Differential Modulation of ��- and ��-Opioid Receptor Agonists by Endogenous RGS4 Protein in SH-SY5Y Cells

Regulator of G-protein signaling (RGS) proteins are a family of molecules that control the duration of G protein signaling. A variety of RGS proteins have been reported to modulate opioid receptor signaling. Here we show that RGS4 is abundantly expressed in human neuroblastoma SH-SY5Y cells that endogenously express μ- and δ-opioid receptors and test the hypothesis that the activity of opioids in these cells is modulated by RGS4. Endogenous RGS4 protein was reduced by ∼90% in SH-SY5Y cells stably expressing short hairpin RNA specifically targeted to RGS4. In these cells, the potency and maximal effect of δ-opioid receptor agonist (SNC80)-mediated inhibition of forskolin-stimulated cAMP accumulation was increased compared with control cells. This effect was reversed by transient transfection of a stable RGS4 mutant (HA-RGS4C2S). Furthermore, MAPK activation by SNC80 was increased in cells with knockdown of RGS4. In contrast, there was no change in the μ-opioid (morphine) response at adenylyl cyclase or MAPK. FLAG-tagged opioid receptors and HA-RGS4C2S were transiently expressed in HEK293T cells, and co-immunoprecipitation experiments showed that the δ-opioid receptor but not the μ-opioid receptor could be precipitated together with the stable RGS4. Using chimeras of the δ- and μ-opioid receptors, the C-tail and third intracellular domain of the δ-opioid receptor were suggested to be the sites of interaction with RGS4. The findings demonstrate a role for endogenous RGS4 protein in modulating δ-opioid receptor signaling in SH-SY5Y cells and provide evidence for a receptor-specific effect of RGS4.μ- and δ-opioid receptors are members of the G protein-coupled receptor family and interact with Gα_i/o proteins (1, 2). This results in signaling to a variety of downstream effectors, including adenylyl cyclase and the mitogen-activated protein kinase (MAPK)² cascade. Signaling of opioid receptors is regulated negatively by regulator of G protein signaling (RGS) proteins (3, 4). These are a family of molecules containing a “RGS consensus” domain that bind to Gα subunits and act as GTPase-accelerating proteins to increase the rate of GTP hydrolysis. This results in a decrease in the lifetime of the active Gα-GTP and free Gβγ subunits and limits signaling to downstream effectors (5–8). The mechanisms by which RGS proteins selectively modulate G protein-mediated receptor signal transduction pathways, especially opioid receptor signaling, are beginning to unfold (9–12). The foundation for the function and selectivity of RGS proteins in regulating opioid signaling lies in their ability to interact with opioid receptors and their cognate G proteins. In general, the selectivity or the preference of an RGS protein for a particular receptor is determined by a variety of factors, including tissue-specific expression and precise interaction with the intracellular domains of receptor proteins, G protein subunits, and effectors as well as other pathway-specific components (13).The effects of RGS proteins on opioid receptor signaling have been examined in several systems. The findings are not always consistent, probably due to the different methodologies used. It has been shown that members of the RZ, R4, and R7 subfamilies (7) of RGS proteins play crucial roles not only in terminating acute opioid agonist action but also in opioid receptor desensitization, internalization, recycling, and degradation (3, 14), thereby affecting opioid tolerance and dependence (15–18). Much work has been performed with RGS4, because it is a smaller RGS protein with a structure consisting of the RGS consensus (box) sequence and a small N terminus (19, 20). It also has a wide distribution in the brain, especially in brain regions important for opioid actions, including the striatum, locus coeruleus, dorsal horn of the spinal cord, and cerebral cortex (21). In vitro RGS4 has been shown to reverse δ-opioid receptor agonist-induced inhibition of cAMP synthesis in membranes prepared from NG108-15 cells (6). Overexpression of RGS4 in HEK293 cells also attenuated morphine-, [d-Ala²,N-Me-Phe⁴,Gly-ol⁵]enkephalin (DAMGO)-, and [d-Pen²,d-Pen⁵]enkephalin (DPDPE)-induced inhibition of adenylyl cyclase (22, 23). Co-expression of RGS4 with GIRK1/GIRK2 channels in Xenopus oocytes