Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi


Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_259


Introduction and Historical Background

Regulator of G protein signaling (RGS) proteins constitute a diverse family with more than 30 members that contain the hallmark RGS domain. Most members serve as negative regulators of G protein signaling by catalyzing the GTP hydrolysis on Gα subunits leading to their inactivation (Ross and Wilkie 2000; Hollinger and Hepler 2002). Based on their structural organization and sequence homology RGS proteins are divided into five to six families (Ross and Wilkie 2000; Hollinger and Hepler 2002). The R7 RGS family (R7 RGS) contains multidomain proteins conserved from C. elegans to humans that, in mammals are represented by four members: RGS6, RGS7, RGS9, and RGS11. R7 RGS proteins play important roles in the nervous system by controlling neurotransmitter action at rhodopsin, μ-opioid, D2 dopamine, and GABA(B) receptors (Anderson et al. 2009a).

The unique feature of this group is that they form obligatory complexes with Gβ5, an atypical member of the G protein beta subunit family (Sondek and Siderovski 2001; Slepak 2009). The stability of all R7 RGS proteins crucially depends on this interaction and knockout of Gβ5 in mice leads to severe downregulation in the levels of all four R7 RGS proteins (Chen et al. 2003). Localization of R7 RGS-Gβ5 complexes in discrete membrane compartments in native cells in parallel with cytoplasmic distribution in heterologous expression systems have prompted speculations that their membrane anchoring is mediated by unidentified proteins (Hu and Wensel 2002; Lishko et al. 2002). This led to searches for additional binding partners.

For R7 RGS proteins, these studies were very productive and resulted in the identification of two homologous binding partners: RGS9 Anchor Protein (R9AP) and R7 Binding Protein (R7BP), novel proteins that now constitute a two-member family. First, proteomics search for RGS9 binding partners in the retina identified a transmembrane protein R9AP (Hu and Wensel 2002). Three years later, a homologous R7BP protein was found as a binding partner of RGS9 in the brain using similar approach (Martemyanov et al. 2005). R7BP was also independently discovered as a universal anchor for all R7 RGS proteins by Ken Blumer’s group by in silico BLAST searches (Drenan et al. 2005).

Structural Organization

Both R7BP and R9AP are small membrane proteins that show distant homology to SNARE proteins involved in the regulation of exocytosis (Keresztes et al. 2003; Martemyanov et al. 2003). N-termini of R9AP and R7BP are predicted to contain 4 alpha helices organized in the bundle with the fourth helix containing prominent heptad repeats, a feature underlying coiled coil interactions (Fig. 1). This helical bundle region mediates binding to the R7 RGS proteins (Anderson et al. 2009a; Jayaraman et al. 2009). From the side of the RGS proteins, N-terminal DEP (Disheveled, Egl-10, Pleckstrin) and DHEX (DEP Helical Extension) domains were found to be essential for the association (Anderson et al. 2009a; Jayaraman et al. 2009) although no high-resolution information of the determinants involved in binding have been reported. The C-termini of R7BP and R9AP contain membrane anchoring elements. In R9AP this is presented by transmembrane region, while R7BP is anchored through palmitoylation of two conserved cysteine residues which are additionally aided by a polybasic stretch with a sequence resembling membrane attachment motif of Ras proteins (Drenan et al. 2005; Song et al. 2006). In summary, while differing in amino acid composition, both R9AP and R7BP contain two domains: the N-terminal RGS binding region and C-terminal membrane localization domain.
R7BP/R9AP, Fig. 1

Schematic structure of R7BP and R9AP proteins. Membrane attachment of R7BP is mediated by palmitoylated cysteines (wavy lines) acting in conjunction with polybasic region (PBR). R9AP is anchored to the membrane by transmembrane segment (TM). Blue cylinders designate predicted alpha helical regions. CC label designates heptad repeats-containing region that is predicted to engage in coiled-coil interactions

