Thesis
Reference
Chronic effects of γ-hydroxybutyrate (GHB) modulation of GIRK channels by regulators of G protein signaling proteins
LOMAZZI, Marta
Abstract
Nous avons démontré que le faible couplage dans les neurones DA est dû à une expression spécifique de canaux hétéromériques GIRK2/3 et à la présence d'un membre de la famille des régulateurs de la signalisation des protéines G, RGS2. On a aussi déterminé que la modulation par RGS2 joue un rôle dans les phénomènes d'adaptation survenant lors d'expositions chroniques aux drogues: nous avons observé une EC₅₀ significativement plus basse dans les souris injectées avec la drogue ainsi qu'une réduction de l'expression du messager RGS2. Finalement, nous avons démontré qu'une auto-administration de GHB, à des doses qui, normalement, induisent une récompense, devenaient aversives après que les animaux avaient été exposés au GHB. Ces résultats suggèrent un mécanisme qui pourrait sous-tendre une forme particulière de tolérance au GHB. Cette découverte offre de nouvelles perspectives quant aux cibles utilisées pour le traitement des addictions.
LOMAZZI, Marta. Chronic effects of γ-hydroxybutyrate (GHB) modulation of GIRK channels by regulators of G protein signaling proteins. Thèse de doctorat : Univ. Genève et Lausanne, 2008, no. Neur. 22
URN : urn:nbn:ch:unige-861
DOI : 10.13097/archive-ouverte/unige:86
Available at:
http://archive-ouverte.unige.ch/unige:86
Disclaimer: layout of this document may differ from the published version.
FACULTE DES SCIENCES DOCTORAT EN NEUROSCIENCES
des Universités de Genève et de Lausanne
UNIVERSITE DE GENEVE FACULTE DES SCIENCES Professeur Christian Lüscher, directeur de thèse
Professeur Ivan Rodriguez, co-directeur de thèse
TITRE DE LA THESE
“Chronic Effects Of γ-Hydroxybutyrate (GHB) Modulation Of GIRK Channels
By Regulators Of G Protein Signaling Proteins”
THESE présentée à la Faculté des Sciences de l’Université de Genève
pour obtenir le grade de Docteur en Neurosciences
par
Marta LOMAZZI d’ Italie
Thèse N° 22 Genève
Atelier d'impression UniMail 2008
A Nicolo’…
pour avoir été à mes côtes à chaque moment
Table of contents:
RESUME EN FRANÇAIS
………...……….…7
INTRODUCTION
………...……….…………...10
1. Definition and historical perspective
………...……….11
2. G-protein mediated signaling
………...………12
3. RGS molecular determinants
………...……….16
4. RGS family members
………...……….…...17
5. Effects on Gα: kinetics and beyond
………...………20
5.1 Activation/Deactivation………...………..
20
5.2 Desensitization………...……….………….
21
6. Macromolecular signaling complex: coupling efficiency
……….22
7. Effects on ion channels
………...……….…23
7.1 GIRK………...……….………...
23
7.1.1 Precoupling GIRK-RGS………...………
24
7.1.2 Role of RGS in GIRK activation/deactivation/coupling efficiency………
25
7.1.3 Role of RGS in GIRK desensitization………...………...
26
7.2 VGCC………...……….……….…
28
7.2.1 Role of RGS in calcium oscillation………...……..
30
7.2.2 Role of RGS in kinetic modulation GPCR-calcium channels……….
31
8. Regulation of RGS: expression levels
………...………...34
8.1 RGS transcription………...………..……….…
34
8.2 RGS translation………...……….……..
35
9. Possible role of RGS in human diseases
………...………..37
9.1 Addiction………...……….…
37
9.1.1 Morphine………..………..……….
39
9.1.2 Cocaine………..…………...………...
39
9.1.3 γ hydroxybutyrate………...………..……….
40
9.1.4 Amphetamines………..…………...……
40
9.2 Heart diseases……….………..…………...…
41
9.3 Pain………..…………...…
41
9.4 Alzheimer’s diseases………..…………...…
41
9.5 Schizophrenia………..………..…………...…
42
9.6 Cancer………..………...…...…
42
10. The particular case of RGS2
………..……43
10.1 Structure………..……….………...…
43
10.2 Expression……….………..…………...…
44
10.3 RGS2-Gα interaction………..………..…………...…
46
10.4 Localization………...…..…………...…
46
10.5 RGS2 knock out mice………..…………....
47
10.6 RGS2 modulated pathways……...………..…………...…
48
10.7 Possible role of RGS2 in human diseases…….………..…………...…
49
PROJECT AIMS
………..…………...………50
RESULTS
………..…………...………..52
11. GABA
BR-GIRKs coupling efficiency modulation
B ………53
11.1 GIRK subunit composition………...…..…………...…
53
11.2 RGS modulation………..…………...……..
54
11.2.1 Inhibition of RGS proteins increases coupling efficiency………...
54
11.2.2 Cell-specific expression of RGS proteins in the VTA………...……
55
11.2.3 Coupling efficiency in RGS2 knockout mice………...……….
56
11.2.4 Modulation of RGS2 expression………..….
59
11.2.5 Loss of disinhibition of DA firing in RGS2–/– mice………..
61
11.2.6 Chronic GHB alters preference for self-administration………….………
63
12. RGS2 transcription in the ventral tegmental area
…………...…64
DISCUSSION
………..…………...………..69
PERSPECTIVES
………..…………...………77
METHODS
………..…………...………...79
REFERENCES
………..…………...………85
ACKNOWLEDGMENTS
………….………..…………...…94
List of abbreviations used in the text:
- AC : Adenylyl Cyclase - Ach : Acetylcholine
- AGS : Activator of G-protein Signaling - CSP : Cysteine String Protein
- CREB : cAMP Response Element-Binding protein - DA : Dopamine
- DEP : Dishevelled/EGL-10/Pleckstrin
- FRET : Florescence Resonance Energy Transfer - G protein : Guanine nucleotide binding protein - GAIP : G Alpha Interacting Protein (=RGS19) - GAP : GTPase Activating Protein
- GDP : Guanosine Diphosphate
- GEF : Guanine nucleotide Exchange Factor - GGL : G protein γ-Like
- GHB : γ-Hydroxybutyrate
- GIPC : GAIP/RGS19 C terminus-Interacting Protein - GIPN : GAIP/RGS19 N terminus-Interacting Protein
- GIRK : G protein-gated Inwardly Rectifying Potassium channels - GoLoco : Gi/o-Loco interaction
- GTP : Guanosine Triphosphate - LC : Locus Coeruleus
- NAc : Nucleus Accumbens - PDZ : PSD-95, Disk-large, ZO-1 - PFC : Prefrontal Cortex
- RGS : Regulator of G protein Signaling - SPL: Spinophilin
- TIRF : Total Internal Reflection Fluorescence microscopy - VGCC : Voltage Gated Calcium Channels
- VTA : Ventral Tegmental Area
RESUME EN FRANÇAIS
Les agonistes des récepteurs GABAB, comme le baclofène et le GHB, exercent un effet bidirectionnel sur l’activité des neurones dopaminergiques (DA) de l’aire tegmentale ventrale (VTA). A faible concentration, ces drogues inhibent préférentiellement les neurones GABA, ce qui enlève leur contrôle inhibiteur sur les neurones DA et augmente la libération de dopamine. De fortes concentrations, en revanche, inhibent directement les neurones DA. Cet effet bidirectionnel s’explique par le fait que le couplage entre les récepteurs GABAB et les canaux GIRK est plus faible dans les neurones DA que dans les neurones GABA.