reduced the basal K⁺ current and accelerated the deactivation of GIRK channels activated by κ-opioid receptor agonist {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 (24). Although these previous studies have provided evidence that RGS4 can negatively regulate opioid receptor signaling, they do not confirm a functional role for endogenous RGS4 in endogenous, nontransfected systems.Human neuroblastoma SH-SY5Y cells endogenously express μ- and δ-opioid receptors and a variety of Gα_i/o proteins (25–27). Here we show that RGS4 is abundantly found at both the mRNA and protein levels in these cells. Consequently, we used SH-SY5Y cells to examine the hypothesis that RGS4 negatively modulates opioid receptor signaling under physiological conditions. The endogenously expressed RGS4 level in SH-SY5Y cells was reduced using lentiviral delivery of short hairpin RNA (shRNA) targeting the RGS4 gene. This resulted in changes in δ- but not μ-opioid receptor-mediated signaling to adenylyl cyclase and the MAPK pathway. These findings argue for a selective interaction of RGS4 with the δ-opioid receptor. To test this, we expressed FLAG-tagged μ- and δ-opioid receptors together with a construct for a stable, proteosome-resistant RGS4 protein in HEK293T cells. Co-immunoprecipitation indicated that the δ-opioid but not the μ-opioid receptor was closely associated with RGS4, providing further evidence for a selective interaction between RGS4 and δ-opioid receptor signaling.     

RGS16 Inhibits Breast Cancer Cell Growth by Mitigating Phosphatidylinositol 3-Kinase Signaling

Aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway supports growth of many tumors including those of breast, lung, and prostate. Resistance of breast cancer cells to targeted chemotherapies including tyrosine kinase inhibitors (TKI) has been linked to persistent PI3K activity, which may in part be due to increased membrane expression of epidermal growth factor (EGF) receptors (HER2 and HER3). Recently we found that proteins of the RGS (regulator of G protein signaling) family suppress PI3K activity downstream of the receptor by sequestering its p85α subunit from signaling complexes. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. RGS16 overexpression in MCF7 breast cancer cells inhibited EGF-induced proliferation and Akt phosphorylation, whereas shRNA-mediated extinction of RGS16 augmented cell growth and resistance to TKI treatment. Exposure to TKI also reduced RGS16 expression in MCF7 and BT474 cell lines. RGS16 bound the amino-terminal SH2 and inter-SH2 domains of p85α and inhibited its interaction with the EGF receptor-associated adapter protein Gab1. These results suggest that the loss of RGS16 in some breast tumors enhances PI3K signaling elicited by growth factors and thereby promotes proliferation and TKI evasion downstream of HER activation.The role of the PI3K³ pathway in cell proliferation and survival, adhesion, metabolism, migration, drug resistance, and cytoskeletal rearrangement is well documented (1–3). Mutations in PI3K and dysregulation of the PI3K pathway have been implicated in many human cancers including lymphoma, multiple myeloma, and melanoma (4–8). Because the PI3K signal is a gatekeeper for tumor growth, an understanding of its regulation is critical for the therapeutic intervention of cancer.PI3K, which catalyzes the production of phosphatidylinositol 3,4,5-trisphosphate from phosphatidylinositol 3,4-bisphosphate (9, 10), is activated by extracellular receptor tyrosine kinases including the EGF receptor (EGFR or HER) family, platelet-derived growth factor receptor, and the insulin growth factor receptor. HER stimulation activates Class IA PI3Ks consisting of dimers of p85α or β and either p110α, β, and δ catalytic subunits (11). Tyrosine phosphorylation of the adapter protein Grb2-associated binder 1 (Gab1) recruits p85 to the EGFR complex through a Src homology 2 (SH2) domain in p85 (12), which co-localizes the catalytic p110 subunit and membrane phospholipid substrates at the plasma membrane. Phosphatidylinositol 3,4,5-trisphosphate generated by PI3K activity recruits phosphoinositide-dependent kinase 1 through its pleckstrin homology domain, which in turn phosphorylates the mitogenic and antiapoptotic kinase Akt. Substrates of Akt include mTOR, BAD, IKK, FOXO, p27, MDM2, and GSK3β, all of which are signaling molecules with vital