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Regulators of G Protein Signaling in the Mammalian Brain Ted Wensel Department of Biochemistry and Molecular Biology Baylor College of Medicine, Houston,

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Presentation on theme: "Regulators of G Protein Signaling in the Mammalian Brain Ted Wensel Department of Biochemistry and Molecular Biology Baylor College of Medicine, Houston,"— Presentation transcript:

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2 Regulators of G Protein Signaling in the Mammalian Brain Ted Wensel Department of Biochemistry and Molecular Biology Baylor College of Medicine, Houston, Texas

3 Effector*     Gt  GG     GG GDP GTP Amplitude and Duration of Downstream Signals Depend on Kinetics of G Protein Activation and Inactivation downstream signals R* GTP binding GTP hydrolysis resting receptor inactivation kinase, &c. k act k inact k R*

4 .05.1.15.3.5 1 2.2 k inact (s -1 ) Effect of GTPase Rate Constant on Effector Activity Time Course 01020304050 Time (s) 0 0.04 0.08 0.12 0.16 Relative Effector Activity R* inactivation rate constant set at: k R* = 0.5 s -1.05.1 2.15.5 G protein inactivation rate constant for G protein activation by R*, k act = 100 s -1

5 Effector*     Gt  GG     GG GDP GTP RGS Proteins are GTPase Accelerating Proteins (GAPs) that regulate the amplitude and Duration of G Protein Signaling downstream signals R* GTP binding GTP hydrolysis resting k act k inact RGS(GAP) PKC, Ca 2+,14-3-3, Ub, PIP 2, syntaxins, effectors, etc.

6 A Complex of Gt ,with the catalytic core of RGS9 Slep et al., Nature, 2001 GGGG RGS domain Residuescontrollingeffectorregulation

7 0 1020 3040 Time (s) 0 20 40 60 80 100 GTP hydrolyzed (percent) RGS9+/+ + PDE  RGS9+/+ RGS9-/- + PDE  RGS9-/- Disruption of the RGS9 gene reduces GTP hydrolysis to basal level, and abolishes PDE  enhancement Chen et al., Nature, 2000

8 Recovery from GPCR Activation is Greatly Slowed in RGS9-knockout Mice Responses to brightest flash Responses to dimmest flash WT KO Chen et al., Nature, 2000

9 The RGS family Zheng B et al, Trends Biochem Sci 1999 100 aa R7 Family

10 RGS7 Characteristic domain structure Present in both brain and retina –One of the most abundant RGS proteins in the brain Multiple splice variants -> different C-termini Regulated by PKC and Ca 2+ /Cam, 14-3-3, Ub spleen brain liver lung retina kidney heart testis 37-118 (82) 256-316 (61) 333-448 (116) 119-255 (137) 464-490 (16-42) DEPGGLRGSC Gβ5 N I domain (Intervening)

11 * * 1819 1 3 4 56789 11 13151617AB 2 Human RGS9-1: Retina, 484 a.a. Human RGS9-2: Brain, 671 a.a. RGS9 mRNA is alternatively processed in the retina and in the striatum retina brain RGS9 Western 57 kda 77 kda stop RGS9 ISH (Gold et al., 1997) 12 14 1 3 4 56789 11 13151617A 2 12 14

12 RGS9 immunostaining in medium spiny cells of mouse striatum 10  m CPu NAc

13 1. Expression Levels Modulated by Drugs -Amphetamines induce expression of RGS2,3, 5, 8, and suppress RGS9-2 Burchett et al., 1998; Burchett et al., 1999 -Dopamine D1 and D2 receptors regulate expression of RGS2,5,8,4, and RGS9-2. D1 and D2 act oppositely on RGS2 and RGS4 Geurts et al., 2003; Taymans et al., 2003. -mu-Opioid receptors regulate expression of RGS2 and RGS4. Gold et al., 2003 Evidence for RGS involvement in drug addiction

14 2. Striatal-specific localization of RGS9-2 -RGS9-2 expression is highly localized to the striatum Gold et al., 1997; Thomas et al., 1998; Rahman et al., 1999; Zhang et al., 1999 3. Knockdowns and Knockout -RGS9 knockout mice show increased susceptibility to cocaine and other drugs of abuse Rahman et al., 2003 -knock-down of RGS9-2 or its binding partner Gbeta5 enhances responses to mu-opioid agonists Garzon et al., 2001; Sanchez-Blazquez et al., 2003; Evidence for RGS involvement in drug addiction

15 Rahman et al. Neuron, 2003

16 Convergent Regulation of Adenylate Cyclase Activity by Neuromodulators in Medium Spiny Cells of the Striatum Burns &Wensel Neuron, 2003

17 G Protein-Activated Potassium Channels in Oocytes Activate and Recover Faster in Response to Muscarinic Activation when RGS9 Is Present

18 GGL (G protein Gamma-Like) domains of RGS proteins bind G  5 G  5S and G  5L are divergent members of the G protein  subunit family- they do not bind G protein  subunits. N-DEP-I-domains C-domain-RGS-domain GGL domain

19 RsrII pXOP-EGFP-RGS9-1 pXOP-G  5L EGFPRGS9-1 G  5L RsrII ApaLI BamHIEcoRI AgeI SacII XOP 650 400 300 650 400 300 PWT2PW T1 G  5L EGFP-RGS9-1 ab cd A B C RGS9 and G  5 Must be Co- expressed for production of stable protein in vivo