Dans cette étude, nous avons démontré que le faible couplage dans les neurones DA est dû en fait à une expression spécifique de canaux hétéromériques GIRK2/3 et la présence d’un membre de la famille des régulateurs de la signalisation des protéines G, RGS2.
Dans les neurones DA de souris, nous avons observé que l’ajout de l’inhibiteur non- spécifique des RGS, PIP3, réduisait significativement la concentration d’agonistes GABAB requise pour induire la moitié d’un courant GIRK maximal (EC50). Nous avons émis l’hypothèse qu’une expression cellulaire spécifique de RGS pouvait moduler l’EC50. En utilisant la technique du «single-cell RT-PCR» pour mesurer l’expression du messager RGS dans les neurones GABA et DA, nous avons observé la présence de RGS4 dans les neurones GABA et la présence de RGS4 et RGS2 dans les neurones DA. En accord avec notre hypothèse, nous avons démontré dans les cellules DA de souris
RGS2-/- que l’EC50 était significativement réduite comparé aux souris contrôles.
Nous avons ensuite examiné si RGS2 pouvait moduler les canaux GIRK2/3. Dans les neurones DA de souris GIRK3-/- RGS2-/-, nous avons observé que l’EC50 était similaire à l’EC50 des souris GIRK3-/-, suggérant une interaction spécifique de RGS2 avec les canaux contenants GIRK3. Cette interaction a pu être confirmée avec des expériences FRET.
De plus, afin de déterminer si la modulation par RGS2 joue un rôle dans les phénomènes d’adaptation survenant lors d’expositions chroniques aux drogues, nous avons injecté des souris avec des doses croissantes de GHB ou de morphine. Chez ces souris, nous avons observé une EC50 significativement plus basse comparé aux souris contrôles injectées avec une solution saline ainsi qu’une réduction de l’expression du messager RGS2.
La baisse de l’EC50 dans les souris RGS2-/- suggère une fenêtre de concentrations réduite dans laquelle les agonistes GABAB produisent une désinhibition. Afin de tester cette hypothèse, nous nous avons mesuré la fréquence de décharge des neurones DA dans les souris RGS2-/-. A faible concentration, nous avons constaté que le baclofène et le GHB augmentait uniquement la fréquence des neurones DA de souris contrôles, mais pas des souris RGS2-/-. A forte concentration, la fréquence était diminuée dans les 2 génotypes.
Finalement, nous avons démontré qu’une auto-administration de GHB, à des doses normalement induisant une récompense, devenait aversive après que les animaux aient été chroniquement exposés au GHB. Ces résultats suggèrent un mécanisme qui pourrait sous-tendre une forme particulière de tolérance au GHB. Ils permettent d’établir un lien entre l’efficacité du couplage entre les récepteurs GABAB et les canaux GIRK et la réponse comportementale au GHB.
Pris dans leur ensemble, ces résultats suggèrent que l’efficacité du couplage entre les récepteurs GABAB et les canaux GIRK dans les neurones DA est due à une expression sélective de sous-unités GIRK et une expression cellulaire spécifique de protéines RGS2.
Cette découverte offre de nouvelles perspectives quant aux cibles utilisées pour le traitement des addictions.
INTRODUCTION
1. Definition and historical perspective
Siderovski and colleagues isolated in 1990 a set of putative G0/G1 switch regulatory genes (which they called G0S genes) by differential screening of a cDNA library prepared from human peripheral blood mononuclear cells. Several of these G0S genes were found to be similar to immediate-early genes differentially expressed in quiescent rodent fibroblasts activated by serum, growth factors, and other agents (Siderovski, Blum et al. 1990). Few years later the same group identified these G0S genes as new family of regulators of G-protein-coupled receptors (Siderovski, Heximer et al. 1994). These regulators were identified genetically by their ability to reduce G-protein signaling in Saccharomyces cervesiae (Dohlman, Song et al. 1996) and Caenorhabditis elegans (Koelle and Horvitz 1996) and named Regulator of G protein Signaling (RGS). Partial nucleotide sequencesfor 15 mammalian genes were then identified from a brain cDNA librarythat shared a conserved 120-amino acid core domain. This domain exhibited GAP (GTPase activating proteins) activity and was termed RGS domain, henceforth recognized as the protein family hallmark (Hollinger and Hepler 2002).
It is now well known that RGS are a family of cellular proteins that play an essential regulatory role in G protein-mediated signal transduction. The highest proportion of RGS genes are expressed in brain tissue, which is in line with the extensive diversity of neuronal and glial G protein-coupled receptors and the signal modulation necessary for proper brain function (Hollinger and Hepler 2002).
2. G-protein mediated signaling
G proteins (guanine nucleotide binding proteins) are a family of proteins involved in second messenger cascades. These molecules work as "molecular switches", alternating between an inactive guanosine diphosphate (GDP) and activated guanosine triphosphate (GTP) bound state, ultimately regulating downstream cell processes. Heterotrimeric G proteins are activated by G protein-coupled receptors (GPCR) and made up of α, β and γ subunits. Four main families exist for Gα subunits: Gαs, Gαi/o, Gαq/11, and Gα12/13. These groups differ primarily in effector recognition, but share a similar mechanism of activation. Gαs stimulates the production of cAMP from ATP that acts as a second messenger interacting with and activating protein kinase A (PKA). PKA can then phosphorylate a lot of downstream targets such as CREB (cAMP Response Element- Binding protein). Gαi/o inhibits the production of cAMP from ATP. Gαq/11 stimulates membrane bound phospholipase C beta (PLCβ) which then cleaves phosphatidylinositol bisphosphate (PIP2) into two second messengers, inositol triphosphate (IP3) and diacylglycerol (DAG). Gα12/13 are involved in Rho family GTPase signaling and regulate cell migration.
Each main α family is characterised by several α subtypes which share amino acid sequence similarity (Simon, Strathmann et al. 1991) (Milligan and Kostenis 2006) (Table1).
Table1. The families and main subfamilies of mammalian heterotrimeric G-protein α subunit. From Simon, Strathamann et al. 1991; Milligan and Kostenis 2006.
α families α subtypes Expression
Gαs Gαs
Gαsolf
Ubiquitous Olfactory neurons
Gαi/o Gαi2/3
GαoA/B
Gαz
Gαt
Neurons, Neuroendocrine cells, Astroglia, Heart Neurons, Neuroendocrine cells, Astroglia, Heart Neurons, Neurosecretory cells, Platelets Transducin photoreceptors, Taste buds cells
Gαq/11 Gαq
Gα11
Gα14
Gα15/16
Ubiquitous Ubiquitous Epithelial cell Haematopoietic cells
Gα12/13 Gα12
Gα13
Ubiquitous Ubiquitous
The signaling cascade starts after activation of the G-protein coupled receptor. This activation allows a conformational change of the receptors itself that function as a guanine nucleotide exchange factor (GEF) exchanging GDP for GTP on the Gα subunit.
The active heterotrimeric G proteins are released from the receptor, and dissociate into free, active GTP-bound α subunit and βγ dimer, both of which activate downstream effectors. The response is terminated upon GTP hydrolysis by the α subunit, which can then bind the βγ dimer and the receptor (Gilman 1987) (Fig.1).
