Malbon Laboratory Research

REGULATION OF HORMONE-SENSITIVE EFFECTOR SYSTEMS

The role of heterotrimeric G proteins in the signaling of Wnts has been established, from flies to mammals. Wnts signal, in part, via heptahelical G protein coupled receptors (GPCR) to effectors systems that control fundamental processes, such as adipogenesis, bone formation, cell progression, cell fate, and other aspects of early development. Dysregulation of Wnt signaling provides a basis for human disease, including cancer. The phosphoprotein Dishevelled (DVD) is essential to Wnt/b-catenin (canonical), Wnt/ca²⁺/cGMP, and Wnt/planar cell polarity (PCP) pathways. Wnts promote mouse F9 embryonic teratocarcinoma cells (F9) to primitive endoderm (PE) and each of these effector pathways, via one/more or 3 mammalian Dvls. We propose 3 specific aims: (i) to define functional roles of mammalian Dvls on each major Wnt-stimulated pathway using siRNA and read outs of b-catenin stability, Lef/Tcf-sensitive transcription, Ca²⁺ imaging and cGMP analysis, JNK activation via Rho, and PE formation; (ii) to establish multivalency of Dvl interactions with GPCR, G proteins, kinases/phosphatases, and adaptor molecules critical to Wnt signaling making use of tandem affinity-based purification (TAP) and advanced proteomics (MALDI, QToF, and nanospray mass spectrometry), siRNA-based knock downs, enzyme inhibitors/dominant negative constructs, and eventual mutagenesis of potential docking sites; and (iii) to establish the functional consequences of spatial localization and trafficking of Dvl in naïve and Wnt-activated F9 cells, focusing on defining the dynamic aspects of activation /deactivation using fluorescence-based strategies (including e photon, fcs, and TIRF microscopy), energy transfer measurements in live cells (via BRET), and cell fractionation coupled with TAP-tagged Dvl followed by proteomics. The overarching hypothesis is that Dvls function as dynamic scaffolds for integration of cell signaling to a spectrum of diverse pathways, much like the AKAP protein family members function. Dvls both dock and are candidate substrates for many kinases/phosphatases, although the details of these interactive regulations remain to be established by proteomics. Real-time measurements of docking in live cells are made possible by BRET and parallel biochemical/proteomics assays that can define the status of Dvl with respect to protein phosphorylation. The functional basis for 3 mammalian Dvls, for their spatial and temporal trafficking, and for cell membrane association are addressed using in vivo and in vitro strategies. Understanding the multivalency and docking of Dvl open opportunity to intervene in the primary signaling pathways controlled by Wnts, including those whose dysregulation leads to cancer, birth defects, and altered bone formation. Dvls, as multivalent dynamic scaffolds, are high-value likely targets for new therapies.

STRUCTURE AND BIOLOGY OF BETA –ADRENERGIC RECEPTORS

A-Kinase Anchoring Proteins constitute a diverse family of scaffold proteins which share a common binding site for the RI/II subunit of PKA and are now recognized as scaffolds for multivalent cell signaling. AKAP250 (a.k.a., AKAP12, gravin, or a human homologue of SSeCKS) plays a critical role in signaling of the b2-adrenergic receptor (b2AR), the prototype for the superfamily of G-protein-coupled receptors (GPCR). We have demonstrated that AKAP250 is multivalent, providing a scaffold for b2AR signaling complexes that include minimally PKA, PKC, PP2B, Src, and the b2AR. Agonist activation of the b2AR stimulates eventual desensitization, sequestration, resensitization, and recycling of the receptor, a process disrupted in the absence of AKAP250. Recently we have mapped the domains of the b2AR as well as those for AKAP250 that provide the basis for protein-protein interactions and a central role of protein phosphorylation in defining this dynamic interaction. The overarching goal of this research plan is to understand at the “meso”-scale, the dynamic role of AKAP scaffold in b2AR signaling complexes. Four specific aims target the goal: namely 1) to probe protein-lipid and protein-protein interactions of AKAP250 in b2AR signaling (focusing on three domains that may dictate scaffold-membrane association); 2) to identify new signaling molecules that constitute AKAP-based b2AR signaling complexes and to establish their function in the complex (making use of yeast-2-hybrid and HTS proteomic analysis of complex pull-downs); 3) to map the spatial organization and trafficking mechanisms of b2AR signaling complexes in response to stimulation by b-agonist (desensitization) and by insulin (counterregulation), using 2-photon confocal and bioluminescence resonance energy transfer (BRET) spectroscopy; and 4) to elucidate the ordered pattern of phosphorylation of key molecules constituting

