Our research focuses on the natriuretic peptide family and their cognate receptors, guanylyl cyclase-A (GC-A) and GC-B (Fig. 1). Both receptors are single membrane-spanning enzymes that consist of an extracellular ligand binding domain and intracellular kinase homology regulatory domain and cGMP synthesizing GC domain. Atrial natriuretic peptide (ANP) and brain natriuretic peptide decrease blood pressure and inhibit cardiac hypertrophy by activating GC-A. C-type natriuretic peptide (CNP) activation of GC-B regulates oocyte meiosis and stimulates long bone growth.
Initial studies on post translational modifications of these enzymes indicated that phosphorylation and dephosphorylation are required for receptor activation and inactivation, respectively (Potter and Garbers, JBC, 1992, Potter, Biochemistry, 1998, Potter and Hunter, MCB, 1998, Potter and Hunter, JBC, 1998). Subsequent studies demonstrated that hormones that antagonize the actions of natriuretic peptides reduce signaling through this pathway by stimulating natriuretic peptide receptor dephosphorylation (Abbey and Potter, JBC, 2002, Abbey-Hosch et. al, Hypertension, 2004, Potthast et al, JBC, 2004). Later, we determined that GC-A, but not GC-B, is inactivated in failed murine and human hearts (Dickey et. al, Endocrinology, 2007, Dickey et. al, JMCC, 2013). We also found that N-linked glycosylation, another post-translational modification, is required for GC-B signaling and that missense mutation that inhibit glycosylation of GC-B cause dwarfism (Dickey et. al, JBC, 2016).
Phosphomimetic forms of GC-A and GC-B that cannot be inactivated by dephosphorylation were engineered (Yoder et al. PLoS One, 2012, Otto et al, Mol. Pharmacol, 2017). In collaboration with Dr. Laurinda Jaffe's group, we produced mice expressing glutamate-substituted natriuretic peptide receptors that cannot be inactivated by dephosphorylation. Initial studies on GC-B7E/7E demonstrated that receptor dephosphorylation mediates luteinizing hormone-dependent resumption of meiosis in oocytes (Shuhaibar et al, Dev. Biol., 2016). The GC-B7E/7E mice also have longer bones than wild type mice! To determine the contribution of GC-B dephosphorylation to human dwarfism, we generated the fibroblast growth factor receptor (FGFR)3G374R/+ mouse line that mimics the mutation that accounts for most cases of human dwarfism (small mouse in Fig. 2). The FGFR3G374R/+ mice are being crossed with the GC-B7E/7E mice to determine the contribution of GC-B dephosphorylation to (FGFR)3G374R/+ dependent dwarfism. In addition, a mouse expressing two alleles of a dephosphorylation resistant form of GC-A called GC-A8E/8E was made and will be tested for resistance to heart failure.
Studies on non-covalent regulation of GC-A and GC-B determined that ATP allosterically activates these enzymes by binding a site in the catalytic domain (Antos et al, JBC, 1995, Antos and Potter, AJP, 2007, Robinson and Potter, JBC, 2011, and Robinson and Potter, 2012). New data indicate that GC-A and GC-B contain a second ATP binding site in their kinase homology domains (Edmund and Potter, in preparation). GC-B mutations that cause skeletal overgrowth take advantage of this type of regulation by mimicking an allosterically activated conformation of the enzyme (Dickey et al, JBC, 2017).
After receiving my B.A. degree in chemistry from Ohio Wesleyan University, (Delaware, OH), I entered the graduate program in the Department of Pharmacology at the University of Minnesota. I worked with Dr. Tim Walseth studying intracellular calcium signaling pathways and received my Ph.D. in 1999. I then completed a postdoctoral fellowship with Dr. Yoji Shimizu in the Center for Immunology at the University of Minnesota studying the signal transduction pathway for adhesion of T cells before joining the Potter lab in December of 2004.
