Protein-regulated Inducer Of Neurite Outgrowth Research Articles

Ferredoxin 1: a gatekeeper in halting lung adenocarcinoma progression through activation of the GPRIN2 signaling pathway.

Lung adenocarcinoma (LUAD) is a highly lethal form of lung cancer. Despite advancements in treatments, managing LUAD is still challenging due to its aggressive behavior. Recent studies indicate that various molecular pathways, including the dysregulation of ferredoxin 1 (FDX1), play roles in LUAD progression. FDX1, a crucial protein in cellular redox reactions and energy metabolism, has been linked to several cancers. However, its exact role in the development of LUAD is not yet fully understood. We investigated the role of ferredoxin 1 (FDX1) in LUAD progression through analysis of its expression in LUAD tissues and its impact on patient survival. Functional assays were performed to assess the effects of FDX1 overexpression on LUAD cell proliferation, migration, and invasion. A xenograft model was employed to evaluate the tumorigenesis potential of LUAD cells with FDX1 overexpression. Mechanistic insights into FDX1 regulation were gained through depletion experiments targeting the G protein-regulated inducer of neurite outgrowth 2 (GPRIN2)/PI3K signaling pathway. FDX1 expression was down-regulated in LUAD tissues, correlating with shorter patient survival. Overexpression of FDX1 suppressed LUAD cell proliferation, migration, and invasion in vitro, and inhibited tumorigenesis in vivo. Mechanistically, the GPRIN2/PI3K signaling pathway was implicated in FDX1 regulation, as depletion of GPRIN2 reversed the effects of FDX1 overexpression on cellular functions. Our findings highlight FDX1 as a potential tumor suppressor in LUAD, acting through modulation of the GPRIN2/PI3K signaling pathway. These results suggest FDX1 as a promising therapeutic target for LUAD treatment, warranting further investigation into its clinical relevance.

MiR-654-5p promotes gastric cancer progression via the GPRIN1/NF-κB pathway.

BackgroundGastric carcinoma (GC) ranks the fifth most common cancer worldwide, with high incidence and mortality rates. Numerous microRNAs (miRNAs), including miR-654-5p, have been implicated in the pathophysiological processes of tumorigenesis. Nevertheless, the mechanism of miR-654-5p in GC is unclear.ObjectivesOur study is devoted to exploring the function and molecular mechanism of miR-654-5p on the malignant cell behaviors of GC.MethodsThe gene expression was detected by reverse transcription quantitative polymerase chain reaction. GC cell proliferation and motion were assessed by colony formation assay and transwell assay. The binding capacity between miR-654-5p and G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) was explored by luciferase reporter and RNA pulldown assays. The protein levels were detected by Western blotting.ResultsmiR-654-5p expression was higher in GC cells and tissues than control cells and tissues. miR-654-5p promoted GC cell growth and motion. Moreover, our findings showed that miR-654-5p was bound with GPRIN1. Importantly, downregulation of GPRIN1 rescued the inhibitory influence of miR-654-5p knockdown on GC cell malignant behaviors. Additionally, miR-654-5p activated the nuclear factor kappa-B (NF-κB) pathway by regulation of GPRIN1.ConclusionsmiR-654-5p facilitated cell proliferation, migration, and invasion in GC via targeting the GPRIN1 to activate the NF-κB pathway.

Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in β-arrestin 2-Akt signaling and dopaminergic behaviors

The G protein-regulated inducer of neurite growth (GRIN) family has three isoforms (GRIN1-3), which bind to the Gαi/o subfamily of G protein that mediate signal processing via G protein-coupled receptors (GPCRs). Here, we show that GRIN3 is involved in regulation of dopamine-dependent behaviors and is essential for activation of the dopamine receptors (DAR)-β-arrestin signaling cascade. Analysis of functional regions of GRIN3 showed that a di-cysteine motif (Cys751/752) is required for plasma membrane localization. GRIN3 was co-immunoprecipitated with GPCR kinases 2/6 and β-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with β-arrestin 2. We also generated GRIN3-knockout mice (GRIN3KO). GRIN3KO exhibited reduced locomotor activity and increased anxiety-like behavior in the elevated maze test, as well as a reduced locomoter response to dopamine stimulation. We also examined the phosphorylation of Akt at threonine 308 (phospho308-Akt), which is dephosphorylated via a β-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-β-arrestin 2 signaling pathway was completely abolished in striatum of GRIN3KO. Our results suggest that GRIN3 has a role in recruitment and assembly of proteins involved in β-arrestin-dependent, G protein-independent signaling.

Axon targeting of the alpha 7 nicotinic receptor in developing hippocampal neurons by Gprin1 regulates growth

Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein-regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth-associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.