AKAP-based b2AR signaling complexes and its function in the dynamic regulation of the complexes (using mass spectrometry in tandem with domain-specific mutagenesis). The targeted convergence of mass spectrometry-based proteomics, sensitive multi-photon confocal microscopy, BRET, and read-outs of localization, function, and protein-protein interactions create an unparalleled opportunity for success.

Atherosclerosis and Peripheral Apoprotein E Synthesis

The long term objective of this research is to understand the functional basis for the expression of lipoprotein (apo) E in peripheral tissues. ApoE is important for regulating systemic cholesterol transport metabolism. Among the plasma apolipoproteins, apoE is unusual in being expressed in many tissues and is involved in cellular processes and diseases that are independent of systemic lipoprotein metabolism. This proposal has two primary objectives. The first is to define the mechanisms by which low-levels of plasma apoE suppress atherosclerotic lesion development. Previous studies showed that levels of transgenic apoE too low to correct hypercholesterolemia in apoE-deficient mice still blocked aortic lesion formation. The second goal is to determine how localized apoE expression in adrenocortical cells regulates the utilization of cholesterol for steroid production. These studies employ apoE-deficient mouse lines that have been engineered to express different levels of transgenic apoE selectively in the adrenal gland. The proposal has 3 aims. Aim 1 has 3 goals focused on how low-levels

(= 10^-8M) of apoE alter the initial stages of lesion formation. Goal 1 will use quantitative real-time PCR to monitor expression of a set of candidate genes during initial stages of leukocyte recruitment to the vascular wall. Goal 2 will determine whether signaling pathways for platelet derived growth factor (PDGF) or those involving the transcription the transcription factors NF-aB and Egr-1 are activated in vascular cells at early stages, and whether low-level apoE alters this activation. Goal 3 will test which receptors of the LDL receptor family are important for the atheroprotective effects of low-level apoE. Airm 2 will use transgenic mice expressing apoE in adrenocortical cells to define mechanisms by which apoE alters cholesterol utilization. Goal 1 will use immunocytochemical approaches to evaluate the variegated pattern of transgenic apoE expression in the adrenal cortex and to test the effect of localized apoE on the expression of key proteins involved in the provision of substrate cholesterol to the steroidogenic pathway. Goal 2 will test which receptors of the LDL receptor family are important for the effects of apoE in adrenocortical cells. Aim 3 has collaborative projects to test the effects of low-level apoE. On neointimal formation after arterial injury and on regression of atherosclerotic lesions. These studies will provide new mechanistic formation about actions of apoE on cholesterol and metabolism and atherosclerosis.

Laboratory Staff

Publications (selected from 1992-2006)

2006 2005 2004 2003 2002 2001 2000 1999 1998 Earlier Publications

2006

Wang, H.-y., Tao, J., Shumay, E., and Malbon, C.C. (2006) G-protein-coupled receptor-associated A-kinase anchoring proteins: AKAP79 and AKAP250 (gravin). Eur J Cell Biol. In press.

Wang, H.-y., Liu, T., and Malbon, C.C. (2006) Structure-function analysis of Frizzleds. Cell Signal. 18: 934-41. PDF

Gavi, S., Shumay, E., Wang, H.-y., and Malbon, C.C. (2006) G protein Coupled Receptors as Substrates for Tyrosine Kinases. Trends in Endocrinology & Metabolism 17, 46-52. PDF

2005

Malbon, C. C. and Wang, H.-y. (2005) Dishevelled: A Mobile Scaffold Catalyzing Development Current Topics in Developmental Biology, 72, 153-166.

Malbon, C.C. and Wang, H.-y (2005) AKAP-based Scaffolds and Insulin Action Cell Science Rev. 17 (2): 1742-1749.