This G-protein mediated signaling could be also activated by non-7 transmembrane GPCR such as Cysteine String Protein (CSP) and Activator of G-protein Signaling (AGS).
CSP are secretory vesicles proteins involved in neurotransmitter release. These proteins contains two G protein binding site, with no homology with established modulators of G proteins. The region between 83–112 amino acids associates with Gβ and/or Gαβγ, while the N-terminal associates only with Gα. CSP proteins modulates G protein activity by
targeting the Gαs-GDP and promoting the GDP-GTP exchange, acting as GEF. For example, CSP associates with G-proteins and promote the Gβγ-mediated inhibition of N- type Ca2+ channels (Natochin, Campbell et al. 2005).
AGS are a family of G-protein modulators that interacts with G protein to promote the G protein mediated signaling. AGS proteins do not share any conserved structural domain and activate different G proteins by distinct mechanisms. While group I AGS (i.e. AGS1) activate G-protein acting as a guanine nucleotide exchange, group II (i.e. AGS3) and group III (i.e. AGS2 and AGS8) activate the G-protein signaling without any apparent nucleotide exchange. Group II AGS interact with G protein via a GoLoco (Gi/o-Loco interaction) domain, while group III binds directly to the Gβγ (Blumer, Cismowski et al.
2005).
AGS1 is a member of the Ras superfamily of small G proteins and selectively activates the Gi/o-protein signaling pathway, acting as a guanine nucleotide exchange. Takesono and colleagues, demonstrated that AGS1 competes with the muscarinic receptors for the pool of available heterotrimeric Gαi/o to activate GIRK channel (Takesono, Nowak et al.
2002). AGS2 binds the Gβγ subunit of the G-protein and function as activator of the Gαi2/3. Apparently, AGS2 activates G-protein mediated signaling by interfering with the interaction between G protein subunits somehow increasing the availability of Gβγ in yeast-based system but its function in mammalian G-protein is still unknown (Blumer and Lanier 2003). AGS3 acts as a GDP-dissociation inhibitor of the Gαi subunit, blocking the reassociation of Gαi with the Gβγ dimer, thus inhibiting the Gαi-dependent pathways but enhancing the Gβγ-regulated signaling. This interaction is mediated by a G protein regulatory GoLoco motif (Takesono, Cismowski et al. 1999). A recently described AGS, AGS8, binds to G protein βγ and forms a complex interacting with Gα and Gβγ simultaneously, without inducing Gα and Gβγ dimer dissociation (Sato, Cismowski et al.
2006; Yuan, Sato et al. 2007). This observation suggests that G-proteins could not dissociate upon activation but rather undergo specific conformational rearrangements and exert their effects remaining in complex. This idea is supported by the fact that a covalently linked Gα-βγ fusion protein is signaling competent in yeast (Klein, Reuveni et al. 2000). Moreover, the kinetic coupling model for G protein activation suggests that, in
the presence of regulators of G protein signaling proteins, GTP hydrolysis is too rapid to allow subunit dissociation prior to effector activation (Ross and Wilkie 2000). A potential caveat of the model is that it is not clear how conformational rearrangements of the subunits would allow for productive interactions with respective effectors.
Despite this possible non-dissociative activation of G-protein, here we will consider mainly the dissociative activation of G-protein and its regulation by modulators such as RGS.
3. RGS molecular determinants
RGS proteins are multi-functional, GTPase-accelerating proteins that promote GTP hydrolysis by the α subunit of heterotrimeric G proteins, thereby inactivating the G protein and rapidly switching off G protein-coupled receptor signaling pathways. As described above, the G-protein mediated signaling is terminated upon GTP hydrolysis by the α subunit, which can then bind the βγ dimer and the receptor. RGS proteins modulate this pathway by markedly reducing the lifespan of GTP-bound α subunits by stabilizing the G protein transition state (Fig.1). This activity is exerted by the RGS domain (also called RGS box), a catalytic GTPase activating proteins (GAPs) domain of about 120 amino acid residues, which is responsible for binding Gα subunits and negatively regulate G protein signaling. Specific RGS proteins interact selectively with particular Gα subunits, which may be determined by specific sequences within the RGS domains and the Gα subunits (Xie and Palmer 2007).
New evidence suggests also a potential interaction between RGS and βγ dimer. We demonstrated that a significant FRET was measured between RGS2 and GIRK3. Since GIRK channels are activated by βγ subunits we hypothesize that somehow RGS2 could interact with βγ in the vicinity of GIRK3 (Labouebe, Lomazzi et al. 2007) (See Results) (Fig.1).
Fig.1 Schematic representation of G-protein cycle G-protein activation via GDP-GTP exchange, leads to dissociation between α-GTP and βγ. The reaction terminates after GTP hydrolysis by α subunit.
Historically, RGS has been described to act at the level of the α subunit of the G-protein increasing its intrinsic GTPase activity thus speeding up the hydrolysis of GTP. Possible other activity could be exert by RGS on βγ dimer.
α α ββ γ γ
G GDDPP
G GTTPP
γ γ β β
RRGGSS
α α
G GDDPP
γ γ β
β
+
Pi
Pi
α α
RGRGSS
4. RGS family members
There are over 20 members in the mammalian RGS family. The RGS domain, which is responsible for the catalytic activity of these proteins, is the characteristic structural element that defines the RGS protein family. Moreover, these proteins could have additional domains that integrate different G protein signaling pathways or anchor scaffolding proteins that link G protein to related signaling proteins (Wang, Zeng et al.
2005) (Xie and Palmer 2007). The N-terminal region of the RGS plays an essential role in plasma membrane targeting, subcellular localizations, contact and specific recognition of GPCRs, ion channels and effector proteins. This region of RGS proteins is one of the most important determinants for their selectivity. Moreover, it’s involved in primary biological activities, such as GAP activity (Zhou, Moroi et al. 2001) (Jeong and Ikeda 2001) (Saitoh, Masuho et al. 2001) (Toro-Castillo, Thapliyal et al. 2007). The GGL (G protein γ-like ) domain contains a G protein γ like subunit that allows a specific interaction with G protein β subunit Gβ5 giving selectivity towards receptors that are coupled to heterotrimeric G proteins consisting of the Gβ5 subunit such as RGS9 and photoreceptors (Blake, Wing et al. 2001) (Makino, Handy et al. 1999). The DEP domain (dishevelled/EGL-10/pleckstrin) confers specificity of interaction with GPCRs and RGS- binding proteins (Kovoor, Seyffarth et al. 2005) (Martemyanov, Yoo et al. 2005). The GoLoco domain allows a specific interaction with GDP-bound Gαi (Kimple, De Vries et al. 2001) and is involved in centrosome/chromosome movement mediated by G protein during cell division (Wilkie and Kinch 2005). The PDZ (PSD-95, Disk-large, ZO-1) domain-links RGS to other proteins and signaling pathways. The PDZ domain of RGS12 interacts with the C-terminal tail of a chemokine G-protein-coupled receptor, contributing to the RGS GAP action (Snow, Hall et al. 1998).
According to the similarity in sequence and the presence of these non-RGS domains, RGS proteins have been classified into nine subfamilies (Xie and Palmer 2007) (Table2).
