Inhibition of Retinal Guanylyl Cyclase by the RGS9-1 N-Terminus

doi:10.1006/bbrc.2001.5346

Biochemical and Biophysical Research Communications

Volume 286, Issue 1, 10 August 2001, Pages 12-19

https://doi.org/10.1006/bbrc.2001.5346 Get rights and content

Abstract

Cyclic GMP plays a key role in retinal phototransduction and its photoreceptor concentration is precisely controlled by the cooperative action of cGMP phosphodiesterase (PDE) and retinal guanylyl cyclase (retGC). However, studies of the relationship between these two systems have focused only on a Ca²⁺-mediated, indirect connection. Using a retinal “regulator of G-protein signaling” (RGS9-1) and its fragments, we show that the N-terminus of RGS9-1 inhibits retGC activity. We also indicate that the GGL domain and/or the RGS domain function as an internal suppressor against the N-terminus, suggesting that proteins bound to these domains regulate the inhibitory activity of the N-terminus. Direct interaction of retGC with RGS9-1 and its N-terminus is also proved by immunoprecipitation and an overlay technique. Since RGS9-1 also controls the lifetime of transducin-activated PDE through regulating GTPase activity of transducin, this study strongly suggests that RGS9-1 mediates the direct interaction between PDE and retGC systems, and that this ingenious mechanism plays an important role in tuning of cGMP concentration in photoreceptors.