Malbon, C. C. (2005) G proteins in Development Nature Reviews: (Molecular Cell Biology), 6, 689-701. PDF

Malbon, C.C. (2005) (Beta) Catenin, Cancer, and G Protein: Not just for Frizzleds Anymore. Science STKE 292, pe35.

2004

Lee, Y.-n., Malbon, C.C., and Wang, H.-y. (2004) Ga₁₃ Signals via p115RhoGEF Cascades Regulating JNK1 and Primitive Endoderm Formation J. Biol. Chem. 279: 54896-54904. PDF

Gavi, S., Yin, D., Shumay, Malbon, C.C., and Wang, H.-y. (2004) Insulin-stimulated Sequestration Requires the Integrity of an SH2 Domain (Y350) and Akt phosphorylation sites (S345,346) of b₂-Adrenergic Receptor. Endocrinology 146: 450-457. PDF

Malbon, C.C., Tao, J., and Wang, H.-y. (2004) AKAPs and Molecules that Compose their GPCR Signaling Complexes. Biochem. J. 379: 1-9. PDF

Shumay, E., Gavi, S., 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 Science 117:593-600. PDF

Wang, H.Y. and Malbon, C.C. (2004) Wnt-Frizzled Signaling to G-protein-Coupled Effectors. Cell. Mol. Life Sci., 61(1): 69-75. 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. 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. PDF 

2003

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. PDF

Wang, H-y. and Malbon, C.C. (2003). Wnt signaling, Ca⁺, and Cyclic GMP: Visualizing Frizzled Functions. Science, 300:1529-1531. PDF

Wang, H-y. and Malbon, C.C. (2003). The Wnt, Calcium, Cyclic GMP Signaling Pathway. Science STKE Connections Map. (http://stke.sciencemag.org/cgi/cm/stkecm;CMP_12420)

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. 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. 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. PDF

2002

Huang, X., Song, X., Wang, H-y. and Malbon, C.C. (2002) Expression of constitutively-activated Q227L Gas in vivo: adaptive changes in expression of regulatory subunits of protein kinase A. Amer. Journal of Physiology, 283(2):C386-95. PDF

DeCostanzo, A., Wang, H-y., and Malbon, C.C. (2002)The Frizzled-1/Beta2-Adrenergic Receptor Chimera: Pharmacological Properties of a Unique G-Protein linked Receptor. Archives of Pharmacology, 365: 341-348.

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. 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. PDF

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. 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. 

2001

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 Heterotrimeric 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. 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. PDF

Tao, J., Malbon, C.C. and Wang^, H-y. (2001) Gai2 enhances insulin signaling via suppression of protein tyrosine phosphatase 1B. J. Biol. Chem., 276: 39705-39712. 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. 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. 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. PDF 

2000

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. PDF

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. PDF

Lin, F., Wang, H-y. and Malbon, C.C. (2000) Gravin-mediated Formation of Signaling Complexes in Beta2-Adrenergic Receptor Desensitization and Resensitization. J. Biol.Chem. 275: 19025-19034. PDF 

Kühl, M., Sheldahl, L., Malbon, C.C, and Moon, R.T. (2000) Ca²⁺/Calmodulin-dependent Protein Kinase II is Stimulated by Wnt and Frizzled Homologs and Promotes Ventral Cell Fates in Xenopus. J. Biol. Chem, 275: 12701-12711 PDF

1999

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/beta-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. PDF

Morris, A.J. and Malbon, C.C. (1999) Physiological Regulation of G-Protein-Mediated Signaling. Physiological Reviews. 79: 1373-1430 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 PDF

Sheldahl, L., Park, M., Malbon, C.C. and Moon, R. (1999) Protein Kinase C is Differentially Stimulated by Wnt and Frizzled Homologs in a G  Protein Dependent Manner. Current Biology, 9: 695-698. PDF 

1998

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. PDF

Guo, Jh., Wang, H-y., and Malbon, C.C. (1998) Conditional, Tissue-specific Expression of Q205L Gai2 in vivo Mimics Insulin  Activation of jun N-Terminal Kinase and P38 Kinase. J. Biol. Chem. 273: 16487-16493. PDF

Malbon, C.C. and Karoor, V. (1998) G-Protein-linked Receptors as Tyrosine Kinase Substrates: News Paradigms in  signal Integration. Cell Signalling  8: 523-527. PDF