Table2 Classification of RGS proteins. RGS proteins are classified in nine subfamilies according to similarity in sequence and features of structural domains:
(From Xie and Palmer 2007)
Selectivity of RGS is improved by a spatiotemporal-specific expression (He, Cowan et al. 1998) (Thomas, Danielson et al. 1998) and by clustering at the genomic level between RGS and G protein related components (Sierra, Gilbert et al. 2002). Moreover, specific adaptors or scaffold proteins bridge the N terminal, C-terminal, or other special regions of RGS proteins with GPCRs, G proteins, or effectors strengthening, modifying, or conveying additional selectivity to RGS proteins. This group of adaptor/scaffold proteins comprehend 14-3-3, GAIP/RGS19 N terminus-interacting protein (GIPN), GAIP/RGS19 C terminus-interacting protein (GIPC) and Spinophilin (SPL). 14-3-3 proteins inhibit the GAP activity of RGS3, RGS7 and RGS8 enhancing GPCR signaling (Benzing, Yaffe et al. 2000). GIPN and GIPC bind respectively the N and the C terminal of GAIP/RGS19 to specific signaling pathways such as dopaminergic D2 and β1-adrenergic pathways (Fischer, De Vries et al. 2003) (Jeanneteau, Guillin et al. 2004). Spinophilin binds
specifically to RGS1, RGS2, RGS4, RGS16, and GAIP/RGS19 and to specific GPCR such as D2 dopaminergic and α2 adrenergic receptors allowing selective receptor recognition for RGS proteins (Wang, Zeng et al. 2005) (Richman, Brady et al. 2001).
This scenario underlines the complexity and the specificity of interaction between RGS and G protein related components that allow RGS to be involved selectively in several pathways achieving different functions. First of all, RGS proteins play an essential role in the G protein-mediated signal transduction (Ross and Wilkie 2000) which implies the regulation of the G protein-coupled receptors and the G protein-regulated ion channels signal transduction systems (Pierce, Premont et al. 2002).
Here, we review the regulatory role of RGS on the G-protein mediated signaling pathways. We first discuss the interaction between RGS and GIRK or Ca2+ channels, and then we focus on the implication of these proteins in different diseases, with particular attention to RGS2 protein.
5. Effect on Gα: kinetics and beyond
5.1 Activation/Deactivation
According to classical kinetic concepts, the activation and the deactivation phase of a channel can be represented through a two-state model (closed and open), where the rate constant for channel current activation (1/τact) is equal to the sum of the macroscopic opening and closing rate constants, kopen and kclose, respectively. The parameter kopen is expected to depend on the agonist concentration and the deactivation rate (1/ τdeact) gives kclose, which is measured upon agonist removal. The activation phase represents the rapid process that opens channels during a depolarization while the deactivation phase is the process that closes the channels during a depolarization; once channels are inactivated, the membrane must be repolarized or hyperpolarized to remove the inactivation. The steady state represents the state of equilibrium of the system, where a same number of channel are in the activated and in the inactivated state (Doupnik, Davidson et al. 1997) (Fig.2).
Deactivation phase Activation
phase
Steady state Ach
Fig.2 Representative traces of Acetylcholine-evoked GIRK currents. Deactivation time constant (τdeact) derives from exponential fits to the GIRK current deactivation phase, while activation time constant (τact)
derives from exponential fits of the GIRK activation phase. Holding potential -80 mV. From Doupnik et al., 1997.
5.2 Desensitization
After a persistent exposure to continuing or increasing doses of drug, receptors loose their responsiveness to the continuously present agonist and the amplitude of the current decreases with time. This phenomenon, called desensitization, represents an adaptative response of the cell to prevent excessive G-protein signaling (Fig.3). Desensitization occurs with different time course, depending on the length of stimulation and can be due to phosphorylation and receptor uncoupling or to receptor internalization followed by down-regulation of receptor/effector (Chuang, Yu et al. 1998).
Fig.3 Representative traces of prolonged Acetylcholine evoked GIRK currents.
Desensitization can be observed after 20 sec of agonist stimulation. Holding potential -80 mV.
From Doupnik et al., 1997.
Since these currents are G-protein mediated via activation of the specific GPCR, the rate of activation, deactivation and desensitization are influenced by G-protein turnover cycle, finally depending on the Gα subunit activity (Chuang, Yu et al. 1998). RGS proteins, acting on the α subunit of the G-protein, speed up the hydrolysis of the GTP through their GAP domain, affecting the G-protein turnover. This RGS activity results in the acceleration of the channel’s kinetics (Doupnik, Davidson et al. 1997; Chuang, Yu et al. 1998)
Desensitization
Steady state Ach
6. Macromolecular signaling complex: coupling efficiency
As described above, the activation of the GPCR leads to the activation of related effectors. In this signaling pathway the strength of interaction between receptor and effector is defined as the coupling efficiency.
The coupling efficiency is measured through the half maximal effective concentration (EC50) which refers to the concentration of agonist necessary to get the 50% of the maximal effect. The EC50 is measuredfrom a dose-response curve which is a simple X-Y graph relating the concentration of the agonist to the response of the receptor. The measured dose is generally plotted on the X axis in a logarithmic scale, and the response is plotted on the Y axis. In such case the curve is typically sigmoidal, with the steepest portion in the middle (Fig.4).
100 80 60 40 20 0
Current(%)
0.1 1 10 100
[Agonist]
EC50
Fig.4. Representative plot of dose-response curve. The EC50 is measured as the concentration on agonist that is needed to obtain 50% of the maximal effect (Current % in this case). From Labouèbe, Lomazzi et al., 2007.
5500
The coupling efficiency modulation can be mediated by both α-GTP and βγ dimers. Both dimers interact with effector molecules to initiate signaling cascades. These effectors can be enzymes such as phospolipase C or adenylyl cyclase, which produce second messengers that regulate intracellular calcium and cyclic AMP concentrations, respectively, or ion channels, such as the G protein–regulated inwardly rectifying K+ channels or voltage gated calcium channels (Neves, Ram et al. 2002).
7. Effects on ion channels
7.1 GIRK
G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart (Stanfield, Nakajima et al. 2002).
The channels are composed by combinations of four subunits GIRK1-4that associate to form homo (GIRK2, GIRK4) or heterotetramers (GIRK1/2, GIRK1/3, GIRK1/4 and GIRK2/3) (Fig.5).
While GIRK 2 and 4 seem to be necessary to form the channel, the other subunits achieve auxiliary functions. These channels have been found in various cell-types with different subcellular localization (Duprat, Lesage et al. 1995; Jelacic, Kennedy et al. 2000).
Fig.5 Schematic representation of GIRK channels.
The channels are composed by four subunits associated to form homo or heterotetramers.
(Adapted from http://www.sfb487.uni-wuerzburg.de)
The main functions of GIRK channels are associated to the control of the resting membrane potential, regulation of cellular excitability and maintenance of potassium homeostasis that leads to hyperpolarization and reduction of membrane excitability. The channels allow K+ ions entering the cells at membrane potentials below the K+ equilibrium potential but outward conductance is limited at membrane potentials above this state. The inward rectification of the channels is due to a voltage-dependent blockade by intracellular polyamines (Kobayashi and Ikeda 2006).
These channels are activated after stimulation of Pertussis toxin (PTX)-sensitive G protein-coupled receptors, such as GABAB receptors, that use the Gi/o family of G
proteins. As described above, upon GPCR stimulation, the Gα of the G protein replaces its bound GDP to GTP, leading to the dissociation between Gα and the Gβγ dimer. The Gβγ dimers bind to and activate GIRK channels (Kofuji, Davidson et al. 1995). Through their catalytic activity on the G-protein, RGS modulate the kinetics and the coupling efficiency of GPCR activated GIRK channels (Fig.6).
