Receptor tyrosine kinases (RTKs) are cell-surface receptors that regulate key cellular processes including cell proliferation, differentiation, migration, survival, and metabolism. RTKs share a general architecture in which an extracellular ligand-binding region is followed by a single membrane-spanning region, a cytoplasmic kinase, and a onglobular C-terminal tail that is frequently the site of autophosphorylation and interactions with downstream effectors. Ligand binding to RTKs stimulates their intracellular kinase activity, which in turn stimulates their cellular effects. Human RTKs are classified into 20 different classes based on the nature of their extracellular regions, and include receptors for insulin, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), fibroblast growth factor (FGF), and Ephrins. Abnormal RTK activity is associated with many human diseases including birth defects, cancer, and diabetes. RTK-targeted therapies are in wide use and include Insulin itself and antibody and small-molecule inhibitors of the Epidermal Growth Factor Receptor (EGFR), its homolog HER2, and the Vascular Endothelial Growth Factor Receptor (VEGFR). In recent years we have pursued X-ray structural and functional studies of the EGFR (ErbB) and Insulin families of RTKs aimed at understanding the molecular mechanisms governing their activity in normal and disease states. The classic mechanism by which RTKs are thought to signal is ?ligandinduced dimerization? although the constitutively dimeric Insulin Receptor (IR) is thought to signal via a liganddependent conformational change. The extracellular region of the EGFR has long been known in addition to autoinhibit EGFR activity in the absence of ligand, and we recently showed that the extracellular region IR homolog IGF1R is also autoinhibitory and that ligand activates IGF1R (and IR) by releasing this autoinhibition. IR/IGF1R autoinhibition involves maintaining a separation between subunit transmembrane regions, which associate in active states of both IR/IGF1R and EGFR. We have developed a system to assess the effects of transmembrane association on RTK activity and propose to (i) determine whether autoinhibition by RTK extracellular regions is a general feature of RTK regulation and if so determine whether transmembrane separation plays a role in autoinhibition as has been observed for IR/IGF1R and EGFR. We also propose to (ii) determine the energetic balance between ligand binding and dimer formation for EGFR/ErbB receptors to uncover the balance between ?release-of-autoinhibition? and ?ligand-induced dimerization? mechanisms for these receptors. Finally, we propose (iii) cell- and FRET-based experiments to determine what, if any, role higher-order oligomers of IR/IGF1R and EGFR homologs may play their signaling function. Modified Specific Aims The 58 Receptor Tyrosine Kinases (RTKs) in the human genome fall into 20 different classes that include receptors for Insulin, Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Ephrins, Nerve Growth Factor (NGF), Vascular Endothelial Growth Factor (VEGF), and their homologs (1,2). RTKs share a general architecture consisting of a ligand-binding extracellular region (ECR) followed by a single membrane-spanning region, a short juxtamembrane region, a tyrosine kinase, and an unstructured C-terminal tail that is frequently the site of autophosphorylation and interactions with downstream effectors (1). Ligand binding to RTK ECDs stimulates the intracellular kinase activity of the receptor thereby transmitting signals across the cell membrane. As is evident from the names of their ligands, RTKs mediate a wide variety of essential cell growth and differentiation events in developing and adult animals. Abnormal RTK function is also associated with many human disorders including birth defects, diabetes, and cancer (3,4), and RTK-targeted therapeutics are widely used (5,6). Understanding the molecular mechanisms governing RTK activity in normal and disease states is of thus interest for both its fundamental importance and its potential impact on the development of RTK-targeted therapeutics. The classical mechanism by which RTKs are thought to regulate their kinase activity is ligand-induced dimerization (2,7). An abundance of structural and biochemical evidence supports the presence of specific RTK dimers in the active state (1), but increasing evidence also suggests the presence of at least a small fraction of inactive, ?preformed? RTK dimers in the absence of ligand (8). Dimerization per se may thus not be sufficient to stimulate signaling in at least some instances. In particular, members of the Insulin Receptor (IR) family are unique among RTKs in forming disulfide-linked dimers (of post-translationally cleaved subunits), and it has long been thought that IR family members signal via a ligand-dependent conformational change (6). In the last award period we combined structural knowledge with biophysical, biochemical, and cell-based assays to show that the ECR of the IR homolog Type I Insulin-like Growth Factor Receptor (IGF1R) autoinhibits IGF1R kinase activity by maintaining a large (>100 ) separation between subunit transmembrane (TM) regions (9). IGF-1 activates IGF1R by releasing this inhibition and unleashing an intrinsic propensity of IGF1R TM and intracellular regions to associate and become activated. The EGF Receptor (EGFR) and Met ECRs have also been shown to be autoinhibitory (10,11), and indirect evidence suggests that the the VEGFR1 (12), PDGFR (13), FGFR (14), and Tie1 (15) RTK ECRs are autoinhibitory. We propose to continue our structural and mechanistic studies of RTK activation and inhibition by determining whether all RTK ECRs are autoinhibitory and investigating the molecular basis for ECR-mediated autoinhibition when present. These studies have the potential to update the ?ligand-induced dimerization? paradigm and shape our understanding of how RTKs evolved and how best to modulate their activities with chemical agents. We further propose specific functional studies of EGFR and IR homologs that will investigate the balance between ?release-of-autoinhibition? and ?dimer-formation? in EGFR activation, and assess the role of higherorder oligomers in regulating the activity of IR and EGFR family members. In addition to providing a better understanding of the molecular mechanisms governing these important receptors, we expect general features of RTK signaling to emerge from the following specific aims:
Aim 1. We will determine whether autoinhibition is a general feature by which RTK ECRs regulate RTK activity by systematically deleting the ECRs of all major classes of RTKs and assessing their activity in cell-based assays. For autoinhibitory RTK ECRs, we will determine the role of TM separation in maintaining autoinhibition using cell-based FRET assays and by fusing the TM and intracellular regions of targeted RTKs to homodimers with known intersubunit spacings. The role of TM twist in regulating RTK activity will be investigated by fusion to leucine zippers truncated at each of the 7 distinct positions of the coiled coil heptad repeat.
