R4 RGS proteins: regulation of G-protein signaling and beyond

The regulators of G-protein signaling (RGS) proteins were initially characterized as inhibitors of signal transduction cascades initiated by G-protein-coupled receptors (GPCR) because of their ability to increase the intrinsic GTPase activity of heterotrimeric G proteins. This GTPase accelerating protein (GAP) activity enhances G protein deactivation and promotes desensitization. However, in addition to this signature trait, emerging data have revealed an expanding network of proteins, lipids, and ions that interact with RGS proteins and confer additional regulatory functions. This review highlights recent advances in our understanding of the physiological functions of one subfamily of RGS proteins with a high degree of homology (B/R4) gleaned from recent studies of knockout mice or cells with reduced RGS expression. We also discuss some of the newly appreciated interactions of RGS proteins with cellular factors that suggest RGS control of several components of G-protein-mediated pathways, as well as a diverse array of non-GPCR-mediated biological responses.     

Regulators of G protein signaling: role in hematopoiesis,megakaryopoiesis and platelet function

Summary. Regulators of G protein signaling (RGS) are intracellular signaling regulators that bind activated G protein α subunits (Gα) and increase their intrinsic GTPase activity via their common RGS homology domain. In addition to their GTPase accelerating activity (GAP), RGS proteins also contain other domains that regulate their receptor selectivity, their interaction with other proteins such as adenylyl cyclase or their subcellular localization via interaction with scaffold proteins such as tubulin, 14‐3‐3 or spinophilin. There are at least 37 different RGS family members in humans and numerous physiological functions have been assigned to these proteins, which have rather a tissue‐specific expression pattern. The role of some RGS proteins was shown to be important for hematopoiesis. More recent studies also focused on their expression in platelets, and for R4 RGS subfamily members RGS2, RGS16 and RGS18, it could be demonstrated that they regulate megakaryopoiesis and/or platelet function. These functional studies mostly comprised in vitro experiments and in vivo studies using small animal models. Their role in human pathology related to platelet dysfunction remains still largely unknown, except for a case report with a RGS2 gain of function mutation. In addition to an introduction on RGS signaling and different effectors with a special focus on the R4 subfamily members, we here will give an overview of the studies related to the role of RGS proteins in hematopoiesis, megakaryopoiesis and platelet function.     

Drugs interacting with G protein α subunits: selectivity and perspectives

Summary— Extracellular signal molecules as diverse as hormones, neurotransmitters and photons use a signal transduction pathway involving a receptor, a G protein and effectors. Compounds that interact directly with G proteins can mimic the receptor-G protein interaction or can block the activation of G proteins by receptors. Several binding sites exist on the G_α protein that may be exploited for the design of synthetic stimulatory or inhibitory ligands. The effector binding site is regulated by endogenous proteins and appears to be a target for selective exogenous ligands. The GTP binding site presents a large homology within the G protein families and therefore the nucleotide analogs might not be considered as a tool to discriminate between the G protein subclasses. In contrast, different experimental strategies have substantiated the specificity in the interaction between a receptor and a G protein, the receptor binding site of G proteins should be considered as potential drug targets. Drugs interfering with this site such as mastoparan and related peptides, GPAnt-2 and suramin, are lead compounds in the design of selective G protein antagonists. Benzalkonium chloride and methoctramine have agonist or antagonist properties, depending on G protein subtypes. Such compounds would be very useful to delineate the functions of G proteins and G protein-coupled receptors, to understand some side effects of drugs used in therapy and to develop new therapeutic agents.     

Signal transduction and drug development]

Extracellular information is translated into the cellular responses by various signal transduction systems such as G protein-coupled receptor-linked and protein tyrosine kinase receptor-mediated pathways. The changes or modifications of theses signaling proteins cause uncontrolled transformation or cell death. One of the features of the signaling molecules is that these proteins contain the domains or motifs for protein-protein or protein-lipid interactions governed by several amino acids. Therefore, these amino acids may become promising targets for the drug design. In contrast with intracellular molecules, the receptors located on cellular surface are still the best candidate for the drugs. The future analysis of cellular signal transduction system allows us to develop new drugs that cannot cause serious side effects in future.     

Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling

Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents and plays an important role in adaptive processes of organs; aberrant processing of signals through these transducing systems is a component of various disease states. In addition to G-protein coupled receptor (GPCR)-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Galphabetagamma heterotrimer or Galpha and Gbetagamma subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Galpha and Gbetagamma) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Galphabetagamma. Such regulatory accessory proteins include the family of regulator of G-protein signaling (RGS) proteins that accelerate the GTPase activity of Galpha and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor-independent activators of G-protein signaling (AGS) proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways, and provide a platform for diverse functions of both the heterotrimeric Galphabetagamma and the individual Galpha and Gbetagamma subunits.     

Direct interactions between the heterotrimeric G protein subunit G beta 5 and the G protein gamma subunit-like domain-containing regulator of G protein signaling 11: gain of function of cyan fluorescent protein-tagged G gamma 3

We used fluorescence resonance energy transfer imaging of enhanced cyan fluorescent protein (CFP)-tagged and enhanced yellow fluorescent protein (YFP)-tagged protein pairs to examine the hypothesis that G protein gamma subunit-like (GGL) domain-containing regulators of G protein signaling (RGS) can directly bind to the Gbeta5 subunit of heterotrimeric G proteins in vivo. We observed that Gbeta5 could interact with Ggamma2 and Ggamma13, after their expression in human embryonic kidney 293 cells. Interestingly, although untagged Ggamma3 did not interact with Gbeta5, CFP-tagged Ggamma3 strongly interacted with YFP-tagged Gbeta5 in FRET studies. Moreover, CFP-Ggamma3 supported Ca(2+) channel inhibition when paired with Gbeta5 or YFP-Gbeta5, indicating a "gain of function" for CFP-Ggamma3. Gbeta5 could also interact with RGS11 and its N-terminal, but not its C-terminal domain. On the other hand, RGS11 did not interact with Gbeta1. These studies demonstrate that the GGL domain-containing N terminus of RGS 11 can directly interact with Gbeta5 in vivo and supports the hypothesis that this interaction may contribute to the specificity of Gbeta5 interactions with cellular effector molecules.     

Activation of sickle red blood cell adhesion via integrin-associated protein/CD47–induced signal transduction

Peripheral human red blood cells (RBCs) are not generally known to become activated and adhesive in response to cell signaling. We show, however, that soluble thrombospondin via integrin-associated protein (IAP; CD47) increases the adhesiveness of sickle RBCs (SS RBCs) by activating signal transduction in the SS RBC. This stimulated adhesion requires occupancy of IAP and shear stress and is mediated by the activation of large G proteins and tyrosine kinases. Reticulocyte-enriched RBCs derived from sickle-cell disease (SCD) patients are most responsive to IAP-induced activation. These studies therefore establish peripheral SS RBCs as signaling cells that respond to a novel synergy between IAP-induced signal transduction and shear stress, suggesting new therapeutic targets in SCD.     

Endogenous regulator of g protein signaling proteins reduce {mu}-opioid receptor desensitization and down-regulation and adenylyl cyclase tolerance in C6 cells

Chronic exposure of cells to mu-opioid agonists leads to tolerance which can be measured by a reduced ability to activate signaling pathways in the cell. Cell signaling through inhibitory G proteins is negatively regulated by RGS (regulator of G protein signaling) proteins. Here we examine the hypothesis that the GTPase accelerating activity of RGS proteins, by altering the lifetime of Galpha and Gbetagamma, plays a role in the development of cellular tolerance to mu-opioids. C6 glioma cells were stably transfected with mu-opioid receptor and pertussis toxin (PTX)-insensitive Galpha(o) that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous Galpha proteins followed by exposure to the mu-opioid agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) or morphine. Receptor desensitization as measured by agonist-stimulated [(35)S]GTPgammaS binding and receptor down-regulation as measured by [(3)H]diprenorphine binding were increased in cells expressing RGS-insensitive Galpha(o). Exposure to high concentrations of morphine or the peptidic mu-opioid agonist DAMGO led to a tolerance to inhibit adenylyl cyclase activity in both cell types with a rapid (30 min) and a slower component. Using a submaximal concentration of DAMGO to induce a reduced level of tolerance, a shift in the concentration-effect curve for DAMGO to inhibit adenylyl cyclase activity was seen in the cells expressing RGS-insensitive Galpha(o), but not in the cells expressing RGS-sensitive Galpha(o), which can be partly explained by an increased supersensitization of the adenylyl cyclase response. The results show that RGS proteins endogenously expressed in C6 cells reduce agonist-induced mu-opioid receptor desensitization, down-regulation, and sensitivity to tolerance to inhibit adenylyl cyclase activity.     

