Signaling by Frizzled Homologs in Development
Research Associate Professor, Physiology and
Biophysics
Ph.D. State University of New York at Stony
Brook
Laboratory
Research
Our long-term goals are to study transmembrane signaling by
heterotrimeric G-proteins, focusing on their role in mediating Frizzled
receptors. The Frizzled 1 gene structure has been solved and the cell-surface
receptor for Wnt ligands is heptihelical, suggestive of coupling by members of
the heterotrimeric G-protein family.
Purification of active Wnt ligands has not been successful and we have
now succeeded in crafting a new strategy with which to study the signaling of
Frizzled receptors, creating chimeric receptors in which the cytoplasmic
domains of Rfz1 and Rfz2 are substituted into the beta-adrenergic receptor
(b2AR). The b2AR/Rfz2 chimera, for example, signals with Rfz2 character to
downstream effectors in Xenopus, zebrafish, and mammalian cells, although now
fully activated by an agonist ligand for the b2AR. With this proof-of-concept, a b2AR/Rfz1
chimera has been created as a tool for study of signaling and biology of Rfz1
in F9 teratocarcinoma stem cells in culture.
Preliminary data demonstrate that activation of the b2AR/Rfz1 chimera
with isoproterenol promotes stabilization of beta-catenin, JNK activation, and
primitive endoderm formation in F9 cells and expression of target gene (Siamois
and Xnr3) in Xenopus.
We propose for
the coming five years to exploit the strategy and use the b2AR/Rfz1 chimera to
answer three basic questions about the Rfz1 signaling and biology. (1) What is
the biology of Rfz1-regulated responses including activation, desensitization,
and agonist-induced down-regulation? We make use of radioligands and specific
antibodies to characterize receptor expression/sequestration. Availability of agonist/antagonists enable
study of the temporal relationships among downstream signaling effectors. (2)
What are the post-receptor mechanisms by which Rfz1 signal, is it a
G-protein-linked receptor, and if so to which G-proteins? Loss-of-function
mutant cells in which G-protein subunits are suppressed by antisense
oligodeoxynucleotides will be studies. Gain-of-function studies will make use
of constitutively-activated G-protein alpha subunits to probe linkages to Rfz1
signaling effectors. (3) What are the structure-function relationships that
govern the biology of Rfz1 compared to Rfz2? By swapping cytoplasmic domains
from Rfz1 to Rfz2 and by alanine-scanning mutagenesis, we will probe the
sequence determinants that support canonical effectors such as beta-catenin
stabilization, JNK activation, and translocation of the phsophoprotein
Dishevelled. In combination, these studies will enable study of the basis of
transmembrane signaling of Frizzled gene products, particularyly Rfz1.
Most Recent Publications
(selected)
Wang, H.Y. and
Malbon, C.C. (2004) Wnt-Frizzled Signaling to G-protein-Coupled Effectors. Cell. Mol. Life Sci., 61(1): 69-75. http://80-www.springerlink.com.proxy.hsclib.sunysb.edu/media/43wvrpyhubrn7qxqmxfr/Contributions/H/3/J/1/H3J1K2JAJ9D0JDG8.pdf
Li,
Hong., Malbon, C.C., and Wang, H.-y (2004) Gene Profiling of Frizzled-1 and
Frizzled-2 Signaling: Expression of
G-protein-coupled Receptor
Chimeras