Distribution, Subcellular Localization, and Interactions with R7 RGS Proteins

In mammals, expression of both R9AP and R7BP proteins appears to be confined to the neuronal tissues (Martemyanov et al. 2005; Grabowska et al. 2008). However, only limited set of non-neuronal tissues have been investigated and it remains possible that the proteins could be expressed more broadly, as for example, ample amounts of R9AP mRNA are found across various tissues in birds (Keresztes et al. 2003). While R7BP is expressed broadly in all regions of central and peripheral nervous system, R9AP is more restricted and appears to be reliably found only in the retina, where it is found in three cell types: rod and cone photoreceptors (Hu and Wensel 2002) and ON-bipolar cells (Cao et al. 2009; Jeffrey et al. 2010).

At the subcellular level, R9AP is targeted to the disc membranes of the outer segments, a ciliated compartment of the photoreceptors and dendritic tips of the ON- bipolar neurons (Hu and Wensel 2002; Cao et al. 2009; Jeffrey et al. 2010). Likewise, R7BP was also found to be localized predominantly in the membrane compartments. Its significant fraction is found in the postsynaptic density and extrasynaptically at the plasma membrane of the spines and dendrites (Anderson et al. 2007b; Grabowska et al. 2008). Although to a lesser extent, some R7BP immunoreactivity is also present pre-synaptically in axons (Grabowska et al. 2008). Subcellular tartgeting of R7BP and R9AP to their membrane compartments requires the membrane attachment sequence at the C-terminus (Drenan et al. 2006; Song et al. 2006).

While plasma membrane compartment is the sole localization site of R9AP, R7BP has been reported to have an alternative destination – nucleus. It possesses two active nuclear localization sequences that are masked by palmitoylation (Drenan et al. 2005; Song et al. 2006). When palmitoylation is abolished, R7BP undergoes translocation to the nucleus, a phenomenon most readily demonstrated in cultured cells (Drenan et al. 2005; Song et al. 2006). However, the fraction of R7BP in the nucleus is very small and no studies have yet reported translocation in vivo under physiological conditions.

R7BP and R9AP anchors show differential selectivity in their association with individual R7 RGS members. This is determined by both their interaction specificity and co-expression patterns. At the biochemical level, R9AP can bind only to RGS9 and homologous RGS11, but not to RGS6 or RGS7 (Martemyanov et al. 2005). In contrast, R7BP associates with all R7 RGS proteins (Drenan et al. 2005; Martemyanov et al. 2005). However, the distribution of R7 RGS protein subtypes does not always overlap with that of R7BP and R9AP making some combinations physiologically irrelevant. For example, although R9AP can bind to RGS9-2, it is not expressed in the striatum, where RGS9-2 is instead found in complex with R7BP. Similarly, RGS11 can bind to R7BP, but because it is present exclusively in bipolar cells where R9AP is more abundant, most of it is found in complex with R9AP (Cao et al. 2008). See Fig. 2 for details on physiologically relevant configurations of R7BP and R9AP complexes with R7 RGS proteins.
R7BP/R9AP, Fig. 2

Physiologically relevant complexes of R7BP and R9AP in vivo

Regulation of the RGS Protein Localization and Activity

Studies with transfected cells indicate that R7 RGS-Gβ5 complexes are predominantly cytoplasmic (Drenan et al. 2005; Song et al. 2006). In contrast, co-transfection with R7BP (or R9AP for RGS9-1) targets R7 RGS proteins to the plasma membrane (Hu and Wensel 2002; Drenan et al. 2005; Song et al. 2006). A similar situation is observed in striatal neurons in vivo for RGS9-2 that becomes mis-localized from post-synaptic densities and plasma membrane compartments upon elimination of R7BP (Anderson et al. 2007b). In striatal neurons, R7BP is also involved in targeting RGS7 to the post-synaptic density (Anderson et al. 2009b). Likewise, the role of R9AP in localization of RGS9-1 to the disc membranes of the photoreceptor outer segments is also well established (Hu and Wensel 2002). Nevertheless, mechanisms governing localization of R7 RGS proteins appear to be complex and anchor-independent targeting has been observed for both RGS7 and RGS11 in the bipolar cells of the retina (Cao et al. 2008, 2009).