I have worked and published papers on multiple aspects of natriuretic peptide receptor signaling ranging from determining ligand specificity requirements to measuring receptor activity in animal disease models. I led work measuring GC-A and GC-B receptor activity in heart-failed mice and in human patients (Dickey et. al, Endocrinology, 2007, Dickey et. al, JMCC, 2013). I also designed and analyzed chimeric and designer forms of natriuretic peptides to developed a better natriuretic peptide-based drug (Dickey et. al, JBC, 2008, Dickey et. al, JBC, 2009, Dickey et. al, Mol. Pharmacol, 2010). To aid in the design of more effective therapeutic natriuretic peptides, I developed a bioassay that measures the ability of normal and proteolytically degraded forms of natriuretic peptides to activate GC-A and GC-B in whole cells (Dickey and Potter, Biochem Pharmacol, 2010, Dickey and Potter, Clin Chem, 2011, Dickey and Potter, JMCC, 2011) I also examined the internalization and degradation mechanisms of GC-A ( Dickey et. al, Mol Pharmacol, 2011). My latest research determined how genetic mutations activate or inactivate GC-B as a means for identifying potential drug targets for bone growth abnormalities (Dickey et. al, JBC, 2016, Dickey et. al, JBC, 2017).
I entered the BMBB PhD program in September 2012 and completed my Preliminary Examinations in June 2014. Before I began my thesis studies, I participated in multiple projects examining the regulation of guanylyl cyclase (GC)-B. The first project demonstrated that luteinizing hormone stimulates GC-B dephosphorylation and inactivation in the rat ovarian follicle (Egbert et. al Development, 2014). The second project involved characterizing the functional effects of mutations in GC-B that cause dwarfism or skeletal overgrowth in humans (Wang et. al, Hum Mutat, 2015). I also participated in the characterization of the GC-B7E/7E "knockin" mouse that expresses a phosphomimetic form of GC-B that is resistant to inactivation by luteinizing hormone (Shuhaibar et. al, Dev. Biol., 2016). Finally, I was second author on a paper showing that most mutations that cause Acromesomelic dysplasia Maroteaux type dwarfism inactivate GC-B by causing incomplete glycosylation of the receptor in the endoplasmic reticulum (Dickey et. al, JBC, 2016).
My initial thesis project investigated the possibility that GC-A and GC-B possess intrinsic kinase activity, but we were unable to develop mutant forms of GC-A and GC-B that would allow us to test this model. My current project investigates how ATP activates GC-A and GC-B. It has been known for many years that ATP and natriuretic peptide together stimulate greater enzymatic activity than either molecule alone, but how ATP activates these receptors has been difficult to understand due to the presence of multiple ATP binding sites. I recently used homology modeling to show that all of the residues that interact with ATP in protein kinase-A except for the catalytic aspartate are conserved in the kinase homology domains of GC-A and GC-B. A structure-function approach is demonstrating the contribution of each of these residues to the ability of ATP to bind and activate GC-A and GC-B.
I received my B.S. with a major in Biochemistry from the University of Minnesota in 2007. I received my Ph.D. under the direction of Dr. Do-Hyung Kim from the Department of Biochemistry, Molecular Biology and Biophysics at the University of Minnesota in 2014 where I worked on Atg8 mammalian homologues and their binding partners (manuscript in preparation). I also designed custom-made TALENs for the generation of several mammalian autophagy gene KO celI lines which have been used to show that ULK1 binds to and phosphorylates Atg14L, an important regulatory step in autophagosome formation (Park et. al, Autophagy, 2016). In other collaborative efforts, I have shown that FIP200 is an integral part of the ULK1 complex responsible for autophagy induction and mTOR inhibition (Jung et. al, Mol Biol Cell, 2009) and (Jung et. al, Autophagy, 2011). I began my post-doctoral studies in Dr. Potter's laboratory in 2014. My initial focus in the lab was to demonstrate that mutant forms of guanylyl cyclase (GC)-B that cause dwarfism are targeted to the plasma membrane by immunofluorescence microscopy, which led to a paper in JBC by Dr. Dickey (Dickey et. al, JBC, 2016). In another collaboration with Dr. Dickey, we demonstrated that three activating mutants of GC-B that cause skeletal overgrowth do so by producing an constitutively allosterically activated conformation of GC-B (Dickey et al, JBC, 2017). More recent work in Dr. Potter’s lab has focused on understanding how phosphorylation regulates guanylyl cyclase (GC)-A and GC-B. Multisite phosphorylation is required for natriuretic peptide activation of GC-A and GC-B but is not required to form an active catalytic site. These studies have shown that single alanine substitutions at known phosphorylation sites reduced maximal velocity (Vmax), while glutamate substitutions have no effect or increased Vmax. I have also generated a dephosphorylated GC-A enzyme that contains single glutamate substitutions at each phosphorylation site (GC-A-8E), which has the same Vmax, Km, and EC50 as the phosphorylated WT enzyme. Adding more glutamates to make GC-A-9E or GC-A-10E had little effect on activity and sequential deletion of individual glutamates in GC-A-8E progressively increased the Km. Double Ala substitutions had a greater effect in GC-A-WT than GC-A-8E, consistent with the idea that one phosphorylation site affects the phosphorylation of other sites. Ultimately, my work is consistent with a model showing that a concentrated region of negative charge, not steric properties, resulting from multiple interdependent phosphorylation sites is required for a natriuretic peptide receptor conformation capable of transmitting the hormone-binding signal to the catalytic domain. The work was recently published in Molecular Pharmacology (Otto et. al, Mol. Pharmacol, 2017).