functions in cell cycle regulation and growth (3). Overexpression of Akt has been shown in several tumors such as ovarian and breast carcinoma and may lead directly to transformation of malignant melanoma (5).Proteins of the RGS (regulator of G protein signaling) family mediate cellular desensitization to G protein-coupled receptor stimulation. RGS proteins act as GTPase-accelerating proteins to reduce the life span of activated (GTP-bound) Gα subunits of the G protein-coupled receptor signal-transducing heterotrimeric G protein (13). The R4 subfamily of RGSs (RGS1, 2, 4, 5, 8, 13, 16, 18, and 21) are the smallest members of the family, containing few residues outside of the ∼120-amino acid RGS domain that mediates binding to Gα proteins and GTPase-accelerating protein activity. We found recently that several R4 RGS proteins interacted with the phosphorylated p85α subunit of PI3K (14). In mast cells, RGS13 inhibited PI3K activation induced by high affinity IgE receptor (FcϵRI) cross-linking by antigen. FcϵRI stimulates PI3K by recruiting its catalytic p110δ subunit through p85 binding to a multi-protein complex that includes Gab2 and Grb2 at the plasma membrane (15). PI3K has an essential function in allergic responses (16). As a result of increased PI3K activation, mice deficient in RGS13 had more IgE-mediated mast cell degranulation and anaphylaxis (14).RGS16, an R4 RGS protein homologous to RGS13, was identified originally as a p53 target gene in breast and colon cancer cells (17, 18). Recent analysis of 222 primary breast cancers found a high rate (50%) of genomic instability at the RGS16 locus (19). Because RGS16 associates with both EGFR (20) and p85α (14), we investigated how it affected the growth and survival of breast cancer cells. We found that RGS16 directly bound the amino-terminal SH2 and inter-SH2 domains of phosphorylated p85α, which mediate p110 and adapter binding and membrane localization (21). RGS16 overexpression in MCF7 breast cancer cells suppressed proliferation and EGF-induced Akt phosphorylation, whereas extinction of RGS16 expression increased cell growth and resistance to TKI treatment. Thus, through regulation of PI3K activity, RGS16 may limit proliferation of mammary cells and render cancer cells more susceptible to TKIs or other therapeutic compounds.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Magnesium Deficiency Causes Loss of Response to Intermittent Hypoxia in Paraganglion Cells

Magnesium deficiency is suggested to contribute to many age-related diseases. Hypoxia-inducible factor 1α (HIF-1α) is known to be a master regulator of hypoxic response. Here we show that hypomagnesemia suppresses reactive oxygen species (ROS)-induced HIF-1α activity in paraganglion cells of the adrenal medulla and carotid body. In PC12 cells cultured in the low magnesium medium and treated with cobalt chloride (CoCl₂) or exposed to intermittent hypoxia, ROS-mediated HIF-1α activity was suppressed. This suppression was due to up-regulation of inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS) that was caused by NF-κB activation, which resulted from ROS and calcium influx mainly through the T-type calcium channels. Induction of tyrosine hydroxylase, a target of HIF-1, by CoCl₂ injection was suppressed in the adrenal medulla of magnesium-deficient mice because of up-regulation of IPAS. Also in the carotid body of magnesium-deficient mice, CoCl₂ and chronic intermittent hypoxia failed to enhance the tyrosine hydroxylase expression. These results demonstrate that serum magnesium levels are a key determinant for ROS-induced hypoxic responses.Hypoxia-inducible factor 1α (HIF-1α)² and its family members are master regulators of hypoxic response (1–3). In hypoxia, the HIF-1, composed of HIF-1α and HIF-1β/Arnt, binds to hypoxia response element (HRE) to induce the gene expression of hypoxia-responsive proteins, such as erythropoietin and vascular endothelial growth factor. In addition to these proteins, tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine biosynthesis, is induced in rat pheochromocytoma-derived PC12 cells and paraganglion cells in the adrenal medulla (AM) and carotid body (CB) in response to hypoxia (4). The CB acts as the primary peripheral chemoreceptor (5), and glomus cells of the CB are responsible for monitoring oxygen levels in arterial blood (5, 6). Through the release of