Rin (Ras-Like Protein in Neurons)
Ras superfamily small GTP (guanosine triphosphate)-binding proteins function as molecular switches, responding to extra- and intracellular stimuli to control the activity of diverse signaling cascades. To date, over 150 different small GTPases have been identified and are classified into six distinct subfamilies – Ras (Rat sarcoma), Rho (Ras homolog gene family), Rab (Ras-related GTP- binding protein), ARF (ADP- ribosylation factor), Ran (Ras-related nuclear protein), and RGK (Rad/Gem/Kir family) – based upon both sequence homology and the regulation of common cellular functions (Colicelli 2004). Rin (Ras-like protein in neurons), along with Rit (Ras-like protein in many tissues) and Drosophila Ric (Ras-related protein which interacted with calmodulin), comprise the Rit subfamily of Ras-related small GTPases (Lee et al. 1996). Rin is expressed exclusively within neurons, including RGCs (retinal ganglion cells) where its expression is regulated by POU4 transcription factors, and has been characterized as regulating neuronal differentiation by controlling distinct signaling cascades (Lee et al. 1996; Zhang et al. 2013; Shi et al. 2005, 2008; Spencer et al. 2002). Rin contributes to NGF (nerve growth factor)-mediated neurite outgrowth via ERK (extracellular signal regulated MAP kinase) and p38 MAP kinase pathways (Shi et al. 2005), whereas in PACAP38 (pituitary adenylate cyclase-activating polypeptide 38)-mediated neuronal differentiation, Rin controls the cAMP (cyclic adenosine monophosphate)/PKA (protein kinase A) signaling cascade (Shi et al. 2008). In addition, Rin is involved in Brn-3a and Plexin signaling and in trafficking of the DAT (dopamine transporter) (Calissano and Latchman 2003; Hartwig et al. 2005; Navaroli et al. 2011). Rin also associates with the polarity protein PAR6 and has been found to display modest transforming ability (Hoshino et al. 2005). Rin expression may be essential for appropriate nervous system function as genetic alterations in and around the gene are associated with SCZ (schizophrenia), PD (Parkinson’s disease), autism, bipolar disorder, and other diseases (Navaroli et al. 2011; Liao et al. 2012; Pankratz et al. 2012; Emamalizadeh et al. 2016).
Biochemical Characterization of Rin GTPase
Recombinant Rin has been shown to specifically bind guanine nucleotides (guanosine triphosphate or guanosine diphosphate, GTP/GDP) in the presence of Mg2+ and to exhibit low intrinsic GTPase activity (Shao et al. 1999). Surprisingly, the guanine nucleotide dissociation rates for purified Rin protein are significantly different when compared with the majority of Ras-related GTPases. Rin displays higher koff values for GTP than GDP (Shao et al. 1999). These GTP dissociation rates are five-to-ten-fold faster than most Ras-like GTPases. Since the cellular concentration of GTP is much higher than that of GDP, these biochemical studies suggest that a relatively high percentage of Rin may remain in the GTP-bound state under basal conditions. Despite these unique biochemical properties, the available data indicate that Rin functions as a nucleotide-dependent molecular switch, and, as predicted, only a very low in vivo level of GTP-bound Rin is found in unstimulated cells (Shi et al. 2008; Spencer et al. 2002). Therefore, these in vitro studies do not appear to accurately represent Rin activity in the normal cellular environment. It is possible that the presence of cellular regulatory proteins contribute to these differences.
Rin Cellular Distribution and Trafficking
For the majority of Ras-related GTPases, association with specific cellular membranes is essential for their biological activities (Colicelli 2004). Conserved C-terminal cysteine-rich motifs are used to direct covalent modification by isoprenoid lipids (prenylation). Prenylation is the initial step in the attachment of these proteins to the cytoplasmic leaflets of a variety of cellular organelles. However, specific membrane localization often requires additional targeting signals, provided either by a cluster of basic amino acids or the palmitoylation of internal cysteine residues (Heo et al. 2006). For example, adjacent to the CAAX prenylation motif (“C” is Cysteine, “A” is an aliphatic amino acid, and “X” is variable), H-Ras and N-Ras have a cysteine residue for palmitoylation, directing the protein to the plasma membrane via the Golgi apparatus. In contrast, K-Ras4A has a lysine-rich polybasic motif, which leads the protein to the plasma membrane more rapidly through a Golgi-independent route (Colicelli 2004).