7.1.1 Precoupling GIRK-RGS
The interaction between RGS, GIRKs and GPCRs seems to be different according to the specific RGS. Jean et al. demonstrated that a degradation-resistant RGS4 mutant (RGS4 (C2V)) co-precipitates with muscarinic M2 receptors forming stable macromolecular complexes. The N-terminal of RGS4 is sufficient for the association but the remaining RGS domain and/or C-terminal are also necessary for an efficient high affinity coupling. The same precoupling has been shown also between RGS4 (C2V) and several different GPCRs such as serotonin 1A or adenosine A1. On the other hand, RGS3s (short isoform) does not interact with any of the GPCR-GIRKs channels complexes tested. These data suggest a “precoupling” (RGS4 (C2V)-GIRK-GPCR) versus a “collision coupling” (RGS3s) with a functional impact of a 100-fold greater
α α ββ γ γ
G
GDDPP α α ββ γ γ
G GTTPP
G GIIRRKKss G
GPPCCRR
R RGGSS
+
K+
Fig.6 GIRK channels activation via GPCRs
After GPCR activation, the signal is transmitted into the cell via G-protein. The interaction between βγ and GIRKs opens the channels leading to K+ efflux and cell hyperpolarization. RGS proteins modulate the kinetic and the coupling efficiency between GPCRs and GIRKs.
potency of the precoupled complex in the acceleration of GPCR-GIRK channel gating kinetics with no attenuation in current amplitude (Jaen and Doupnik 2006). Fowler et al.
measured this interaction through florescence resonance energy transfer (FRET) combined with total internal reflection fluorescence microscopy (TIRF). They showed a significant FRET between RGS4 and GABABR1 or R2 subunits, between Gαo and GABABR1 or R2 and between Kir3.2a/Kir3.4 and GABABR1 or R2. FRET was neither detected between Kir3.2a and RGS4 nor between Kir3.2a and Gαo. These data suggest proximity of the RGS4, GIRK and G-protein to the GPCR, even in absence of direct interactions of RGS4 with GIRK or G-protein, thus allowing a rapid and specific activation/modulation of Kir3 channels (Fowler, Aryal et al. 2007). Using the same technique, we showed a specific interaction between RGS2 and GIRK3, but not GIRK2, suggesting a selective modulation of RGS2 on GIRK3-containing channels (Labouebe, Lomazzi et al. 2007) (See Results).
7.1.2 Role of RGS in GIRK activation/deactivation/coupling efficiency
GIRK activation and deactivation kinetics are modulated by GPCRs via the G protein and are accelerated up to 100 fold by RGS. In some cases this phenomenon is associated with a reduction of the peak amplitude of GPCR-stimulated GIRK currents (Mark and Herlitze 2000) .
Doupnik et al. demonstrated that the time course for receptor-mediated GIRK current activation/deactivation is dramatically accelerated by co-expression of RGS1, RGS3 and RGS4 but not by RGS2 proteins with muscarinic M2 or serotonin A1 receptors (Doupnik, Davidson et al. 1997). In the same line, Herlitze et al. demonstrated that co-expression of RGS2, RGS5 and RGS8 accelerates the speed for Ach-mediated activation and deactivation of GIRK 1/2 and GIRK 1/4 in a concentration-dependent manner, suggesting that the receptor/channel/RGS ratio determines the amount of current amplification.
Moreover they showed that an excess of RGS to channel reduces Ach-elicited currents, while equivalent RGS and channel concentrations have no effect on the current amplification (Herlitze, Ruppersberg et al. 1999). Analogue observations were reported
by Saitoh and colleagues after co-expression of RGS7 or RGS8 with GIRK1/2 in Xenopus oocytes. Both RGSs accelerate the activation of GIRK current similarly but the RGS7 acceleration effect on deactivation is significantly weaker than that of RGS8 (Saitoh, Kubo et al. 1999). Moreover, Keren-Reifman et al., starting from the observation that RGS4 and RGS7 accelerate the kinetics of GIRKs channels in Xenopus oocyte system, revealed that RGSs modulate signaling by mechanism supplementary to their GTPase-activating protein activity. In fact, they demonstrated a biphasic effect of RGS4 that inhibits basal GIRK activity at low concentration while at high concentration stimulates it. They also showed a regulation of RGS7 by Gβ5 through an increase of RGS7 activity and expression (Keren-Raifman, Bera et al. 2001). Taken together, these data demonstrate a clear involvement of RGS proteins in the GIRK kinetic modulation.
Moreover functional domains other than the GAP domain could be involved in this modulation.
Jaen and colleagues demonstrated that RGS3 reduce the coupling efficiency between different GPCR such as M2 muscarinic and 5HT receptors and GIRK in vitro system (Jaen and Doupnik 2005). Our recent study demonstrated for the first time ex vivo RGS modulation of the coupling efficiency of GABAB receptors to GIRKs channels in the ventral tegmental area. In fact, the cell-type specific expression of RGS2 only in DA neurons but not in GABA neurons confers the lower coupling to DA neuron as confirmed by its genetically silencing. Moreover this modulation seems to be selective only for GIRK3 containing channels (Labouebe, Lomazzi et al. 2007) (See Results).
7.1.3 Role of RGS in GIRK desensitization
The endogenous level of RGS proteins plays a role in the modulation of GABAB
receptor-dependent desensitization of GIRK currents (Mutneja, Berton et al. 2005).
Chuang et al. demonstrated that coexpression of RGS4 proteins in oocytes enhances GIRK current desensitization after opioid stimulation, without suppressing the ability of G-protein subunits to activate GIRK channels (Chuang, Yu et al. 1998). RGS4 also modulates the voltage-dependent relaxation of GIRK currents after hyperpolarization to
negative membrane potentials (Ishii, Inanobe et al. 2002). Acute desensitization of GIRK current is also observed in the presence of RGS8, while it is not induced by RGS8 lacking the NH terminus suggesting the involvement of this specific area in desensitization (Saitoh, Masuho et al. 2001) and an action on channel modulation independently of GTPase acceleration (Jeong and Ikeda 2001).
7.2 VGCC
Voltage-gated calcium channels (VGCC) are transmembrane ion channels with permeability for Ca2+ ions which are activated by change in the electrical potential difference near the channel itself. They exert a crucial role in excitable neuronal tissues allowing fast and coordinated depolarization after voltage triggering, thus propagating electrical signals. These channels play essential roles in neurosecretion and other functions (Catterall 1998).
The channels are formed as a complex of several different subunits: α1, α2δ, β1-4, and γ.
The α1 subunit forms the ion conducting pore which contains voltage sensing machinery and the drug/toxin binding sites. The associated subunits have several functions including modulation of gating (Fig.7). The α2δ gene forms two subunits α2 and δ linked to each other. The α2 is the extracellular subunit that interacts mostly with the α1 subunit, while the δ subunit anchors the protein in the plasma membrane. The β subunit stabilizes the final α1 subunit conformation and delivers it to the cell membrane. Moreover, it is involved in the regulation of the activation and inactivation kinetics. Finally, the γ subunit works as an auxiliary subunit of a voltage-dependent calcium channel (Catterall 1998) (Abramow-Newerly, Roy et al. 2006) (Dolphin 2006).