in Mouse F9 Teratocarcinoma Embryonal
Cells. Molecular Pharmacology, 65: 45-55.http://80-molpharm.aspetjournals.org.proxy.hsclib.sunysb.edu/cgi/reprint/65/1/45.pdf
Shumay, E., Wang, H.Y., and Malbon, C.C. (2004)
Trafficking of Beta2-Adrenergic Receptors: Insulin and Beta-agonists Regulate Internalization by
Distinct Cytoskeletal Pathways. J. Cell Sci., 117,
593-600. http://80-jcs.biologists.org.proxy.hsclib.sunysb.edu/cgi/reprint/117/4/593.pdf
Tao, J., Wang,
H-y., and Malbon, C.C. (2003)Protein Kinase A Regulates AKAP250 (Gravin)
Scaffold Binding to the b2-Adrenergic Receptor. EMBO
Journal, 22(24):6419-29. http://embojournal.npgjournals.com/cgi/reprint/22/24/6419.pdf
Wang, H-y. and
Malbon, C.C. (2003). Wnt signaling, Ca+, and Cyclic GMP: Visualizing Frizzled
Functions. Science 300:1529-1531. http://www.sciencemag.org/cgi/reprint/300/5625/1529.pdf
Ahumada,
A., Slusarski, D., Liu, X., Moon, R.T.,
Malbon, C.C. and Wang, H-y. (2002). Activation of Frizzled-2 signals via
cyclic GMP. Science,
298:
2006-2010. http://www.sciencemag.org/cgi/reprint/298/5600/2006.pdf
Shumay, E.,
Song, X., Wang, H-y. and Malbon,
C. C. (2002) p60Src mediates
insulin-stimulated sequestration of the b2-adrenergic receptor:
Molecular Biology of the Cell , 13, 11: 3943-54. http://www.molbiolcell.org/cgi/reprint/13/11/3943.pdf
Liu,
T., Lee, Y-N., Malbon, C.C. and Wang, H.Y. (2002) Activation of the b-catenin/Lef-Tcf
pathway is obligate for formation of primitive endoderm by mouse F9 totipotent,
teratocarcinoma cells in response to retinoic acid. J.
Biol. Chem., 277, 30887-30891. http://www.jbc.org/cgi/reprint/277/34/30887.pdf
Doronin,
S., Shumay, E., Wang, H-y., and Malbon, C.C. (2002) Akt mediates sequestration of the b2-adrenergic
receptor in response to insulin.
J. Biol. Chem., 277: 15124- 15131. http://www.jbc.org/cgi/reprint/277/17/15124.pdf
Doronin, S.,
Wang, H-y., and Malbon, C.C. (2002) Insulin
stimulates phosphorylation of the b2-adrenergic receptor by the insulin receptor,
creating a potent feedback inhibitor of its tyrosine kinase. J. Biol. Chem., 277: 10698-10703. http://www.jbc.org/cgi/reprint/277/12/10698.pdf
Wang, H-y., Song, X., Huang, X-P. and Tao, J. (2002) Gene Profiling of
Transgenic Mice with Targeted Expression of Activated Heterotrimeric G Protein
Alpha Subunits using DNA Microarray. Methods
in Enzymology, 345:529-539.
Wang, H.-y., Kanungo, J., and Malbon, C.C. (2002)
Expression of Galpha13(Q226L) induces P19 stem cells to primitive endoderm via
MEKK1/4. J. Biol. Chem., 277: 3530-3535.http://www.jbc.org/cgi/reprint/277/5/3530.pdf
Song X, Tao J, Huang XP, Rosenquist TA, Malbon CC, Wang HY. (2002) Targeted, regulatable expression of
activated heterotrimeric G protein alpha subunits in transgenic mice. Methods
in Enzymology 344:309-318.
Huang XP, Rosenquist TA,
Wang HY, Malbon CC. (2002) Inducible, tissue-specific suppression
of heterotrimeric G protein alpha subunits in vivo.
Methods in
Enzymology 344:318-327.
Doronin, S.,
Shumay, E., Wang, H-y. and Malbon, C.C. (2001)Lithium Suppresses Signaling and
Induces Rapid Sequestration of Beta2-Adrenergic Receptors Biochem. Biophys. Res. Commu.,288:150-155.
Malbon, C.C.,
Wang, H-y. and Moon, R.T. (2001) Wnt Signaling and Hetertrimeric G-proteins:
Strange Bedfellows or a Classic Romance. Biochem.