Aim 2. We will parse the energetic coupling between ligand binding and formation of doubly- and singly ligated ErbB dimers and assess the contribution of ErbB ECRs to negative cooperativity and dimer formation using isothermal titration calorimetry and our ability to form tethered heterodimers of EGFR family (ErbB) ECRs by fusing them to the heavy and light chains of an Fab.
Aim 3. We will determine whether higher-order oligomers play in a role in IR/IGF1R activation using cellbased activity assays with kinase-impaired IGF1R variants and investigate the role and nature of higher-order oligomers in EGFR family homo- and hetero-dimers using a combination of cell-based assays and FRET experiments.
Receptor Tyrosine Kinases (RTKs) are cell surfaces receptors that mediate response to hormones and growth factors including among others insulin, epidermal growth factor, vascular endothelial growth factor, and fibroblast growth factor. Abnormal RTK function is associated with birth defect, diabetes, and cancer, and RTK- targeted therapies are widely used. By elucidating the molecular mechanisms governing the activity of RTKs in normal and disease states we will learn how these receptors mediate their biological functions and guide development of therapeutic modulators of RTK activity. From: Flicker, Paula (NIH/NIGMS) [E] To: Urdaneta, Angela (NIH/NIGMS) [E] Subject: FW: Revised Aims for 2 R01 GM099321 Date: Thursday, July 30, 2015 3:53:04 PM Attachments: Leahy_SF424_RR_Guide_ModifiedScopeTemplate2.pdf I approve of the revised aims. The PI has removed the original aim 3 and now the former aims 4 is the new aim 3. Paula Paula Flicker, Ph.D. Program Director NIGMS 301-594-0828 Fax:301-480-2004 From: Marisa Bailey [mailto:mabailey@jhu.edu] Sent: Thursday, July 30, 2015 3:34 PM To: Urdaneta, Angela (NIH/NIGMS) [E] Cc: Daniel Leahy; Rhea Dubs; Flicker, Paula (NIH/NIGMS) [E] Subject: RE: Revised Aims for 2 R01 GM099321 Ms. Urdaneta, Please see attached revised Aims, Abstract and Relevance as requested. Let me know if anything additional is needed. Thank you, Marisa Bailey Grants Associate Johns Hopkins University SOM Office of Research Administration 733 N. Broadway, Suite 117 Baltimore, MD 21205 Email: mabailey@jhu.edu From: Marisa Bailey [mailto:mabailey@jhu.edu] Sent: Friday, July 24, 2015 1:45 PM To: Flicker, Paula (NIH/NIGMS) [E]; Urdaneta, Angela (NIH/NIGMS) [E] Cc: Daniel Leahy; Rhea Dubs Subject: FW: Revised Aims for GM099321 Ms. Urdaneta, Good afternoon, please see attached the requested documents from Dr. Leahy for grant # GM099321. Let me know if anything additional is needed. Marisa Bailey Grants Associate Johns Hopkins University SOM Office of Research Administration 733 N. Broadway, Suite 117 Baltimore, MD 21205 Email: mabailey@jhu.edu From: FLICKERP@NIGMS.NIH.GOV Subject: revised aims instructions Date: July 22, 2015 2:42:25 PM EDT To: dleahy@jhmi.edu Dan Instructions for sending us revised aims are in the notice whose link is below. Please had the business official send to Angela and/or me. You are welcome to send me a draft first if you?d rather do so. http://grants.nih.gov/grants/guide/notice-files/not-od-09-088.html thank you Paula From: urdanetaa@nigms.nih.gov Subject: Grant Number: 2R01GM099321 - 14 PI Name: LEAHY, DANIEL J Date: July 16, 2015 4:34:30 PM EDT To: dleahy1@johnshopkins.edu Cc: FLICKERP@NIGMS.NIH.GOV Dear Dr. Leahy, I?m the grants management specialist assigned to your competing continuation and would like to bring to your attention the approved recommended funding direct cost is ~ 73% of what the application is requesting. Since the reduction is considered significant, meaning over 25%, per Grants Policy Statement, revised aims and objectives should be reflected in the folder to sustain the cut. Sometimes grantees waive the right to submit revised aims and objectives and prefer to continue with the original proposed ones. I?m not sure if you would like to discuss this with your Program Officer about the upcoming funding for year 14 and possibly opt for keeping the same aims/objectives. Whether you choose to keep the same aims/objectives despite the significant reduction, or submit revised ones, I would appreciate receiving a response from the Authorized business official at your institution in a short time. Best regards, and let me know if you have any questions, Angela Urdaneta, MBA | Grants Management Specialist | National Institutes of Health | National Institute of General Medical Sciences | T: 301-451-2005 | F: 301-480-2554 | Email: urdanetaa@mail.nih.gov
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