effects of Cripto on activin/TGF-Beta-induced growth inhibition in three mammary epithelial-derived cell lines: MCF-7; MCF-10A; and CID-9 cells. Together, these studies will lead to an improved understanding of the mechanisms of Cripto action and will help illuminate therapeutic targets for the management of neoplastic disease.

Activins and certain BMPs are members of the TGF-beta superfamily that regulate FSH production by the anterior pituitary. They and their receptors also have diverse endocrine, paracrine, and autocrine actions throughout the reproductive, endocrine and other systems and play key roles in development and in pathophysiologic processes. Inhibins oppose some, but not all actions of activin and are crucial for survival as illustrated by the lethal phenotype of inhibin alpha null mice, which suffer gonadal and adrenal tumors and liver necrosis. To exert cellular effects, activins (beta-beta dimers) bind their type II receptor serine kinases (ActRII or ActRIIB) and then recruit the type I receptor serine kinase (ALK4) to form active complexes that initiate downstream signaling. Inhibins (alpha-beta dimers) compete with activins for binding to ActRII but the inhibin/ActRII complexes do not recruit ALK4 or promote signaling. Inhibins are potent functional antagonists of activin yet the relatively low affinity of inhibins compared to activin for ActRII was not in keeping with a simple competition model. Thus other components were sought and during the past grant period we identified betaglycan (TGF-beta type III receptor) as a high affinity inhibin binding protein (co-receptor) that facilitates inhibin binding to ActRII and, thereby, greatly increases the potency of inhibin to antagonize activin signaling. In model systems we have demonstrated that inhibin can antagonize responses to some BMPs. Under Aim I, these findings will be extended to test if inhibins antagonize the effects of BMPs on pituitary cells. We will continue to characterize the binding interactions between inhibins, betaglycan, and the type II receptors, ActRII, ActRIIB and BMPRII. We will identify the betaglycan residues and regions required for inhibin binding and will produce minimized soluble betaglycan forms that will be used for biochemical and structural (with Project II) studies. Identification of the critical interactive surfaces and regions of these proteins will facilitate efforts to devise strategies for modulating inhibin responses. Aim II proposes to block betaglycan or disrupt its expression in inhibin-responsive cell types including pituitary gonadotropes. In vitro and in vivo experiments will test the importance of betaglycan as an obligate mediator of inhibin actions. Finally, under Aim III we will search for additional inhibin co-receptors. We and others have evidence for the existence of multiple inhibin binding proteins including probable variants of betaglycan, which we propose to characterize. These studies will improve our understanding of the mechanisms of inhibin action and will help identify targets for the control of fertility and management of reproductive disorders.

PROJECT SUMMARY (See instructions): The primary goal of the Administrative Core is to provide overall scientific, operational, and financial coordination of this Program Project. The Program Director and Core personnel work closely to ensure that the administrative needs of this diverse program are met in a timely and cost effective manner. The Core personnel provide comprehensive secretarial assistance to the Project and Core Directors. Core personnel will organize the conferences of the Scientific Advisory Panel. From this Core, the Program Director will maintain the balance between the Program and needs of each Project and Core through formal and informal meetings, email and telephone conversations. Core A personnel have a long-term association with this Program Project and have demonstrated the ability to efficiently and smoothly manage the operation of this large, diverse program.

We are exploring the hypothesis that the Corticotropin Releasing Factor (CRF)/urocortin family of ligands and their cognate receptors play specific and critical roles in the modulation of adaptive responses to stress and other circumstances. In previous granting periods, this Project, with collaborators throughout the Program, characterized CRF, cloned 2 CRF receptors, CRFR1 and CRFR2 and identified urocortin (Ucn) 1, which had high affinity for both receptors. During the last period, we and the Sawchenko Project have found two additional ligands, Ucn 2 and 3, which are highly selective for CRFR2. We have focused on exploring the roles of the N-terminal, first extracellular domain (ECD1) of the CRFRs, which include critical binding sites for peptide agonists and antagonists. We have designed and expressed soluble forms of the ECD1's of CRFR1 and CRFR2beta with retained affinity for appropriate ligands and collaborated with Roland Riek, Salk Institute, to solve the structure of the CRFR2beta-ECD1 by NMR. In the next period, we will work closely with a new project headed by R. Riek, Core C and the Rivier Project to solve the structures of receptor ECD1- ligand complexes for CRFR1, CRFR2alpha and CRFR2beta as well as a novel soluble splice variant comprising mainly the ECD1 of R2alpha. The identification of a protein-protein interaction motif in the ECD1 spawned the testable hypothesis that the functional interaction that we had seen between CRFR2beta and erbB2 in cardiomyocytes may in part depend upon an interaction between the two receptors. Results with mice null for CRFR2 have suggested a variety of roles for this receptor and its ligands in the CNS, cardiovascular system and pancreatic islets. Mice deficient in each of the 3 urocortins have been developed in collaboration with the original Lee project and will be analyzed in this project with respect to the regulation of pituitary/adrenal and pancreatic hormones as well as the expression of CRF-related receptors and remaining ligands. Finally, we will examine the significance of Ucn 3 (and later, Ucn 2), shown to be produced in beta cells and to stimulate insulin and glucagon secretion, as local regulators of islet functions. In the context of the Program, progress in this Project should continue to yield information concerning the molecular nature, regulation and physiologic roles of ligands and receptors of the CRF family and provide insight regarding endocrine, metabolic and stress-related diseases.

PROJECT SUMMARY (See instructions): This Program Project focuses on the role of CRF superfamily peptides and their binding proteins, receptors and modulators in the integration of endocrine, autonomic, behavioral and immune responses to stress. The first aim of this Program is to characterize physical interacfions between CRF family ligands, their receptors and binding proteins and to define motifs required to activate downstream signaling events. The second aim is to explore the physiologic and pathophysiologic significance of these molecules at the cellular and system levels and to study the control of protein expression and secretion as well as modes of action. Addressing questions of a complex ligand/receptor system is facilitated by the availability of key scientific tools. This core application is therefore designed to provide transgenic mice, antibodies, and immunoassays necessary to conduct the integrated studies proposed by the individual Projects. Transgenic mice over expressing components of the CRF system, or null-mutant mice lacking one or more CRFRs and/or ligands, are essential for elucidating the physiologic roles of these ligand and receptors in normal development and in disease states. Detection and/or neutralization of gene products in vitro and in vivo are dependent on high affinity and high titer antibodies of appropriate specificity against CRF family peptides and their soluble binding proteins and membrane bound receptors. Measurement of factors modulated by the CRF receptor-ligand system, including the pituitary hormone ACTH, the adrenal steroid corticosterone, and the pancreatic hormones insulin and glucagon, are also required. This Core can provide transgenic mice, high quality antibodies and assay services that require highly trained personnel and specialized equipment which would otherwise be unavailable to individual projects due to high cost. The establishment of a Core permits reduced costs, improved quality control, efficiency, specialization of technical personnel, standardization of protocols and the dedication of equipment and space. Core B is jointly directed by Drs. K.-F. Lee (transgenic mice unit, 5% effort) and W. Vale (antibody production and assay services, 3% effort).