Surprisingly, analysis of an over-expressed GFP ( green fluorescent protein)-tagged protein indicates that Rin primarily localizes to the nucleus, although a detectable amount of GFP protein is also found at the plasma membrane in these studies (Heo et al. 2006). Investigation of the Rin C-terminus has provided key insights into this unique subcellular distribution. Distinct from the majority of G-proteins, the Rit and RGK family GTPases lack a CAAX motif and instead contain a polybasic cluster at the C-terminus (Heo et al. 2006). Interestingly, sequence analysis of the Rin polybasic motif reveals a canonical NLS (nuclear localization signal) (K-K/R-x-K/R). Additionally, Heo et al. have demonstrated the importance of the polybasic C-terminal region in targeting the protein to the plasma membrane via interactions with PI4,5P2 (phosphatidylinositol 4,5-bisphosphate) and PI3,4,5P3 (phosphatidylinositol 3,4,5-trisphosphate) lipids (Heo et al. 2006). Navaroli et al. also showed that Rin co-localized with lipid rafts, at least in the presence of the DAT and that Rin is internalized in vesicles with the DAT PKC (protein kinase C)-dependently. Thus, Rin appears capable of shuttling between the nucleus and plasma membrane in a PIP-lipid dependent fashion, allowing for a novel cellular trafficking pattern, which may contribute to the physiological function of Rin.
The C-terminus of Rin has also been reported to direct Ca2+-dependent interactions with CaM (Lee et al. 1996). Although CaM binding has been shown to be important for neurite outgrowth mediated by Rin (Hoshino and Nakamura 2003), the exact signaling mechanism, as well as the physiological significance of CaM binding, still needs to be elucidated. The Rin homolog Drosophila Ric was originally identified as a CaM binding protein, and genetic studies suggest that CaM association contributes to the regulation of Ric signaling (Harrison et al. 2005).
Regulation of Rin Activity
Like most Ras GTPases, Rin is activated by exchanging GDP with GTP in the nucleotide binding pocket, which results in conformational changes that expose the effector domain and promote the recruitment of proteins responsible for signal transduction cascade activation. This classic GTPase cycle is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which are responsible for the release of bound GDP and the induction of intrinsic GTPase activity, respectively. To date no Rin-specific GEFs or GAPs have been identified. However, Rin was found to associate with mSOS, and SynGAP and GAP1 are capable of decreasing cellular GTP Rin levels.
Several Rin point mutations have been reported to block intrinsic GTPase activity or GTP/GDP exchange, leading to a protein locked in the active or inactive state, respectively. Mutation of Gln78 to Leu (RinQ78L), equivalent to the oncogenic RasQ61L mutation, causes complete inhibition of GTP hydrolysis, resulting in a constitutively active Rin mutant. Meanwhile, the RinS34N mutation, equivalent to dominant-negative RasS17N, is predominantly GDP-bound (Shao et al. 1999) and disrupts signal transduction when over-expressed (Shi et al. 2005). Both of these mutations have proved useful in the analysis of Rin GTPase function.
The G2 domain of Ras GTPases is known as the “effector domain” and serves as the primary region of interaction responsible for directing downstream effector protein binding following G-protein activation. Rin shares a conserved effector domain (HDPTIEDAY) with Rit, and this domain is evolutionarily conserved within the subfamily, as there is only a single amino acid substitution in the Drosophila Ric effector loop (HDPTIEDSY). In addition, the Rin effector domain shares seven out of nine residues with the Ras effector loop (YDPTIEDSY) (Shao et al. 1999). This high rate of conservation has led to the suggestion that Rin is likely to control distinct, but perhaps partially overlapping, downstream signaling pathways compared to Ras.