neurotransmitters, including dopamine, the CB delivers information to the respiratory and cardiovascular networks in the brainstem, resulting in increases of ventilatory frequency and volume and also raising cardiac output.HIF-dependent hypoxic response is also caused by chronic intermittent hypoxia (CIH), which is a common feature of obstructive sleep apnea (OSA). There is accumulating evidence that CIH is associated with an increased oxidative stress (7, 8). Peng et al. (9) have shown that CIH induces reactive oxygen species (ROS) generation, thereby increasing HIF-1α expression, which is critical for eliciting CIH-induced cardiorespiratory responses by the CB. CIH also increases ROS generation and TH expression in the AM, although it is less sensitive than the CB (10).Recent studies have identified that IPAS, which is one of the alternatively spliced variants of HIF-3α, acts as a dominant negative inhibitor of HIF-1α by a direct interaction with HIF-1α and prevents its DNA binding (11). IPAS is predominantly expressed in the Purkinje cells of the cerebellum and corneal epithelium. In addition, because the IPAS gene has an HRE sequence in its promoter, IPAS can be induced by hypoxia in the heart and lung. Therefore, IPAS acts as a negative feedback inhibitor of HIF-1α (12).Magnesium deficiency is believed to be related to many diseases, such as hypertension, ischemic heart disease, and diabetes mellitus (13–16). However, the molecular mechanisms underlying the role of magnesium in the pathogenesis of these diseases have been largely undefined. Our analyses here demonstrate that magnesium deficiency causes a loss of ROS-induced HIF-1α activity by inducing IPAS gene expression.     

Structural Determinants of G-protein �� Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2)

“Regulator of G-protein signaling” (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of Gα subunits (known as GTPase-accelerating protein or “GAP” activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for Gα_q subunits. As many vasoconstrictive hormones signal via G_q heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for Gα_q over Gα_i/o substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits Gα_i-directed binding and GAP activities without perturbing its association with Gα_q. Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of Gα_i at 2.8-Å resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/Gα complexes highlighted the roles of these residues in wild type RGS2 that weaken Gα_i subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective Gα_q GAP that modulates cardiovascular function.G protein-coupled receptors (GPCRs)⁴ form an interface between extracellular and intracellular physiology, as they convert hormonal signals into changes in intracellular metabolism and ultimately cell phenotype and function (1–3). GPCRs are coupled to their underlying second messenger systems by heterotrimeric guanine nucleotide-binding protein (“G-proteins”) composed of three subunits: Gα, Gβ, and Gγ. Four general classes of Gα subunits have been defined based on functional couplings (in the GTP-bound state) to various effector proteins. G_s subfamily Gα subunits are stimulatory to membrane-bound adenylyl cyclases that generate the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP); conversely, G_i subfamily Gα subunits are generally inhibitory to adenylyl cyclases (4). G_12/13 subfamily Gα subunits activate the small G-protein RhoA through stimulation of the GEF subfamily of RGS proteins, namely p115-RhoGEF, LARG, and PDZ-RhoGEF (5). G_q subfamily Gα subunits are potent activators of phospholipase-Cβ enzymes that generate the second messengers diacylglycerol and inositol triphosphate (6); more recently, two additional Gα_q effector proteins have been described: the receptor kinase GRK2 and the RhoA nucleotide exchange factor p63RhoGEF (7, 8).The duration of GPCR signaling is controlled by the time Gα remains bound to GTP before its hydrolysis to GDP. RGS proteins are key modulators of GPCR signaling by virtue of their ability to accelerate the intrinsic GTP hydrolysis activity of Gα subunits (reviewed in Refs. 9 and 10). RGS2/G0S8, one of the first mammalian RGS proteins identified (11) and member of the R4-subfamily (10), has a critical role in the maintenance of normostatic blood pressure both in mouse models (12, 13) and in humans (14, 