I earned a BS in biology and chemistry at Berry College in 2006, followed by earning a Ph.D. in Pharmacology at the University of Minnesota in 2013. My graduate work with Professor Lincoln Potter focused on understanding how ATP allosterically activates GC-A and GC-B. To that end, I published several papers that determined that there is an ATP binding site in the catalytic domain of these enzymes that increases their affinity for GTP (Robinson et al., BrJPharm 2011, Robinson and Potter, JBC 2011, Robinson and Potter, SciSig 2012) I also published work demonstrating that an activating mutation in GC-B causes skeletal overgrowth by mimicking an ATP and ligand bound receptor that fails to desensitize (Robinson et al., Bone 2013).
In addition to my thesis work, Dr. Potter and I collaborated extensively with Professor Laurinda Jaffe’s lab at the University of Connecticut Health Center. We investigated how luteinizing hormone inactivates GC-B to initiate the resumption of meiosis in the murine oocyte (Robinson et al., Dev Biol 2012). We also found that LH induced both GC-B dephosphorylation-dependent inactivation and upregulated phosphodiesterase activity (Egbert et. al, Development, 2014).
My first post-doctoral position was with Professor Roberto Pacifici at Emory University from April 2013 to July 2015. With Dr. Pacifici, I investigated how parathyroid hormone, when administered intermittently, activates the PTH receptor on CD4+ T cells and ultimately increases osteoblast activity and bone growth due to an increase in regulatory T cell (Treg) maturation (Robinson et al., JBMR 2015). A second study investigated the bone loss induced by chronic PTH signaling in CD4+ T cells in people with primary hyperparathyroidism (Li et al., Cell Metab 2015) due to an increase in inflammatory Th17 cells. My last project investigated the contribution of the anti-inflammatory effects of hydrogen sulfide to preventing estrogen-induced bone loss (Grassi, Tyagi et al., JBMR 2016).
Upon returning to the Potter lab for a second post-doctoral, I collaborated with Dr. Deborah Dickey on a project showing that dwarfism causing mutations in GC-B prevent ER-dependent glycosylation of GC-B (Dickey et al., JBC 2016). My current research focuses on determining how phosphorylation-dependent regulation of GC-A and GC-B affects cardiovascular and bone growth diseases, respectively. Specifically, I am using two different mouse models in which the phosphorylation sites of GC-A and GC-B are mutated to glutamate to mimic the negative charge of phosphoserine or phosphothreonine. These mice are essential to determining how phosphorylation of GC-A and GC-B regulates physiology. Our preliminary data demonstrates that the GC-B7E/7E mice have a longer appendicular skeleton than the GC-BWT/WT littermates. We now have the GC-A8E/8E mice and plan to test their ability to adapt to heart failure in the near future. Finally, I am using novel cell lines and genetically engineered mice (Fig. 2) to investigate how FGFR3 inhibits GC-B to cause dwarfism. A manuscript describing the cellular studies was recently submitted for publication.