A variety of proteins have been implicated as candidate Rin effectors using yeast two-hybrid screens (Shao et al. 1999). These studies first identified the RBD (Ras-binding domain) from the Raf kinases as binding partners for constitutively active RinQ78L. Recent in vivo co-immunoprecipitation analyses in PC6 (pheochromocytoma 6) cells support these initial findings by demonstrating that active Rin preferentially associates with B-Raf, suggesting that B-Raf functions as a valid downstream target of Rin (Shi et al. 2005). However, whether B-Raf directly binds Rin in vivo, thus acting as a true effector, has yet to be determined. Rin also interacts with the RID (Ras interacting domains) of two Ral exchange factors, RalGDS and RLF, in yeast two-hybrid screens, suggesting that Rin may regulate Ral GTPase signaling (Shao et al. 1999), although this hypothesis has not been formally tested. While Rin appears capable of associating with a number of known Ras effectors, in agreement with Rin’s distinct G2 domain sequence, not all known Ras effectors demonstrate Rin binding. For example, active Rin does not bind to RIN1 or p110, the catalytic subunit of PI3K (Shao et al. 1999). More interestingly, Rin directly associates with the PDZ domain of PAR6, a cell polarity-regulating molecule, in a GTP-dependent fashion. This interaction requires an intact Rin G2 effector domain, suggesting that PAR6 may represent the first authentic Rin effector that does not also associate with Ras (Hoshino et al. 2005).
The identification of effectors for Ras-like small GTPases is not only important for understanding their physiological functions, but the minimum GTPase binding domain of select effector proteins have been adapted for use in pull-down activity assays. In the case of Rin, both the Raf-RBD and the RalGDS-RID have been used to examine in vivo Rin activity (Spencer et al. 2002).
Functions of Rin GTPase
Rin can be activated by a variety of extracellular stimuli, including NGF, EGF (epidermal growth factor), ionomycin, and tocopherol acetate (Spencer et al. 2002), suggesting that Rin controls the activation of a variety of signaling pathways and cellular functions. Indeed, a significant amount of work has been performed to understand the physiological role of Rin GTPase-mediated signaling.
The exclusive expression of Rin in neurons indicates a critical role for Rin in the central nervous system. Indeed, over-expression of a constitutively active Rin mutant alone is capable of inducing neurite outgrowth in PC6 cells (Hoshino and Nakamura 2003; Shi et al. 2005), suggesting a physiological role for Rin signaling in neuronal differentiation and development. shRNA-mediated knockdown of Rin dramatically reduces NGF-mediated neurite outgrowth in PC6 cells, suggesting that Rin is a pivotal component of NGF signaling by regulating both ERK and p38 MAPK pathways (Shi et al. 2005). Further detailed mechanistic studies have revealed that Rin activates ERK through B-Raf. Rin selectively regulates the p38α MAPK isoform in vitro; however, the mechanism of Rin-mediated p38 activation has yet to be determined (Shi et al. 2005). The identification of a Rin-PAR6-Rac/Cdc42 complex suggests a putative pathway (Hoshino et al. 2005), since Rac and Cdc42 are known to serve as upstream regulators of p38 MAPK signaling.
PACAP38 potently induces neuronal differentiation through the activation of PACR1, a heterotrimeric GPCR (G-protein-coupled receptor). Rin is activated downstream of PACAP38 and appears to play a critical role in PACAP38-mediated neuronal differentiation. Typically, PACAP38-mediated PACR1 activation results in stimulation of a Gαs-AC (adenylate cyclase)-cAMP signaling cascade. However, PACAP38-dependent Rin activation is mediated by Src downstream of Gαs/i and functions upstream of the cAMP/PKA signaling cascade, which in turn regulates HSP27 (heat shock protein 27) phosphorylation (Shi et al. 2008). HSP27 serves as a chaperone to facilitate correct protein folding, has been reported to regulate cytoskeleton stability, and has known roles in neuronal morphology and survival signaling. Indeed, RNAi-mediated silencing of HSP27 is sufficient to block PACAP38- and RinQ78L-induced neurite outgrowth (Shi et al. 2008), further supporting the notion that HSP27 serves as a critical downstream target of Rin in neuronal differentiation.
Rin has also been found to associate with the intracellular domain of Plexin B3, a member of the Plexin family of semaphorin receptors (Hartwig et al. 2005). Semaphorins are secreted or membrane-bound proteins that provide critical guidance signals to redirect or inhibit axonal growth. Given the ability of Rin to promote neurite outgrowth, an intriguing possibility is that Rin signaling contributes to Plexin B3-mediated axonal extension/retraction. Indeed, Plexin B1 has been shown to function as a GAP, down-regulating R-Ras GTPase activity to induce axonal retraction. Despite the report of the direct interaction of Rin with Plexin B3, it remains to be determined whether Plexin B3 functions as a RinGAP.