15); additionally, Rgs2-deficient mice exhibit impaired aggression and increased anxiety (16, 17), behavioral phenotypes with potential human clinical correlates (18, 19).Although many RGS proteins are promiscuous and thus act on multiple Gα substrates in vitro (e.g. Ref. 20), RGS2 exhibits exquisite specificity for Gα_q in biochemical binding assays and single turnover GTPase acceleration assays (20, 21). Consistent with this in vitro selectivity,⁵ mice deficient in RGS2 uniquely exhibit constitutive hypertension and prolonged responses to vasoconstrictors, as would be expected upon loss of a potent negative regulator of Gα_q that mediates signaling from various vasoconstrictive hormones such as angiotensin II, endothelin, thrombin, norepinephrine, and vasopressin (22). In addition, RGS2-deficient mice respond to sustained pressure overload with an accelerated time course of maladaptive cardiac remodeling (23), a pathophysiological response that evokes myocardial hypertrophy known to be critically dependent on Gα_q signaling (24, 25).To gain insight into the structural basis of the unique Gα substrate selectivity exhibited by RGS2, a series of point mutants in RGS2 were evaluated that enable this protein to bind and accelerate GTP hydrolysis by Gα_i; we subsequently delineated the structural determinants of the Gα_i/mutant RGS2 interaction using x-ray crystallography. Three key positions, first identified by Heximer and colleagues (21) and highlighted in our structural studies as key determinants of RGS2 substrate selection, were also found to be conserved throughout the evolution of the RGS2 protein in a manner suggestive of specialization toward cardiovascular signaling modulation.     

Role of Molecular Chaperones in G Protein ��5/Regulator of G Protein Signaling Dimer Assembly and G Protein �¦� Dimer Specificity

The G protein βγ subunit dimer (Gβγ) and the Gβ5/regulator of G protein signaling (RGS) dimer play fundamental roles in propagating and regulating G protein pathways, respectively. How these complexes form dimers when the individual subunits are unstable is a question that has remained unaddressed for many years. In the case of Gβγ, recent studies have shown that phosducin-like protein 1 (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gβ and mediate its interaction with Gγ. However, it is not known what fraction of the many Gβγ combinations is assembled this way or whether chaperones influence the specificity of Gβγ dimer formation. Moreover, the mechanism of Gβ5-RGS assembly has yet to be assessed experimentally. The current study was undertaken to directly address these issues. The data show that PhLP1 plays a vital role in the assembly of Gγ2 with all four Gβ1–4 subunits and in the assembly of Gβ2 with all twelve Gγ subunits, without affecting the specificity of the Gβγ interactions. The results also show that Gβ5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gβγ. PhLP1 seems to stabilize the interaction of Gβ5 with CCT until Gβ5 is folded, after which it is released to allow Gβ5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gβγ combinations and suggest a CCT-dependent mechanism for Gβ5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way.Eukaryotic cells utilize receptors coupled to heterotrimeric GTP-binding proteins (G proteins)³ to mediate a vast array of responses ranging from nutrient-induced migration of single-celled organisms to neurotransmitter-regulated neuronal activity in the human brain (1). Ligand binding to a G protein-coupled receptor (GPCR) initiates GTP exchange on the G protein heterotrimer (composed of Gα, Gβ, and Gγ subunits), which in turn causes the release of Gα-GTP from the Gβγ dimer (2–4). Both Gα-GTP and Gβγ propagate and amplify the signal by interacting with effector enzymes and ion channels (1, 5). The duration and amplitude of the signal is dictated by receptor phosphorylation coupled with arrestin binding and internalization (6) and by regulators of G protein signaling (RGS) proteins, which serve as GTPase-activating proteins for the GTP-bound Gα subunit (7, 8). The G protein signaling cycle is reset as the inactive Gα-GDP reassembles with the Gβγ dimer and Gαβγ re-associates with the GPCR (5).To fulfill its essential role in signaling, the G protein heterotrimer must be assembled post-translationally from its nascent polypeptides. Significant progress has