The nucleus has been repeatedly reported to be an alternative destination for relatively minor fraction of several R7 RGS proteins (Burchett 2003). Consistent with its nuclear shuttling, R7BP is capable of targeting R7 RGS to the nucleus of the transfected cells upon de-palmitoylation (Drenan et al. 2005). Furthermore, knockout of R7BP abolishes nuclear localization of a significant fraction of RGS7 in the central nervous system neurons (Panicker et al. 2010). However, the functional significance of plasma membrane – nuclear shuttling of R7 RGS proteins or their functional role at this location is currently unknown.

In addition to localization, association with R9AP and R7BP influences the efficiency of R7 RGS to catalyze G protein GTPase (GAP) activity. For instance, R9AP has been shown to potentiate the ability of RGS9-1 and RGS11 to stimulate GTPase of Gat and Gao, respectively (Hu et al. 2003; Masuho et al. 2010). The most straightforward explanation for the stimulatory effects is facilitation of the R7 RGS complex compartmentalization with membrane bound G proteins and receptors. The restriction of the diffusion of the complex from the three-dimensional cytoplasm to the two-dimensional plane of the plasma membrane is expected to speed up Gα-GTP encounter. However, the mechanism is likely to be more complex, as at least R9AP action was shown to provide an allosteric modulation of the RGS9 and RGS11 complexes (Baker et al. 2006; Masuho et al. 2010).

Effects on the Proteolytic Stability of the R7 RGS Complexes

Perhaps the most pronounced effects of membrane anchors are on regulation of post-translational stability of R7 RGS proteins. These effects are observed only with two RGS proteins: RGS9 and RGS11. Studies with genetic knockouts indicate that elimination of R9AP severely compromises proteolytic stability of RGS9 (Keresztes et al. 2004) and RGS11 (Cao et al. 2008) in the retina. Likewise, knockout of R7BP leads to destabilization of RGS9-2 in the brain (Anderson et al. 2007a). This explains why loss-of-function mutations in R9AP produce the same phenotype as RGS9-1 mutations – slow adaptation to both light and dark conditions and difficulty in tracking moving objects (Nishiguchi et al. 2004). Similarly, knockout of R7BP causes motor co-ordination deficits characteristic of severe reduction in levels of RGS9-2 (Anderson et al. 2010). Loss of R7BP has been shown to facilitate recruitment of the destabilizing chaperone Hsc70 (Posokhova et al. 2010) to RGS9-2 and trigger its proteolysis by cellular cysteine proteases (Anderson et al. 2007b). Association of RGS9-Gβ5 with R7BP is controlled dynamically and is sensitive to changes in oxygenation and neuronal excitability (Anderson et al. 2009b). Because the abundance of R7 RGS proteins controls the extent of the G protein signaling and has direct behavioral implications, regulation of R7 RGS degradation and coupling to R7BP can be viewed as a plasticity mechanism.

Summary and Conclusions

In summary, R7BP and R9AP proteins play very important roles in controlling stability, localization and functional activity of R7 RGS proteins and as a result critically contribute to the regulation of G protein signaling (Fig. 3). There are currently a number of outstanding questions pertaining to the role of R7BP and R9AP in regulation of (1) G protein signaling selectivity, (2) RGS catalytic activity and (3) physiological processes in the nucleus. Finding answers to these questions would help gain a better understanding of G protein signaling.
R7BP/R9AP, Fig. 3

Role of membrane anchors R7BP and R9AP in G protein signaling regulation


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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of NeuroscienceThe Scripps Research InstituteJupiterUSA
  2. 2.Department of PharmacologyUniversity of MinnesotaMinneapolisUSA