Fig.7 Schematic representation of calcium channel.
While α1 subunit is involved in pore formation, the other subunits mainly modulate the gating.
From Abramow-Newerly, Roy et al. 2006
There are several different types of voltage-gated calcium channels (L, P/Q, N, and R- type). These channels are structurally homologous among varying types but not structurally identical. Moreover, they are differently inhibited by toxins.
The activities of these channels are increased by voltage, but decreased by G protein- mediated signals, which in turn are modulated by RGS proteins. GPCR activation causes Gβγ to directly inhibit channel opening by binding to specific regions of the α1 subunits of N-, P/Q-, and R-type Ca2+ channels (Fig.8). Both the on and off rates of current inhibition by activated Gαz, Gαq and Gαi/o proteins are increased by RGS proteins (Abramow-Newerly, Roy et al. 2006). Moreover, regulating specificity of GPCR by RGS proteins extends to single receptor types. However, interaction of RGS proteins with the receptor complex appears to confer specificity of action (Xu, Zeng et al. 1999). How receptor recognition is achieved by RGS is not known. Evidences suggest that the scaffold protein spinophilin, by binding the third intracellular loop of several GPCRs and the N-terminal domain of RGS2 but also RGS1, RGS4, RGS16 and GAIP, is involved in this mechanism. In fact, SPL expression in Xenopus oocytes markedly increased specific inhibition of α-adrenergic receptor-Ca2+ signaling by RGS2 (Wang, Zeng et al. 2005).
α α ββ γ γ
GDGDPP α α
GGTTPP
C Caa22++ chchaannnneellss GPGPCCRR
RGRGSS
+ β βγγ
CCaa22++
X
Fig.8 Ca2+ channels inhibition via GPCR
After GPCR activation, Gβγ binds to specific regions of the α1 subunits directly inhibiting Ca2+ channel opening. RGS proteins increase the on and the off rates of current inhibition.
7.2.1 Role of RGS in calcium oscillation
GPCR-evoked Ca2+ signaling generates second messengers, which lead to Ca2+ fluxes into and out of the cytosol. Ligand binding to GPCRs results in activation of Gαq that activates PLCβ inducing the hydrolysis of phosphatidylinositol bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3). IP3 releases Cai2+ from the endoplasmic reticulum (ER), followed by Ca2+ influx in the cytosol. The increase in Ca2+ leads to activation of the plasma membrane Ca2+ ATPase pumps to remove Ca2+ from the cytosol.
The overall signal is a transient change in [Ca2+]i (Wang, Huang et al. 2004) (Fig.9).
Fig.9
GPCR activation leads to G-protein dissociation and to PLCβ activation via Gαq
dimer. PLCβ hydrolyzes PIP2 into IP3. IP3 induces release of Ca2+ from the ER causing Ca2+ influx in the cytosol. The increase in Ca2+ activates plasma membrane Ca2+ ATPase pumps that remove Ca2+
from the cytosol. The signal is a transient change in [Ca2+]i .
PIP2 PLCβ IP3
Ca2+
Ca2+
αα
GGTTPP RRGGSS
As the negative regulators of GPCRs signaling, RGS proteins play a central role in determining the duration of the stimulated state and, thus, can play a central role in controlling [Ca2+]i oscillations. Luo et al., demonstrated that [Ca2+]i oscillations evoked by GPCR requires the action of RGS and that cyclical activation and inactivation of RGS protein activity generates [Ca2+]i oscillations in pancreatic acini and salivary gland cells (Luo, Popov et al. 2001). Few years later, the same group confirmed this observation in vivo providing also evidence for the role of RGS proteins and the kinetic of IP3
production in triggering [Ca2+]i oscillations: in fact, deletion of RGS2 increases the steady-state concentration of IP3 that can result from an increased frequency of oscillations in the concentration of IP3 and, consequently, increased the frequency of [Ca2+]i oscillations. Moreover, persistent rapid termination of Ca2+ signaling by the muscarinic antagonist atropine indicates that RGS proteins in general play a critical role in controlling Ca2+ signaling in native cells in vivo. Finally, deletion of RGS2 modifies the kinetic of IP3 production without affecting the peak level of IP3, but rather increases the steady-state level of IP3 at all muscarinic agonist carbachol concentrations. The increased steady-state level of IP3 leads to an increased frequency of [Ca2+]i oscillations (Wang, Huang et al. 2004). Moreover, RGS4, RGS1 and RGS16 inhibit phospolipase C activity and calcium signaling in a receptor-selective manner in both permeabilized cells and cells dialyzed with RGS4 through a patch pipette in acini prepared from the rat and mouse pancreas (Xu, Zeng et al. 1999). The N-terminal domain of RGS4 discriminates among receptor signaling complexes coupled via Gαq. The lack of the N-terminal domain of RGS4 eliminates receptor selectivity and reduces potency by 104-fold (Zeng, Xu et al.
1998)
7.2.2 Role of RGS in desensitization and kinetic modulation of GPCR-calcium channels
RGS proteins show selectivity in regulating Ca2+ ion channels at different levels.
Blocking endogenous RGS4 proteins using specific antibodies slows down the rate of desensitization of Gαi pathways acting on presynaptic Ca2+ channels in chick dorsal root ganglia (DRG) in a selective manner (Diverse-Pierluissi, Fischer et al. 1999) .
Transfection of HEK 293T cells with RGS5 accelerates the catalytic rate of GTP hydrolysis of Gαi3 subunit and suppressed angiotensinII-and endothelin1–induced intracellular Ca2+ transient in a concentration dependent manner: a 10 fold increase in amount of RGS5 induces about 20-25% reduction on Ca2+ signaling (Zhou, Moroi et al.
2001).
RGS2 splice variant from chick DRG (RGS2L) reduces muscarinic and bradykinin- mediated inhibition of neuronal L and N type channels and accelerates their recovery
from inhibition (Kammermeier and Ikeda 1999; Melliti, Meza et al. 2001; Tosetti, Parente et al. 2003). Moreover, RGS2L reduces the inhibition mediated by both the Pertussis toxin (PTX)-sensitive (Gαi/o-coupled) and the PTX-insensitive (Gαq/11-coupled) pathways in a RGS concentration dependent manner: at low concentration, RGS2L preferentially reduces the inhibition mediated by the PTX-insensitive pathway, whereas at 100-fold higher concentration, it attenuates both pathways equally (Tosetti, Parente et al. 2003). On the contrary, RGS2 accelerates the fast inhibition and recovery of presynaptic P/Q type Ca2+ channels controlled by muscarinic M2 receptors (Mark, Wittemann et al. 2000).
Coexpression of N-type or R-type Ca2+ channels in HEK293 cells with G protein- coupled muscarinic M2 receptors and full-length RGS3, short splice variant RGS3T, or RGS8 significantly attenuates the magnitude of receptor-mediated Ca2+ channel inhibition. This signaling activity of RGS3 is unaffected by its extended amino-terminal domain (Toro-Castillo, Thapliyal et al. 2007) (Melliti, Meza et al. 1999). The attenuation of Ca2+ current inhibition results primarily from a shift in the steady state dose-response relationship to higher agonist concentrations. The kinetics of Ca2+ channel inhibition are also modified by RGS3T which induces a slower decay and a faster recovery of Ca2+
channels from inhibition after agonist removal (Melliti, Meza et al. 1999).