20 Chen et al. PNAS, 2003 0 2 4 6 8 10 12 14 16 18 13579111315171921232527 Age (day) Body Weight (g) G  5+/+ G  5-/- G  5 knockouts have almost no RGS7 or RGS9 and feed poorly G  5-/- G  5+/+ 81 50 RGS9 G  5-/- G  5+/+ 81 50 RGS7

21 Alternative splicing of the RGS7 gene GGL DEP mRNA (2.3 kb) Alternatively spliced Exon1- 16 a1 a2 a3 Exon 17 RGS 5’- * -3’ a.a. after the RGS domain a1a2 End of RGS domain a3 54 bp81 bp78 bp Currently identified isoforms 35 a1 a3 * A 42 a2a1 * B * 34 a1 C 17 a3 * D 24 a2 * E 16 * F

22 Calmodulin has an inhibitory effect on RGS7 phosphorylation by PKC  Time (min) ATP / Protein GST-S7DC (RGS domain and C-terminus) GST-S7DC + 0.6 μM CaM GST-S7DC + 3 μM CaM GST-S7DC + 15 μM CaM Commassie Phospho-Image 5 10 15 20 30 40 Interactions of Calmodulin with RGS7 and RGS9 Weiwei Liu, Sun. AM, F16, Prog. # 162.10

23 Differential phosphorylation of RGS7 isoforms by PKC Phospho-Image DDEEBBCCAAFF -+-+-+-+-+-+ CaM Commassie a3a1 A a2 B a1 C a3 D a2 E F

24 b 11 2 b 11 2 -H 3 PO 4 y2y2 GLQND-NH 3 b 6/ y 7 y5y5 a 6 -H 3 PO 4 TRKSVYGLQNDIR y5y5 y7y7 b 11 a 6,b 6 b 8 -H 3 PO 4 y2y2 b8b8

25 b3b3 y 12 2 -H 3 PO 4 TRKSVYGLQNDIR y 10 y 12 b3b3 b2b2 y 10 b2b2

26 GAP activity of RGS9 is stimulated by PI, but inhibited by PIP, PIP 2 and PIP 3 0.03 0.01 0.02 0.03 0.04 RGS9 +PI +PI3P +PI4P +PI(4,5)P2 +PI(3,4,5)P3  K inact (s -1 ) The I (Intervening) domain of RGS9 is a novel phosphoinositide binding domain Muling Mao, Wed. AM, G66, Prog. # 817.10

27 G  5L G  -GTP Effector RGS Multiple Interactions Regulate R7 RGS Proteins R9AP PKC  PIP 2 Ca 2+, Cal- modulin Protein Levels, Ub, proteasome Alternative Splicing Membrane Anchors Effector Enhancement Phosphorylation Inhibition by phosphoinositides 14-3-3

28 Thanks to: Baylor College of Medicine Wensel lab: Muling Mao, Weiwei Liu, James Mancuso, Wei He, Chris Cowan, Xue Zhang, Lisha Lu, Feng He, Guang Hu, Julie Vargas Milan Jamrich, Heithem El- Hodiri Caltech Mel Simon, Jason Chen (U. Utah) Henry Lester, Abe Kovoor Univ. of Utah Wolfgang Baehr Univ. of Washington Kris Palczewski, Geeng-Fu Jang, Izabel Sokal Yale the late Paul Sigler, Kevin Slep, Michele Kercher Yale/UTSW Eric Nestler, Zia Rahman, Stephen Gold Stanford Denis Baylor, Marie Burns NIH (NIDA, NEI, NLM, NIGMS, NIDDK), Welch Foundation

29 Test of S434D StartBeadsSupWashStart Beads SupWash CaEGTA S434D wt + + - - S434D - - + + CaM + - + - In Vitro Phosphorylation CaM-Agarose binding S434 is not the major phosphate incorporation site in vitro. Commassie Phospho-Image

30 9N, 1-219 9NG, 1-295 9GDC, 214-484 9DC, 291-484 9D, 276-431 9G, 214-280 7NGDC, 1-469 7DC, 318-469 + GST, his 6 GST GST, none GST, his 6 GST GST, his 6, none GST + his 6, his 6 GST GST, none G  5S, 43-395 G  5L, 1-395 b b b b E E E b b/X Mixture = "G  5 " b/X + + + his 6 N-dom. GGLRGS C-dom. 9NGDC, 1-484 Copur. G  5 Tag GST, his 6, GFP b/X Express. System Name, residues + 9NGD, 1-431 GST b + DEP Expression of RGS9-1/ G  5 Modules b = baculovirus, E= E. coli, X = X. laevis RGS9 RGS7 no

31 GGLdomain RGS domain RGS9-C domain Binds G protein  5L subunit Binds anchor protein R9AP Accelerates hydrolysis of GTP-G t  & encodes effector specificity Different in RGS9-1 & RGS9-2 N 484 or 671 a.a. Domain Functions in RGS9 S475 phos- phorylation RGS9-N domain DEP domain Suppresses Basal GAP Activity Helps PDE  Enhancement of GAP Activity Required for Effector Enhancement of GAP Activity I domain Binds phosphoinositides


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