Plexins are transmembrane receptors that make cell guidance decisions in vascular and neuronal development, disease, and regeneration. Targeting plexins and their co-receptors, especially neuropilin, is a novel route to treating retinal diseases such as age-related macular degeneration (AMD) and diabetic retinopathy, but the mechanistic details of plexin activation are poorly understood. We will characterize the interactions between plexin and membrane lipids. Plexins are unique transmembrane receptors in that their intracellular regions interact directly with membrane-anchored small GTPases. These Ras and Rhofamily GTPases, in turn, are associated with cell migration in the retina. The preliminary results in aim 1 indicate that protein-membrane interactions play a key role in the signaling mechanisms of plexins, for example, rationalizing the different functional roles of the GTPases in the system. Our work will be the first to study the interactions of plexin domains with the lipid bilayer at the molecular level. This includes, in aim1, the plexin-B1 transmembrane region which has been predicted to assume three states of inter helix contacts. Altogether the results support a multi-state model which incorporates previous and the new results, but also points to critical new interactions between the proteins and the lipid bilayer.
Aim 2 seeks to resolve the structure and function of the transmembrane regions of plexin coreceptors, neuropilin and semaphorins. We hypothesize that such interactions set up cross signaling with ligands and co-receptors. In order to address this hypothesis, we will examine receptor homo- and hetero-oligomerization using time resolved fluorescence spectroscopy and functional experiments, both using live cells. Together with NMR and molecular dynamics, these complementary tools will address the hypothesis at different levels of complexity, ranging from domain level to in-cell behavior. Plexin function is modulated by interaction with co-receptors, such as neuropilins which are also single pass transmembrane (TM) proteins. TM regions are recently recognized as an effective target for peptides designed to disrupt receptor signaling.
In aim 2, we will characterize the structure and function of plexin TM interactions with the TM domains of neuropilins. Our data suggest that TM interactions between plexins and certain of their semaphorin ligands can also occur in cis. Such TM based regulation would be a novel mechanism in the plexin field, and one that can be targeted to inhibit the receptors in the retina. Specifically, expanding the successful use of neuropilin peptides as anti-tumor agents, TM regions are a target for anti-angiogenesis therapy in eye disease such as AMD and diabetic retinopathy.
Diseases affecting the retina, such as age related macular degeneration (AMD), diabetic retinopathy and macular edema (DR/ DME) are responsible for a large proportion of blindness, affecting approximately 10 million people in the United States alone. Pathological angiogenesis is central to this group of diseases because the inappropriate formation of new blood vessels leads to a disruption and swelling of the retinal pigment epithelium. Some blood vessels can even become leaky or burst. Protein receptor families (such as neuropilins, plexins, VEGFR2) and their ligands (incl. semaphorins) are involved in cell adhesion and regulate the cell migration of growing blood vessels as well as nerve cells (guiding both axons and dendrites). The collaboration of the receptors and ligands between different endothelial, epithelial and neuronal cell types is of paramount importance but is not yet understood at the fundamental level. Despite this lack of knowledge, a monoclonal antibody against the VEGFR2 ligand (VEGF-A), Ranibizumab (Lucentis; FDA approved since 2006) decelerates visual loss in 90% of cases and allows about 40% of treated patients to regain some visual acuity. The administration of such antibodies, which can block proteins involved in blood vessel formation, has become a principal mode of treatment. What effects these therapeutics have on normal neuronal and blood vessel development/maintenance over the longer term (on the order of decades) is not yet understood. Furthermore, retina cells could become resistant against single agent treatments, as has been observed in anticancer therapy where resistance against VEGF blockade treatments has become a serious problem. Furthermore, this antibody needs to be injected directly into the eye every month, leading to a low level of compliance, esp. in elderly patients. Thus there is an urgent need for the development of therapeutic agents that can be administered orally, as eye drops, or intravenously. Neuropilin-1 (Nrp1) is a single-pass transmembrane receptor that interacts with many receptor-ligand complexes and adhesion molecules. Nrp1 binds two different ligands: vascular endothelial growth factors (VEGFs) and semaphorins (Semas). The major role of Nrp1 is in angiogenesis; in association with plexins it functions as a receptor for cell guidance cues to guide axon, dendrite and blood vessel development. Endothelial/epithelial cells and neurons form extensive networks especially in the human eye; here the same guidance cues and receptors are utilized for both retinal/optic nerve and ocular blood vessel development. Plexin and Nrp1 receptors are implicated in retinal nerve and vascular damage. Lately, Nrp1 and its co-receptor plexin have become diagnostic markers for cancer progression and a promising target for therapy against pathological angiogenesis. An anti Nrp1 antibody has been developed and small molecule ligands have been discovered that target the extracellular domains. However, an understanding of the molecular mechanisms and structures is critical for the development of highly targeted therapeutics that are needed to alter pathways selectively or in combination. The proposed project will lay the basic scientific groundwork for the further development of peptide inhibitors against the transmembrane receptors that are involved in retinal diseases. This would include inhibitors against the ligands, which as we show for the transmembrane ligand, semaphoring, can act in cis and effectively become a co-receptor. These peptides will likely have therapeutic potential. One such agent is a peptide inhibitor against neuropilin-1 which has already shown promise in preclinical trials. Moreover, basic knowledge of the plexin-semaphorin-neuropilin system is still lacking at the molecular level, especially for the protein regions in and near the cellular membrane. The protein-protein interactions of these regions, also with co-receptors, such as cMet, are not yet characterized. The other aim of the proposal focuses on the interaction of plexin domains with the membrane and with small GTPases, including Rac1 and Rap1 which are emerging as key players in retinal vascularization. Utilizing a combined experimental and computational approach, these regions will be studied for the first time in membrane model systems as well as in cells. This derived knowledge will also allow the future development of therapeutic agents.