Tosetti et al., showed the selectivity of chick GAIP/RGS19 on Go-mediated forms of Ca2+ channel inhibition produced by γ-aminobutyric acid in DRG neurons, testing the voltage-independent inhibition (mediated by Goα) and voltage-dependent inhibition (mediated by Goβγ). Dialyzing chick GAIP in these cells selectively reduces voltage independent inhibition without affecting voltage dependent inhibition (Tosetti, Turner et al. 2002).
Finally, RGS12 regulates the GABA inhibited N-type calcium channel Cav2.2 in chick DRG. This voltage-independent inhibition is mediated by tyrosine kinase that phosphorylates the α1 subunit of the channel and thereby recruits RGS12. RGS12 binds to the SNARE (soluble Nethylmaleimide-sensitive factor attachment protein receptors)- binding region of α1, altering the kinetics of termination of GABA-mediated inhibition of calcium channels (Schiff, Siderovski et al. 2000) (Richman, Strock et al. 2005).
In summary, RGS proteins are involved in the Ca2+ channels kinetics modulation, exerting high specificity in the regulation of different pathways. This specificity could be due not only to the different kind of RGS but also to splice variants and different concentrations of these proteins.
8. Regulation of RGS: expression levels
8.1 RGS transcription
Since RGS proteins undercome fast and transient changes of gene expression after different stimuli (Ingi, Krumins et al. 1998) (Burchett, Bannon et al. 1999), we can hypothesize that these proteins are involved in early neuronal response to a stimulus. But their regulation is not completely understood. Few evidences suggest than RGS2 and RGS4 expression could be under the control of dopaminergic system (Taymans, Leysen et al. 2003).
Pepperl et al. demonstrated that RGS2 expression in vitro is strongly induced by one hour of treatment with forskolin (Pepperl, Shah-Basu et al. 1998). This observation was later confirmed in vivo by Taymans and colleagues that demonstrated that RGS2 and RGS4 expression level is mediated by D1 and D2 receptor in the rat striatum, with opposite gene expression changes. They showed an increase of RGS2 by D1 receptor stimulation or D2 receptor blockade, and a decrease of RGS2 by D1 receptor blockade or D2 receptor stimulation. Both subtypes of dopamine receptors in the striatum are linked to adenylyl cyclase, but while D1 receptors stimulate AC5 via Gαs, D2 receptors inhibit AC5 via Gαo. According to these signaling cascades, cAMP levels will be increased by a D1 agonist or a D2 antagonist while cAMP will decrease with a D1 antagonist or a D2 agonist (Taymans, Leysen et al. 2003). RGS2 expression seems to follow cAMP levels.
This is explained by the presence of a cAMP responsive element (CRE) in the human RGS2 promoter region (Siderovski, Blum et al. 1990) and by cAMP-dependent activation of the RGS2 promoter in rat and mice (Thirunavukkarasu, Halladay et al. 2002). When cAMP is induced, protein kinases such as PKA become activated and phosphorylate CREB which migrates to the cell nucleus and binds to CRE elements in the promoter region, inducing gene transcription (Taymans, Leysen et al. 2003) .
RGS4 expression is more controversial. It seems to be dependent on the dopaminergic system, but its expression change inversely to the cAMP concentration if compared to
RGS2 regulation. Taymans and colleagues observed an increase of RGS4 induced by D2 receptor stimulation or by D1 receptor blockade. RGS4 is upregulated at low cAMP levels and RGS4 levels are unchanged after in vivo stimulation of adenylyl cyclase by forskolin (Taymans, Leysen et al. 2003).
8.2 RGS translation
After RGS mRNA translation into protein, posttranslational modification such as palmitoylation, myristoylation and phosphorylation can occur modifying RGS cellular localization, membrane attachment, stability and conformation. These posttranslational changes are RGS-type specific and depend on the residues that are present on each RGS protein (De Vries, Zheng et al. 2000).
Palmitoylation is the covalent attachment of fatty acids to cysteine residues of membrane proteins to enhance the hydro-phobicity of proteins, contributing to their subcellular trafficking and membrane association (Chen and Manning 2001).
Myristoylation is an irreversible covalent bound between a myristoyl group of the protein and an N-terminal glycine residue of a nascent polypeptide. This modification is involved in membrane targeting and signal transduction (Chen and Manning 2001).
Phosphorylation is the addition of a phosphate (PO4) group on serine, threonine, and tyrosine residues of proteins. It is a reversible reaction catalysed by enzymes called kinases for phosphorylation and phosphatases for dephosphorylation. Many enzymes and receptors are activated and inactivated by phosphorylation and dephosphorylation (Chen and Manning 2001). Several RGS present potential phosphorylation sites for kinases such as protein kinase C (PKC) in the RGS domain as well as outside their RGS domain.
Fischer and colleagues demonstrated that GAIP/RGS19 is phosphorylated only when it’s associated to the membrane, suggesting that the membrane association of GAIP may be regulated by phosphorylation. Since these phosphorylation sites are located in GAIP’s RGS domain and in its N terminus, which could modify membrane association, phosphorylation could represent an important regulatory event in controlling not only its
localization but also the effect of RGS proteins on G protein signaling (Fischer, Elenko et al. 2000). Phosphorylation is also the base of feedback mechanism to regulate RGS function. It can either enhance or inhibit GAP activity, based on the RGS and kinase involved. PKC, for instance, phosphorylates RGS2 and reduces its GAP activity, with the net effect of augmenting the Gαq/11 signals (Cunningham, Waldo et al. 2001). Rather than modifying GAP activity directly, phosphorylation seems to be involved in the RGS stability directly or by regulating interaction with binding partners. In the case of RGS3 and RGS7, phosphorylation is necessary for interaction with the intracellular scaffold protein 14-3-3 (Benzing, Yaffe et al. 2000).
Finally, specific residues such as aspartate, glutamate, or cysteine at position 2 in the N-terminal of RGS enhance ubiqutination, labelling proteins for proteosomal degradation. For example, RGS4 quickly succumbs to this proteosomal pathway when expressed in cell lines, with a half-life of less than 1 h (Davydov and Varshavsky 2000).
Considering the regulatory functions of RGS on the G protein signaling, any changes in RGS protein expression levels are expected to influence somehow receptor mediated signaling cascades. Pathological changes in the RGS expression levels have been observed in association with different kind of diseases, involving various systems. Here we will review some of the RGS related diseases focusing mainly to the diseases associated to the mesolimbic reward system.
9. Possible role of RGS in human diseases
Many signaling systems relay on GPCR to convert internal and external stimuli to intracellular responses. Moreover, these receptors are found ubiquitously in the body and are virtually involved in every physiological process. It is not surprising that any abnormal change in their regulation at the level of the G protein signaling could lead to pathology. GPCR mediated signaling are involved in all major disease areas such as neurodegenerative, cardiovascular, metabolic, psychiatric, cancer and infectious diseases and comprise the largest family of drug targets (Lundstrom 2006). As described above, RGS proteins serve important roles as modulators and integrators of G protein signaling.
Non-physiological changes in their expression level could be involved in the etiology of different diseases (Hollinger and Hepler 2002). Nowadays, RGS proteins have become new candidates for therapeutic intervention (Zhong and Neubig 2001).
Here we review the main human disease associated to pathological changes in RGS expression levels.