Biophys. Res. Commu. 287:589-593.
Song, X., Zheng, X.,
Malbon, C.C. and Wang, H-y.
(2001) Gai2 enhances in vivo activation of and
insulin signaling to GLUT4. J. Biol.
Chem., 276: 34651-34658. http://www.jbc.org/cgi/reprint/276/37/34651.pdf
Tao, J., Malbon,
C.C. and Wang, H-y. (2001) Insulin
stimulates tyrosine phosphorylation and inactivation of protein tyrosine
phosphatase 1B in vivo. J. Biol. Chem., 276:29520-29525. http://www.jbc.org/cgi/reprint/276/31/29520.pdf
Liu, T., DeCostanzo, A. J., Liu, X., Wang, H-y.,
Hallagan, S., Moon, R.T. and Malbon, C.C. (2001) Heterotrimeric G-proteins
Go and Gq mediate
signaling from activation of rat
Frizzled-1 to the beta-catenin/Lef Tcf pathway in development. Science, 292: 1718-1722.
http://www.sciencemag.org/cgi/reprint/292/5522/1718.pdf
Fan, G-f.,
Shumay, E., Wang, H-y., and Malbon, C.C. (2001) The Scaffold Protein Gravin
(AKAP250) binds the Beta2-adrenergic Receptor Via the Receptor Cytoplasmic R329
to L413 Domain and Provides a Mobile Scaffold During Desensitization. J. Biol. Chem., 276: 24005-24014. http://www.jbc.org/cgi/reprint/276/26/24005.pdf
Fan, F., Shumay, E., Malbon, C. C. and Wang,
H-y. (2001) c-Src tyrosine kinase binds the b2-adrenergic
receptor via phospho-Tyr350, phosphorylates G-protein –linked Receptor Kinase
2, and mediates agonist-induced receptor desensitization. J. Biol. Chem., 276:
13240-13247. http://www.jbc.org/cgi/reprint/276/16/13240.pdf
Wang,
H-y., Doronin, S., and Malbon, C.C. (2000) Insulin Activation of
Mitogen-activated Protein Kinases Erk1,2 is Amplified via Beta-adrenergic
Receptor Expression and Requires the Integrity of the Tyr350 of the Receptor. J. Biol. Chem., 275: (46) 36086-36093. http://WWW.JBC.ORG/cgi/reprint/275/46/36086.pdf
Lin,
F., Wang, H-y. and Malbon, C.C. (2000) Gravin-mediated Formation of Signaling
Complexes in G-protein-linked Receptor
Desensitization and Resensitization. J. Biol. Chem., 275, 19025-19034.
http://WWW.JBC.ORG/cgi/reprint/275/25/19025.pdf
Wang,
H-y., Moxham, C. and Malbon, C.C. (2000) Antisense RNA-mediated Inhibition of
Gene Expression. Genetic Manipulations
of Receptor Expression & Function (D. Accili, editor), 11, 189-204.
Kanungo, J., Potapova, I., Malbon, C. C., and Wang, H-y.
(2000) Retinoic Acid-induced Differentiation of P19 Embryonal Carcinoma Stem
Cells is Mimicked by Constitutively-active MEKK4 and MEKK1, but Blocked Only by
the Dominant Negative Mutant of MEKK4. J.
Biol. Chem., 275, 24032-24039.
http://www.jbc.org/cgi/reprint/275/31/24032.pdf
Liu, X., Liu, T., Slusarski, D.C., Yang-Snyder, J., Malbon, C.C., Moon, R.T., and Wang, H-y. (1999) Activation of a Frizzled-2/b-Adrenergic Receptor Chimera promotes Wnt-Signaling and Differentiation of Mouse F9 Teratocarcinoma Cells via Gao and Gat. Proc. Natl. Acad. Sci. U.S.A., 96, 14383-14388.
http://www.pnas.org/cgi/reprint/96/25/14383.pdf
Shih, M., Lin,
F. Scott, J.D., Wang, H-y. and Malbon, C.C. (1999) Dynamic
Complexation of b2-adrenergic receptors with Protein Kinases and
Phosphatases. J. Biol. Chem., 274, 1588-1595.
http://WWW.JBC.ORG/cgi/reprint/274/3/1588.pdf
Liu, X., Malbon, C.C., and Wang, H-y.