Recent Peer-Reviewed Publications from the Potter Lab
1. Shuhaibar, L. C., Robinson, J. W., Shuhaibar, N. P., Egbert, J. R., Vigone, G., Valentina, B., Kaback, D., Siu-Pok, Y., Feil, R., Fisher, M. C., Dealy, C. N., Potter, L. R*., Jaffe, L. A. (2017) Dephosphorylation of the NPR2 guanylyl cyclase contributes to inhibition of bone growth by fibroblast growth factor, eLife Dec. 4:6 pii: e31343, doi: 10.7554/eLife.31343
2. Robinson JW, Egbert JR, Davydova J, Schmidt H, Jaffe LA, Potter LR (2017) Dephosphorylation Is the Mechanism of Fibroblast Growth Factor Inhibition of Guanylyl Cyclase-B, Cellular Signalling, 40, 222-229, PMID: 28964968
3. Dickey DM, Otto NM, Potter LR (2017) Skeletal Overgrowth-causing Mutations Mimic an Allosterically Activated Conformation of Guanylyl Cyclase-B that is Inhibited by 2,4,6-trinitrophenyl ATP, J. Biol. Chem. June 16; 292:10220-10229, PMID: 28450398
4. Otto NM, McDowell WG, Dickey DM, Potter LR (2017) A Glutamate-Substituted Mutant Mimics the Phosphorylated and Active Form of Guanylyl Cyclase-A, Mol. Pharmacol. 92: 67-74, PMID: 28416574
5. Dickey DM, Edmund AB, Otto NM, Chaffee TS, Robinson JW, Potter LR (2016) Catalytically Active Guanylyl Cyclase-B Requires ER-mediated Glycosylation and Mutations that Inhibit this Process Cause Dwarfism, J. Biol. Chem. 291, 11385-11393, PMID: 26980729
6. Shuhaibar LC, Egbert JR, Edmund AB, Uliasz TF, Dickey DM, Yee SP, Potter LR, Jaffe LA (2016) Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone. Dev. Biol. doi: 10.1016/j.ydbio.2015.10.025. Epub 2015 Oct 30. PMID: 26522847
7. Wang SR, Jacobsen CM, Carmichael H, Edmund AB, Robinson JW, Olney RC, Miller TC, Moon JE, Mericq V, Potter LR, Warman ML, Hirschhorn JN, Dauber A. (2015) Heterozygous Mutations in Natriuretic Peptide Receptor-B (NPR2) Gene as a Cause of Short Stature. Hum Mutat. 2015 Feb 23. doi: 10.1002/humu.22773.
8. Egbert J. R, Shuhaibar L. C, Edmund A. B, Van Helden D. A, Robinson J. W, Uliasz TF, Baena V, Geerts A, Wunder F, Potter L. R. and Jaffe L. A. (2014) Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes, Development, Sep;141(18):3594-604. doi: 10.1242/dev.112219, PMID: 25183874
9. Buys, E. S., Potter, L. R., Pasquale, L. R. and Ksander, B. R. (2014) Regulation of intraocular pressure by soluble and membrane guanylyl cyclase and their role in glaucoma, Front Mol Neurosci, May 19:7:38
10. Robinson, J. W., Dickey, D. M., Miura, K., Michigami, T., Ozono, K. and Potter, L. R. (2013) A Human Skeletal Overgrowth Mutation Increases Maximal Velocity and Blocks Desensitization of Guanylyl Cyclase-B, Bone, Oct:56(2):375-82
11. Robinson, J. W. and Potter, L. R. (2012) Guanylyl Cyclase A and B Are Asymmetric Dimers that Are Allosterically Activated by ATP Binding to the Catalytic Domain, Science Signaling 5, 240, ra65.
12. Yoder, A. R., Robinson, J. W., Dickey, D. M, Andersland, J. Rose, B. A., Stone, M. D., Griffin, T. J. and Potter, L. R. (2012) A Functional Screen Provides Evidence for a Conserved Regulatory, Juxtamembrane Phosphorylation Site in Guanylyl Cyclase A and B, PLoS One 7(5): e36747.doi.1371/journal.pone.0036747
13. Robinson, J. W., Zhang, M., Shuhaibar, L. C., Norris, R. P., Geerts, A., Wunder, F., Eppig, J. J., Potter, L. R. and Jaffe, L. A. (2012) Luteinizing Hormone Reduces the Activity of the NPR2 Guanylyl Cyclase in Mouse Ovarian Follicles, Contributing to the Cyclic GMP Decrease that Promotes Resumption of Meiosis in Oocytes, Developmental Biology, 366, 308-316
14. Dickey, D. M, Dries, D. L., Margulies, K. B and Potter, L. R. (2012) Guanylyl Cyclase-A and -B Activities in Ventricles and Cardiomyocyte from Failed and Non-failed Human Hearts: GC-A is Inactive in the Failed Cardiomyocyte. J. Molecular and Cellular Cardiology, 52, 727-32
15. Potter, L. R. (2011) Guanylyl cyclase structure, function and regulation. Cellular Signalling, 23, 1921-26