Up-regulation of regulator of G protein signaling 4 expression in a model of neuropathic pain and insensitivity to morphine

We hypothesized that the up-regulated expression of one or more members of the regulator of G protein signaling (RGS) family can cause an attenuation of signaling via Gi/Go-coupled opioid receptors, and thereby play a role in the development of hyperalgesia and accompanying insensitivity to morphine observed in animal models of neuropathic pain. Accordingly, we examined the mRNA expression of several RGS genes in a rat model of chronic neuropathic pain induced by partial ligation of the sciatic nerve. During the development of hyperalgesia, RGS4 was the only isoform examined whose mRNA levels increased significantly (up to 230%) in the lumbar spinal cord. In situ hybridization studies confirmed that RGS4 is present in the dorsal horn of the spinal cord where mu-opioid receptors (MORs) are also expressed. Overexpression of RGS4 in human embryonic kidney 293 cells stably expressing mu-opioid receptors predictably attenuated opioid agonist-induced inhibition of adenylyl cyclase. This inhibitory effect was overcome partially at high agonist concentrations, supporting the view that morphine insensitivity is promoted by RGS4 overexpression. These studies provide evidence that the up-regulation of RGS4 expression may contribute to changes in pain signal processing that lead to the development of hyperalgesia, and further affect its modulation by morphine.     

Regulators of G-protein signalling: multifunctional proteins with impact on signalling in the cardiovascular system

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 alpha-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 alpha-subunits, but are G-protein-regulated effectors, Gbetagamma 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 betagamma-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.     

骨形成蛋白信号转导及调控

背景:骨形成蛋白属于转化生长因子β超家族成员,具有很强的诱导成骨能力。目的:对骨形成蛋白功能、信号转导及调控机制及其信号通路异常与人类疾病的关系进行综述。方法:由第一作者用计算机检索中国期刊全文数据库(CNKI:2010)Medline(1999/2010)数据库,检索词分别为"骨形成蛋白、骨形成蛋白受体、Smad蛋白、信号转导、信号调控"和"Bone morphogenetic proteins,Bone morphogenetic proteins receptors,Smads,Signal transduction,Signaling regulation"。从骨形成蛋白的结构与功能,骨形成蛋白信号传导通路及调控,骨形成蛋白信号通路异常与人类疾病3方面进行总结。共检索到110篇文章,按纳入和排除标准对文献进行筛选,共纳入47篇文章。结果与结论:骨形成蛋白主要通过Smad依赖性和Smad非依赖性2条信号转导途径发挥作用,此过程受到细胞内外许多蛋白的调控,此外骨形成蛋白信号通路异常与人类某些疾病密切相关。     

RGS4 causes increased mortality and reduced cardiac hypertrophy in response to pressure overload

RGS family members are GTPase-activating proteins (GAPs) for heterotrimeric G proteins. There is evidence that altered RGS gene expression may contribute to the pathogenesis of cardiac hypertrophy and failure. We investigated the ability of RGS4 to modulate cardiac physiology using a transgenic mouse model. Overexpression of RGS4 in postnatal ventricular tissue did not affect cardiac morphology or basal cardiac function, but markedly compromised the ability of the heart to adapt to transverse aortic constriction (TAC). In contrast to wild-type mice, the transgenic animals developed significantly reduced ventricular hypertrophy in response to pressure overload and also did not exhibit induction of the cardiac "fetal" gene program. TAC of the transgenic mice caused a rapid decompensation in most animals characterized by left ventricular dilatation, depressed systolic function, and increased postoperative mortality when compared with nontransgenic littermates. These results implicate RGS proteins as a crucial component of the signaling pathway involved in both the cardiac response to acute ventricular pressure overload and the cardiac hypertrophic program.     

Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice

Signaling by hormones and neurotransmitters that activate G protein-coupled receptors (GPCRs) maintains blood pressure within the normal range despite large changes in cardiac output that can occur within seconds. This implies that blood pressure regulation requires precise kinetic control of GPCR signaling. To test this hypothesis, we analyzed mice deficient in RGS2, a GTPase-activating protein that greatly accelerates the deactivation rate of heterotrimeric G proteins in vitro. Both rgs2+/- and rgs2-/- mice exhibited a strong hypertensive phenotype, renovascular abnormalities, persistent constriction of the resistance vasculature, and prolonged response of the vasculature to vasoconstrictors in vivo. Analysis of P2Y receptor-mediated Ca2+ signaling in vascular smooth muscle cells in vitro indicated that loss of RGS2 increased agonist potency and efficacy and slowed the kinetics of signal termination. These results establish that abnormally prolonged signaling by G protein-coupled vasoconstrictor receptors can contribute to the onset of hypertension, and they suggest that genetic defects affecting the function or expression of RGS2 may be novel risk factors for development of hypertension in humans.     

Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.     

Structure, function and pathophysiology of protease activated receptors

Discovered in the 1990s, protease activated receptors(1) (PARs) are membrane-spanning cell surface proteins that belong to the G protein coupled receptor (GPCR) family. A defining feature of these receptors is their irreversible activation by proteases; mainly serine. Proteolytic agonists remove the PAR extracellular amino terminal pro-domain to expose a new amino terminus, or tethered ligand, that binds intramolecularly to induce intracellular signal transduction via a number of molecular pathways that regulate a variety of cellular responses. By these mechanisms PARs function as cell surface sensors of extracellular and cell surface associated proteases, contributing extensively to regulation of homeostasis, as well as to dysfunctional responses required for progression of a number of diseases. This review examines common and distinguishing structural features of PARs, mechanisms of receptor activation, trafficking and signal termination, and discusses the physiological and pathological roles of these receptors and emerging approaches for modulating PAR-mediated signaling in disease.     

Platelet Gs hypofunction and abnormal morphology resulting from a heterozygous RGS2 mutation

Summary. Background: Regulator of G‐protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known. Objective: To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology. Methods: Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild‐type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics. Results: The mutation leads to reduced cAMP production after stimulation of Gs‐coupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild‐type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal α‐granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change. Conclusions: We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.     

Endogenous regulator of G protein signaling proteins suppress Galphao-dependent, mu-opioid agonist-mediated adenylyl cyclase supersensitization

Chronic mu-opioid agonist treatment leads to dependence with withdrawal on removal of agonist. At the cellular level withdrawal is accompanied by a supersensitization of adenylyl cyclase, an effect that requires inhibitory Galpha proteins. Inhibitory Galpha protein action is modulated by regulator of G protein signaling (RGS) proteins that act as GTPase activating proteins and reduce the lifetime of Galpha-GTP. In this article, we use C6 glioma cells expressing the rat mu-opioid receptor (C6mu) to examine the hypothesis that Galphao alone can mediate mu-opioid agonist induced adenylyl cyclase supersensitivity and that endogenous RGS proteins serve to limit the extent of this supersensitization. C6mu cells were stably transfected with pertussis toxin (PTX)-insensitive Galphao that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous Galpha proteins followed by exposure to the mu-opioid agonists [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin or morphine. Supersensitization was observed in cells expressing wild-type Galpha, but this was lost on PTX treatment. In cells expressing PTX-insensitive Galphao supersensitization was recovered, confirming that Galphao alone can support supersensitization. In cells expressing the RGS-insensitive mutant Galphao, there was a greater degree of supersensitization and the concentration of micro-agonist needed to achieve half-maximal supersensitization was reduced by 10-fold. The amount of supersensitization seen did not directly relate to the degree of acute inhibition of adenylyl cyclase. These results demonstrate a role for Galphao in adenylyl cyclase supersensitization after mu-agonist exposure and show that this action is modulated by endogenous RGS proteins.     