(1998) Identification of Amino Acid Residues of Gsalpha Critical to Repression
of Adipogenesis. J. Biol Chem. 273, 11685-11694. http://WWW.JBC.ORG/cgi/reprint/273/19/11685.pdf
Guo, Jh., Wang, H-y., and Malbon,
C.C. (1998) Conditional, Tissue-specific Expression of Q205L Galphai2 in vivo Mimics Insulin Activation
of jun N-Terminal Kinase and P38 Kinase. J. Biol. Chem. 273, 16487-16493.
http://WWW.JBC.ORG/cgi/reprint/273/26/16487.pdf
Zheng, X., Guo, Jh., Wang, H-y. and
Malbon, C.C. (1998) Expression of Q205L Galphai2 in vivo Ameliorates
Streptozotocin-induced Diabetes. J. Biol. Chem. 273, 23649-23651.
http://WWW.JBC.ORG/cgi/reprint/273/37/23649.pdf
Karoor, V., Wang, L., Wang, H-y. and Malbon, C.C. (1998) Insulin stimulates sequestration of beta-adrenergic receptor and enhanced association of beta-adrenergic receptors with Grb2 via tyrosine 350. J. Biol. Chem., 273, 33035-3304. http://WWW.JBC.ORG/cgi/reprint/273/49/33035.pdf
Earlier Publications (selected)
Sivaraman, V.S.,
Wang, H.-y., Nuovo, G., and Malbon, C.C. (1997),
Hyperexpression of Mitogen-activated Protein Kinase is Associated
with Breast Cancer. J. Clin. Invest., 99, 1478-1483.
Wang, H.-y.,
Goligorsky, M.S., and Malbon, C.C. (1997) Temporal Activation of Ca2+-calmodulin-sensitive
Protein Kinase type II is Obligate for
Adipogenesis. J. Biol. Chem. 272,
1817-1822.
Chen,
J.-F., Guo, J.H., Moxham, C.M., Wang, H.-y. and Malbon, C.C. (1997) Expression
of Constitutively-active (Q205L) Gi
2 in vivo. J. Mol. Med.
75, 283-289.
Wang, H.-y.,
Johnson, G.L., and Malbon, C.C. (1996) Repression of adipogenesis by Gs is
expressed within region 146-220. J.
Biol. Chem. 271, 22022-22029.
Wang, H.-y., and
Malbon, C.C. (1995) The Gs /Gi 2 Axis Controls Adipogenesis Independently of
Adenylylcyclase.
Int. J. Obesity, 20, 26-31.
Moxham, D.M.,
Wang, H.-y., and Malbon, C.C. (1994) G-proteins Controlling Differentiation,
Growth, and Development: Analysis by
Antisense RNA/DNA Technology. Methods in
Neurosciences, 26, 553-571.
Lin, J.-H., Wang, H.-y., Fong, J.C., Pan, J.-T., and Wang, F.-F, (1993) Correlation Between Prolactin Secretion and Gs Protein Expression During Sustained Cholera-Toxin Stimulation. Biochem. J., 296, 335-340.
Wang, H.-y.,
Watkins, D.C., and Malbon, C.C. (1992) Gs Activity Modulates the
Differentiation of Mouse 3T3-L1 Fibroblasts to Adipocytes. Nature, 358, 334-337.
http://physiology.pnb.sunysb.edu/
http://www.pharm.sunysb.edu/faculty/malbon/lab/