9.1 Addiction
Some drugs of abuse activate GPCRs increasing the dopaminergic signaling in regions of the mesolimbic system.
The mesolimbic reward system is the neural pathways in the brain involved in the pleasure system, or reward circuit, one of the major sources of incentive and behavioural motivation. Moreover, this system is the target of many drugs of abuse (Adinoff 2004).
This system originates in the ventral tegmental area (VTA) which is part of the midbrain, close to the substantia nigra. This area consists mainly of dopamine (DA) and GABA neurons. While GABA neurons exert an inhibitory control on DA neurons, DA neurons are excitatory and their activation leads to the release of dopamine neurotrasmitter in the projecting areas (Cameron, Wessendorf et al. 1997) (Fig.10).
Fig.10
Dopamine
The midbrain DA neurons receive excitatory and inhibitory inputs from different areas of the brain. The excitatory afferences are glutamatergic and arise mainly from the prefrontal cortex (PFC), the subthalamic, laterodorsal- (LDTg) and pedunculopontine (PPTg) tegmental nuclei, and bed nucleus of the stria terminalis (BNST). These inputs regulate the firing activity, in particular burst firing through activation of ionotropic glutamate receptors of the NMDA-type (Grillner and Mercuri 2002) (Aston-Jones and Harris 2004). The inhibitory inputs originate mainly in the Nucleus Accumbens (NAc), pallidum and from intrinsic GABAergic interneurons of the midbrain (Grillner and Mercuri 2002).
The VTA DA neurons projects mainly to the limbic area of the ventral striatum such as the NAc, to PFC and amigdala, where VTA DA neurons release dopamine after their activation (Grillner and Mercuri 2002) (Fig.11).
Fig.11 Schematic representation of the major structures involved in the rewarding system.
The ventral tegmental area, the nucleus accumbens, and the prefrontal cortex.
The VTA is connected to both the nucleus accumbens and the prefrontal cortex via this pathway and it sends information to these structures by releasing dopamine in these areas. From http://www.nida.nih.gov/pubs/teaching/
- Schematic representation
of DA and GABA neuron in the VTA.
DA GABA
neuron neuron
GABA neurons exert a inhibitory control on DA neuron activity (inhibition of dopamine release).
Drugs of abuse such as opioids and stimulants share this common dopaminergic reward pathway increasing the dopaminergic signaling. This increased signaling underlies the rewarding effects of the abused drugs and contributes to drug-seeking behavior. After prolonged exposure to drug of abuse, adaptive changes in neuronal systems lead to tolerance, sensitization, and dependence. As RGS proteins regulate GPCR signaling, drug-induced changes in RGS protein levels over a long period could contribute to these chronic effects (Traynor and Neubig 2005).
9.1.1 Morphine
RGS9, through its modulatory activity on the μ-opioid receptor, represents a potent negative modulator of opiate action. Acute administration of morphine increases the RGS9-2 mRNA expression level in nucleus accumbens, striatum and thalamus but not in the cortex while chronic exposure reduced its expression level (Zachariou, Georgescu et al. 2003) (Lopez-Fando, Rodriguez-Munoz et al. 2005).
The same increase could be observed in RGS4 mRNA in the locus coeruleus (LC), reticulotegmental pontine nucleus and dorsal central gray, while chronic injection of morphine leads to an increase of RGS4 only in the dorsal central gray (Gold, Han et al.
2003) (Bishop, Cullinan et al. 2002). Naltrexone (opioid receptor antagonist)-induced withdrawal cause an increase in RGS2 and RGS4 mRNA with a pick 6h after withdrawal.
Moreover the expression level of RGS4 protein increases after chronic morphine and decreases 6 hours after withdrawal, suggesting a role of RGS4 in the maintenance of the homeostasis in the LC (Gold, Han et al. 2003).
Finally, RGS2 mRNA decreases of about 45% after morphine chronic injection in the VTA (Labouebe, Lomazzi et al. 2007) (See Results).
9.1.2 Cocaine
Acute administration of cocaine causes a reduction in RGS4 mRNA (Bishop, Cullinan et al. 2002), while induction of RGS4 is observed in different areas of the brain after
repeated cocaine exposure (Zhang, Zhang et al. 2005) or in response to stimuli that evoke plasticity (Ingi, Krumins et al. 1998).
Overexpression of RGS9-2 in NAc reduced the locomotor responses to cocaine and to D2 but not to D1 receptor agonist while RGS9 KO showed augmented locomotor and rewarding responses to cocaine. These observations suggested a compensatory adaptation of psychostimulant-induced-RGS9 to diminish drug responsiveness (Rahman, Schwarz et al. 2003).
9.1.3 γ-hydroxybutyrate (GHB)
Chronic injection of GHB causes a reduction of RGS2 mRNA in the VTA leading to a stronger coupling between GABAB receptors and GIRK channels. This observation suggests that if RGS2 is expressed at low levels, GHB at recreationally doses may abrogate the rewarding properties of the drug. Moreover, chronic injections of GHB shift the rewarding properties of GHB in mice during GHB self-administration test (Labouebe, Lomazzi et al. 2007) (See Results).
9.1.4 Amphetamines
The effect of amphetamines on RGS regulation is still controversial. Burchett et al.
demonstrated that a single injection of amphetamine or methamphetamine increases RGS2, RGS3 and RGS5 mRNA in rat striatum by two- to four folds. Prior repeated treatments with amphetamines suppress induction of RGS5 only suggesting an implication of RGS2 and RGS3 in tolerance (Burchett, Bannon et al. 1999). On the other hand, Schwendt and colleagues observed a decrease of RGS4 in rat forebrain in an inverse dose-dependent manner after single amphetamines administration (Schwendt, Gold et al. 2006).
9.2 Heart disease
Alterations in RGS protein levels produce cardiovascular phenotypes. RGS2 KO mice exert a strong hypertensive phenotype and both copies of the gene are necessary for normal cardiovascular function (Riddle, Schwartzman et al. 2005). RGS2-/- mice are hypertensive (Doggrell 2004), exhibit increased vasoconstriction in vitro in response to Gq-coupled agonists and decrease relaxation in response to cGMP (Heximer, Knutsen et al. 2003). Moreover, expression levels of RGS2 and RGS3 are increased in failing hearts (Takeishi, Jalili et al. 1999).
9.3 Pain
The regulation of nociception and opioid responsiveness via µ-opioid receptors is under RGS modulation. Blocking the expression of RGS4, RGS7, RGS9 and RGS12 through oligonucleotide antisense increases the duration of acute morphine antinociception (Garzon, Rodriguez-Diaz et al. 2001). Moreover, RGS4 is over expressed up to 230% in the lumbar spinal cord in a model of neuropathic pain. This increase is associated with the development of hyperalgesia and may be involved in the aberrant response to morphine associated to development of neuropathic pain (Garnier, Zaratin et al. 2003).
9.4 Alzheimer’s disease
Alzheimer’s disease (AD) is an age-related neurodegenerative disease with deficits in multiple neurotransmitter systems. Deficits in M1 muscarinic receptor system signaling are associated to the etiology of AD. Since M1 receptors stimulate non-amyloidogenic processing of the amyloid precursor protein via Gq/11, which is modulated by RGS4, any change in the expression level of these components could lead to the pathology. Muma and colleagues observed a reduction in the total level of RGS4 and Gq/11 expression in human brain autopsy from AD parietal cortex (Muma, Mariyappa et al. 2003). Moreover,