Calissano et al. used a yeast two-hybrid screen to identify a putative interaction between Rin and the N-terminus of Brn-3a, a transcription factor widely expressed in the peripheral nervous system. Surprisingly, Brn-3a preferentially associated with GDP-bound Rin (Calissano and Latchman 2003), and only GDP-bound Rin was shown to induce Brn-3a-mediated gene transcription. However, unstimulated neurons contain high basal levels of GDP-Rin, and additional studies are needed to determine whether transient in vivo fluctuations in GDP-Rin levels contribute to Brn-3a-mediated neuronal gene transcription.
More recently, Rin has been shown to directly interact with the C-terminal endocytic sequence of the DAT (Navaroli et al. 2011). The DAT serves as a primary mechanism for terminating DA (dopamine) signaling by removing excess DA from synapses. PKC activation promotes the Rin-DAT association, which appears to be essential for appropriate PKC-mediated DAT internalization and downregulation. Appropriate DA signaling is critical in the regulation of movement and cognition, and its dysfunction can lead to a number of human disorders, including PD. Rin is reported to be enriched in dopaminergic neurons (Navaroli et al. 2011), and genome-wide association studies have identified the Rin gene as associated with PD susceptibility (Emamalizadeh et al. 2016; Pankratz et al. 2012). Similarly, Rin expression was significantly reduced in the substantia nigra of patients with PD (Pankratz et al. 2012; Bossers et al. 2009). Whether this link to PD is due to Rin’s ability to regulate DAT trafficking, and thus DA levels, will require additional studies.
Along these same lines, the Rin gene has been implicated in the pathogenesis of other neurological disorders. One report describes an enrichment of deletions near the Rin gene in patients with SCZ, while another study identified a family with SCZ whose members have a duplication in 18q12.3 that had no known gene within the duplicated region but disrupted the Rin gene (Navaroli et al. 2011; Liao et al. 2012). Loss of Rin was also linked to language delay, mental retardation, and behavioral abnormalities, such as hyperactivity and autism (Navaroli et al. 2011). Two Rin SNPs (single-nucleotide polymorphisms) have been shown to differentially associate with a variety of neurological disorders, including PD, SCZ, essential tremor, autism, and bipolar disorder (Emamalizadeh et al. 2016). Given the importance of calcium signaling and synaptic development to appropriate wiring of the brain, and Rin’s contributions to neuronal differentiation signaling and potential regulation by calcium/CaM, it seems logical that altered Rin function could contribute to nervous system dysfunction, but additional studies are necessary to determine the biological effects of Rin signaling in the brain.
To our knowledge, Rin is the only Ras subfamily GTPase to be expressed exclusively in neurons, including RGCs. In keeping with this unique expression pattern, in vitro studies indicate that Rin may be regulated by POU4 transcription factors and serves as a critical mediator of pheochromocytoma cell neurite outgrowth, acting to couple diverse extracellular stimuli (e.g., NGF, PACAP38) to the activation of ERK and p38 MAPK pathways, cAMP/PKA cascade signaling, and the PAR6-Rac/Cdc42 polarity pathway. In addition, preliminary reports suggest that Rin contributes to both Plexin- and Brn-3a-mediated neuronal differentiation and DAT trafficking. Alteration or loss of Rin function is associated with neurological disorders as varied as PD, SCZ, and autism. Despite this progress, much remains to be elucidated concerning the biological effects of Rin signaling and the molecular mechanisms that govern these diverse actions. Our current understanding of Rin function comes from over-expression and RNAi silencing studies in mammalian cell lines, and future studies must investigate the neuronal effects of Rin ablation making use of genetically engineered Rin null animals. A deeper understanding of in vivo Rin regulation, together with the identification of additional Rin effector proteins, will be critical to define the physiological function of this novel neuronal regulatory protein.
This work was supported by Public Health Service grant NS045103 from the National Institute of Neurological Disorders and Stroke and 14-1 from the Kentucky Spinal Cord and Head Injury Research Trust.
- Navaroli DM, Stevens ZH, Uzelac Z, Gabriel L, King MJ, Lifshitz LM, Sitte HH, Melikian HE. The plasma membrane-associated GTPase Rin interacts with the dopamine transporter and is required for protein kinase C-regulated dopamine transporter trafficking. J Neurosci. 2011;31(39):13758–70.PubMedPubMedCentralCrossRefGoogle Scholar