been made recently regarding the mechanism by which this process occurs. It has been clear for some time that the Gβγ dimer must assemble first, followed by subsequent association of Gα with Gβγ (9). What has not been clear was how Gβγ assembly would occur given the fact that neither Gβ nor Gγ is structurally stable without the other. An important breakthrough was the finding that phosducin-like protein 1 (PhLP1) functions as a co-chaperone with the chaperonin containing tailless complex polypeptide 1 (CCT) in the folding of nascent Gβ and its association with Gγ (10–15). CCT is an important chaperone that assists in the folding of actin and tubulin and many other cytosolic proteins, including many β propeller proteins like Gβ (16). PhLP1 has been known for some time to interact with Gβγ and was initially believed to inhibit Gβγ function (17). However, several recent studies have demonstrated that PhLP1 and CCT work together in a highly orchestrated manner to form the Gβγ dimer (10–15).Studies on the mechanism of PhLP1-mediated Gβγ assembly have focused on the most common dimer Gβ1γ2 (10, 13, 14), leaving open questions about the role of PhLP1 in the assembly of the other Gβγ combinations. These are important considerations given that humans possess 5 Gβ genes and 12 Gγ genes with some important splice variants (18, 19), resulting in more than 60 possible combinations of Gβγ dimers. Gβ1–4 share between 80 and 90% sequence identity and are broadly expressed (18, 19). Gβ5, the more atypical isoform, shares only ∼53% identity with Gβ1, carries a longer N-terminal domain, and is only expressed in the central nervous system and retina (20). The Gγ protein family is more heterogeneous than the Gβ family. The sequence identity of the 12 Gγ isoforms extends from 10 to 70% (21), and the Gγ family can be separated into 5 subfamilies (21–23). All Gγ proteins carry C-terminal isoprenyl modifications, which contribute to their association with the cell membrane, GPCRs, Gαs, and effectors (9). Subfamily I Gγ isoforms are post-translationally farnesylated, whereas all others are geranylgeranylated (22, 24).There is some inherent selectivity in the assembly of different Gβγ combinations, but in general Gβ1–4 can form dimers with most Gγ subunits (25). The physiological purpose of this large number of Gβγ combinations has intrigued researchers in the field for many years, and a large body of research indicates that GPCRs and effectors couple to a preferred subset of Gβγ combinations based somewhat on specific sequence complementarity, but even more so on cellular expression patterns, subcellular localization, and post-translational modifications (18).In contrast to Gβ1–4, Gβ5 does not interact with Gγ subunits in vivo, but it instead forms irreversible dimers with RGS proteins of the R7 family, which includes RGS proteins 6, 7, 9, and 11 (26). All R7 family proteins contain an N-terminal DEP (disheveled, Egl-10, pleckstrin) domain, a central Gγ-like (GGL) domain, and a C-terminal RGS domain (8, 26). The DEP domain interacts with the membrane anchoring/nuclear shuttling R7-binding protein, and the GGL domain binds to Gβ5 in a manner similar to other Gβγ associations (27, 28). Like Gβγs, Gβ5 and R7 RGS proteins form obligate dimers required for their mutual stability (26). Without their partner, Gβ5 and R7 RGS proteins are rapidly degraded in cells (26, 29). Gβ5-R7 RGS complexes act as important GTPase-accelerating proteins for G_i/oα and G_qα subunits in neuronal cells and some immune cells (26).It has been recently shown that all Gβ isoforms are able to interact with the CCT complex, but to varying degrees (15). Gβ4 and Gβ1 bind CCT better than Gβ2 and Gβ3, whereas Gβ5 binds CCT poorly (15). These results suggest that Gβ1 and Gβ4 might be more dependent on PhLP1 than the other Gβs, given the co-chaperone role of PhLP1 with CCT in Gβ1γ2 assembly. However, another report has indicated that Gγ2 assembly with Gβ1 and Gβ2 is more PhLP1-dependent than with Gβ3 and Gβ4 (30). Thus, it is not clear from current information whether PhLP1 and CCT participate in assembly of all Gβγ combinations or whether they contribute to the specificity of Gβγ dimer formation, nor is it clear whether they or other chaperones are involved in Gβ5-R7 RGS dimer formation. This report was designed to address these issues.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.