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Citation Excerpt :
It has also been suggested that the Gβ5•RGS complex might bind to inactive Gα-subunits and then joining them to a larger GPCR complex that positions the RGS protein in the area where its GAP activity is required (Siderovski and Willard, 2005; Sondek and Siderovski, 2001). The GGL domain has been also associated with other functions namely: RGS6 GGL domain interaction with SCG10 neuronal protein to enhance differentiation (Liu et al., 2002), and to inhibit a transcriptional repressor named DMAP1 (Liu and Fisher, 2004), to suppress the RGS9-1 inhibitory ability of guanylyl cyclase activity (Yu et al., 2001). The R7 family members of RGS proteins also interact with the membrane anchoring proteins R7BP and R9AP.
Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on.
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The regulators of G protein signaling (RGS) are a family of cellular proteins that play an essential regulatory role in G protein-mediated signal transduction. There are multiple RGS subfamilies consisting of over 20 different RGS proteins. They are basically the guanosine triphosphatase (GTPase)-accelerating proteins that specifically interact with G protein α subunits. RGS proteins display remarkable selectivity and specificity in their regulation of receptors, ion channels, and other G protein-mediated physiological events. The molecular and cellular mechanisms underlying such selectivity are complex and cooperate at many different levels. Recent research data have provided strong evidence that the spatiotemporal-specific expression of RGS proteins and their target components, as well as the specific protein–protein recognition and interaction through their characteristic structural domains and functional motifs, are determinants for RGS selectivity and specificity. Other molecular mechanisms, such as alternative splicing and scaffold proteins, also significantly contribute to RGS selectivity. To pursue a thorough understanding of the mechanisms of RGS selective regulation will be of great significance for the advancement of our knowledge of molecular and cellular signal transduction.
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Regulators of G-protein signalling (RGS) proteins are a large and diverse family initially identified as GTPase activating proteins (GAPs) of heterotrimeric G-protein Gα-subunits. At least some can also influence Gα activity through either effector antagonism or by acting as guanine nucleotide dissociation inhibitors (GDIs). As our understanding of RGS protein structure and function has developed, so has the realisation that they play roles beyond G-protein regulation. Such diversity of function is enabled by the variety of RGS protein structure and their ability to interact with other cellular molecules including phospholipids, receptors, effectors and scaffolds. The activity, sub-cellular distribution and expression levels of RGS proteins are dynamically regulated, providing a layer of complexity that has yet to be fully elucidated.
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The intracellular regulator of G protein signalling (RGS) proteins were first identified as GTPase activating proteins (GAPs) for heterotrimeric G proteins, however, it was later found that they can also regulate G protein–effector interactions in other ways that are still not well understood. There is increasing evidence that some of the effects of RGS proteins occur due to their ability to interact with multiprotein signalling complexes. In this review, we will discuss recent evidence that supports the idea that RGS proteins can bind to proteins other than Gα, such as G protein coupled receptors (GPCRs, e.g. muscarinic, dopaminergic, adrenergic, angiotensin, interleukin and opioid receptors) and effectors (e.g. adenylyl cyclase, GIRK channels, PDEγ, PLC-β and Ca²⁺ channels). Furthermore, we will investigate novel RGS binding partners (e.g. GIPC, spinophilin, 14-3-3) that underlie the formation of signalling scaffolds or govern RGS protein availability and/or activity.
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Regulator of G-protein signalling (RGS) proteins form a superfamily of at least 25 proteins, which are highly diverse in structure, expression patterns, and function. They share a 120 amino acid homology domain (RGS domain), which exhibits GTPase accelerating activity for α-subunits of heterotrimeric G-proteins, and thus, are negative regulators of G-protein-mediated signalling. Based on the organisation of the Rgs genes, structural similarities, and differences in functions, they can be divided into at least six subfamilies of RGS proteins and three more families of RGS-like proteins. Many of these proteins regulate signalling processes within cells, not only via interaction with G-protein α-subunits, but are G-protein-regulated effectors, Gβγ scavenger, or scaffolding proteins in signal transduction complexes as well. The expression of at least 16 different RGS proteins in the mammalian or human myocardium have been described. A subgroup of at least eight was detected in a single atrial myocyte. The exact functions of these proteins remain mostly elusive, but RGS proteins such as RGS4 are involved in the regulation of G_i-protein βγ-subunit-gated K⁺ channels. An up-regulation of RGS4 expression has been consistently found in human heart failure and some animal models. Evidence is increasing that the enhanced RGS4 expression counter-regulates the G_q/11-induced signalling caused by hypertrophic stimuli. In the vascular system, RGS5 seems to be an important signalling regulator. It is expressed in vascular endothelial cells, but not in cultured smooth muscle cells. Its down-regulation, both in a model of capillary morphogenesis and in an animal model of stroke, render it a candidate gene, which may be involved in the regulation of capillary growth, angiogenesis, and in the pathophysiology of stroke.
Signal-dependent translocation of transducin, RGS9-1-Gβ5L complex, and arrestin to detergent-resistant membrane rafts in photoreceptors
2002, Current Biology
Citation Excerpt :
Since PDEγ has affinity for the transition state of transducin, and a portion of PDE is associated with raft, it could potentially act as an anchor to recruit the RGS9-1 complex [8, 10]. We can also speculate that RGS9-1 is anchored to the raft via another molecule, for example, guanylate cyclase, which is permanently raft associated (Figure 1B) and is reported to bind RGS9-1 [11, 12]. Another possibility is that RGS9-1 phosphorylation [13, 14] plays a role in its association with rafts.
Many lines of evidence show that membranes contain microdomains, “lipid rafts”, that are different from the rest of the membrane in specific lipid and protein composition [1]. In several biological systems, they were shown to be necessary for trafficking and signal transduction. Here, we investigate if lipid rafts have a role in the regulation of the G protein-mediated pathway underlying vertebrate phototransduction. Photoreceptor membranes contain detergent-resistant membrane (DRM) rafts. Rhodopsin and cGMP phosphodiesterase are found in raft and nonraft portions of the membrane; guanylate cyclase is found exclusively in the raft. Distribution of these proteins does not change in the light or dark. In contrast, the G protein transducin, the RGS9-1-Gβ5L complex, and the p44 isoform of arrestin undergo dramatic translocation to the raft upon illumination. Phosphorylation of RGS9-1 occurs exclusively in the raft. GTPγS or pertussis toxin prevent the light-mediated translocation of transducin and RGS9-1, whereas AlF⁻₄ causes both proteins to move to the raft in the dark. This shows that the Gαt-RGS9-1-Gβ5L complex has the highest affinity to rafts in the transition state of the GTPase. GTPγS binds to transducin at a significantly slower rate in the raft, indicating that this translocation results in a reduced rhodopsin-transducin coupling. Thus, an external signal can rearrange components of a G protein pathway in specific domains of the cell membrane, changing its signaling properties. These findings could reveal a novel mechanism utilized by the cells for regulation of G protein-mediated signal transduction.

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^☆: This work was supported by a National Institutes of Health Grant EY09631 and an unrestricted grant from Research to Prevent Blindness.

¹: To whom correspondence should be addressed at Kresge Eye Institute, Wayne State University, School of Medicine, 4717 St. Antoine Street, Detroit, MI 48201. Fax: 313-577-0238. E-mail: [email protected].

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Regular ArticleInhibition of Retinal Guanylyl Cyclase by the RGS9-1 N-Terminus☆

Abstract

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Regular Article
Inhibition of Retinal Guanylyl Cyclase by the RGS9-1 N-Terminus☆