A region-specific increase in Galphaq and Galpha11 proteins in brains of rats during cocaine withdrawal

Serotonin 2A (5-HT2A) receptor-mediated increases in plasma hormone levels become supersensitive after 42 h of withdrawal from cocaine treatment. The present study investigated which components of the 5-HT2A receptor signaling system are associated with this supersensitivity. Rats were injected daily for 14 days with either saline or cocaine (15 mg/kg i.p.) twice a day or were injected using a "binge" protocol (three injections per day, 1 h apart). Rats were sacrificed 2 or 7 days after the last cocaine injection, and the levels of membrane and cytosol-associated 5-HT2A receptors, Galphaq, Galpha11, regulators of G protein signaling (RGS)4, and RGS7 proteins were assayed in the hypothalamic paraventricular nucleus, amygdala, and frontal cortex using Western blot analysis. Two days of withdrawal from cocaine, administered twice a day or using a binge protocol, produced an increase in membrane-associated Galphaq and Galpha11 proteins in the paraventricular nucleus and the amygdala (but not in the frontal cortex). This effect was reversible after 7 days of withdrawal. The protein levels of the 5-HT2A receptor, Galphaz protein, and RGS4 or RGS7 proteins were not altered by cocaine withdrawal in any of the above-mentioned brain regions. These findings suggest that the supersensitivity of the 5-HT2A receptors, during withdrawal from chronic cocaine, is associated with an increase in membrane-associated Galphaq and Galpha11 proteins and not with changes in the expression of 5-HT2A receptors.     

RGS5在肿瘤抗血管生成的研究进展

G蛋白信号调节蛋白(RGS)是能够直接与激活的Gα亚基结合,负性调节G蛋白信号的一组多功能蛋白家族。在肿瘤领域,RGS5作为RGS家族一员,在多种肿瘤组织的血管周细胞中有高表达,在肿瘤形成过程中对血管的成熟和重构发挥着重要作用。RGS5基因的缺失可使肿瘤血管正常化并改善肿瘤血供,既可促进自体免疫细胞进入肿瘤也有利于改善乏氧增加放化疗敏感性。这可能为我们提供了肿瘤治疗的新靶点。     

Formation of protein kinase C(epsilon)-Lck signaling modules confers cardioprotection

The epsilon isoform of protein kinase C (PKCepsilon) is a member of the PKC family of serine/threonine kinases and plays a critical role in protection against ischemic injury in multiple organs. Functional proteomic analyses of PKCepsilon signaling show that this isozyme forms multiprotein complexes in the heart; however, the precise signaling mechanisms whereby PKCepsilon orchestrates cardioprotection are poorly understood. Here we report that Lck, a member of the Src family of tyrosine kinases, forms a functional signaling module with PKCepsilon. In cardiac cells, PKCepsilon interacts with, phosphorylates, and activates Lck. In vivo studies showed that cardioprotection elicited either by cardiac-specific transgenic activation of PKCepsilon or by ischemic preconditioning enhances the formation of PKCepsilon-Lck modules. Disruption of these modules, via ablation of the Lck gene, abrogated the infarct-sparing effects of these two forms of cardioprotection, indicating that the formation of PKCepsilon-Lck signaling modules is required for the manifestation of a cardioprotective phenotype. These findings demonstrate, for the first time to our knowledge, that the assembly of a module (PKCepsilon-Lck) is an obligatory step in the signal transduction that results in a specific phenotype. Thus, PKCepsilon-Lck modules may serve as novel therapeutic targets for the prevention of ischemic injury.