Shih, M and Malbon, C.C. (1998) Serum and Insulin-induced Grb2-dependents Shift in Agonist Affinity for b- adrenergic Receptor. Cell Signalling, 8: 575-582. 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. 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. PDF

For further references, please see http://www.ncbi.nlm.nih.gov/PubMed/

Earlier Publications (selected)

Karoor, V. and Malbon, C.C. (1996)  IGF-1 stimulates phosphorylation of the beta 2-adrenergic receptor in vivo on sites  distinct from those phosphorylated in response to insulin.   J. Biol. Chem.  271: 29347-29352. PDF

Hockerman, G.H., Girvin, M.E., Malbon, C.C. and Ruoho, A.E. (1996)  Antagonist Conformations Within the Beta-Adrenergic Receptor Ligand Binding  Pocket.   Mol. Pharmacol.  49: 1021-1032.

Moxham, C.M., and Malbon, C.C. (1996) G_ialpha2-deficiency Impairs Insulin Action in vivo.  Nature 379: 840-844.

Baltensperger, K., Karoor, V., Paul, H., Czech, M.P., Ruoho, A., and Malbon, C.C. (1996)  The  -Adrenergic Receptor is a Substrate for the Insulin Receptor Kinase. J. Biol. Chem. 271: 1061-1064. PDF

Moxham, C.M., Hod, Y., and Malbon, C.C. (1993) Induction of G_ialpha2-specific Antisense RNA in vivo Inhibits Neonatal Growth. Science 260: 991-995.

Watkins, D.C., Johnson, G.L., and Malbon, C.C. (1992) Regulation of the Differentiation of Teratocarcinoma Cells into Primitive Endoderm by G_ialpha2.  Science 258: 1373-1375.

Wang, H.-y., Watkins, D.C., and Malbon, C.C. (1992) Antisense Oligodeoxynucleotides to G_s Protein alpha-subunit Sequence Accelerate Differentiation of Fibroblasts to Adipocytes. Nature 358: 334-337. 

Postdoctoral Research Training Program

National Institutes of Health, National Research Service Award

The institutional NRSA program sponsored by the Diabetes & Metabolic Diseases Research Center (DMDRC) offers multidisciplinary post-graduate training opportunities to the scientifically-trained (Ph.D.) and clinically-trained (M.D.) to acquire expertise in the study of metabolic diseases using state-of-the-art approaches of biochemistry, cell and molecular biology. Opportunities for training include diabetes & insulin action, protein metabolism, G-protein-coupled receptor action in disease states, cell signaling, Ras and MAP kinase regulation. Expertise is derived from 27 trainers from 9 departments with basic/clinical research in 5 major disciplines (physiology, pharmacology, biochemistry, molecular biology & cell biology). The training faculty support the tenet that a successful research career in the diverse and multifaceted area of endocrine and metabolic diseases requires a broadly-based background founded in these five major disciplines as well as a hybrid perspective which is receptive to strategies transcending the limits of one's immediate specialty. Training is principally as participants in vigorous, supportive research programs of individual trainers as well as more-broadly-based training as DMDRC members. Trainees actively participate in weekly interdepartmental seminars, minisymposia, journal clubs in specialized areas (endocrinology, cell signaling), and periodic scientific meetings where reports on original research are presented. The trainees (8 per year) will be principally medical (M.D.) or Ph.D. graduates who demonstrate a keen interest in taking advantage of these opportunities. Emphasis is placed on the vigorous recruitment of women and underrepresented minorities. Trainees are selected based upon their ability to conduct original research in a careful and critical manner, the nature and quality of their thesis and/or prior work, and recommendations by referees. Competitive applicants visit the campus and present a seminar. Facilities include modern laboratories (>80,000 n.s.f.) equipped for original research endeavors. Unique opportunities exist for advanced training in transgenic and KO mice use, molecular biology, proteomics (MALDI and QToF mass spectrometry) & structural biology, microscopy & imaging analysis, DNA microarray, and iRNA use. The DMDRC T32 program sponsors bioethics training, career building, and planning. The DMDRC training program enjoys strong University-wide support.

Transgenic mouse models of human G-protein-based disease

Organization