Theα4 Nicotinic Receptor Promotes CD4+T-Cell Proliferation and a Helper T-Cell Immune Response

Smoking is a common addiction and a leading cause of disease. Chronic nicotine exposure is known to activate nicotinic acetylcholine receptors (nAChRs) in immune cells. We demonstrate a novel role for α4 nAChRs in the effect of nicotine on T-cell proliferation and immunity. Using cell-based sorting and proteomic analysis we define an α4 nAChR expressing helper T-cell population (α4(+)CD3(+)CD4(+)) and show that this group of cells is responsive to sustained nicotine exposure. In the circulation, spleen, bone marrow, and thymus, we find that nicotine promotes an increase in CD3(+)CD4(+) cells via its activation of the α4 nAChR and regulation of G protein subunit o, G protein regulated-inducer of neurite outgrowth, and CDC42 signaling within T cells. In particular, nicotine is found to promote a helper T cell 2 adaptive immunologic response within T cells that is absent in α4(-/-) mice. We thus present a new mechanism of α4 nAChR signaling and immune regulation in T cells, possibly accounting for the effect of smoking on the immune system.

G Protein-regulated Inducer of Neurite Outgrowth (GRIN) Modulates Sprouty Protein Repression of Mitogen-activated Protein Kinase (MAPK) Activation by Growth Factor Stimulation

Gα(o/i) interacts directly with GRIN (G protein-regulated inducer of neurite outgrowth). Using the yeast two-hybrid system, we identified Sprouty2 as an interacting partner of GRIN. Gα(o) and Sprouty2 bind to overlapping regions of GRIN, thus competing for GRIN binding. Imaging experiments demonstrated that Gα(o) expression promoted GRIN translocation to the plasma membrane, whereas Sprouty2 expression failed to do so. Given the role of Sprouty2 in the regulation of growth factor-mediated MAPK activation, we examined the contribution of the GRIN-Sprouty2 interaction to CB1 cannabinoid receptor regulation of FGF receptor signaling. In Neuro-2A cells, a system that expresses all of the components endogenously, modulation of GRIN levels led to regulation of MAPK activation. Overexpression of GRIN potentiated FGF activation of MAPK and decreased tyrosine phosphorylation of Sprouty2. Pretreatment with G(o/i)-coupled CB1 receptor agonist attenuated subsequent FGF activation of MAPK. Decreased expression of GRIN both diminished FGF activation of MAPK and blocked CB1R attenuation of MAPK activation. These observations indicate that Gα(o) interacts with GRIN and outcompetes GRIN from bound Sprouty. Free Sprouty then in turn inhibits growth factor signaling. Thus, here we present a novel mechanism of how G(o/i)-coupled receptors can inhibit growth factor signaling to MAPK.

G Protein-regulated Inducer of Neurite Outgrowth (GRIN) Modulates Sprouty Protein Repression of Mitogen-activated Protein Kinase (MAPK) Activation by Growth Factor Stimulation

G Protein-regulated Inducer of Neurite Outgrowth (GRIN) Modulates Sprouty Protein Repression of Mitogen-activated Protein Kinase (MAPK) Activation by Growth Factor Stimulation

34 DIFFERENTIALLY EXPRESSED PROTEINS OF EARLY-STAGE PLACENTA DERIVED FROM CLONED CAT EMBRYOS USING PROTEOMICS ANALYSIS

We have previously demonstrated that differentially expressed proteins affect abnormal development and function of cloned term placenta. This is associated with cloned fetus morbidity and mortality. We also frequently observed loss of the cloned fetus and failed development during early pregnancy periods. To confirm the pattern of important gene expression in cloned placenta during pre- and post-implantation, we investigated expression pattern of proteins in early stage (21 days) domestic cat placentas of cloned embryo transfer (CEP; n = 2) and artificial insemination (CP; n = 4) derived pregnancy. The differentially expressed proteins were investigated by 2-DE and MALDI-TOF/MS. Twenty-three proteins were up- and down-regulated at least 1.5-fold in the CEP (P < 0.05) compared with the CP. Differentially expressed proteins were analysed using PDQest program and statistically analysed by 1-way ANOVA using the SPSS software. In CEP, 13 proteins were up-regulated, such as 78-kDa glucose-regulated protein (GRP78), annexin A2 (ANXA2), protein DJ-1 (DJ1), adenylate kinase isoenzyme 1 (AK1), protein disulfide-isomerase A3 (PDIA3), heat shock protein β-1 (HSPB1), actin, cytoplasmic 1 (ACTB), serum albumin (ALB), protein disulfide-isomerase A6 (PDIA6), G protein-regulated inducer of neurite outgrowth 1 (GRIN1) and triosephosphate isomerase (TIM). In contrast, 10 proteins were down-regulated, such as vinculin (VCL), triosephosphate isomerase (TIM), heterogeneous nuclear ribonucleoprotein H (hnRNPH), tropomyosin α-4 (TPM4), 60-kDa heat shock protein, mitochondrial (Hsp60), serum albumin (ALB), calumenin (CALU), keratin type 1 (CK1) and prohibitin (PHB). To validate the identified proteins in the CEP compared with the CP, we investigate a peptide sequences using MALDI-TOF/TOF tandem mass spectrometry. The sequence information obtained a high ions score from NCBI and Swiss-Prot databases. In conclusion, we did identify abnormal expression of proteins that might be associated with impaired development of CEP, which may endanger the cloned fetus during early pregnancy. This work was partly supported by the BK21 program and the KOSEF (10525010001-05N2501-00110) and the Next-generation BioGreen21 program (No. PJ007990012011).

Comparative analysis of serum proteomes of degenerative scoliosis

Degenerative scoliosis (DS) is an important degenerative lumbar disease causing spinal dysfunction and affecting the quality of life of the elderly, and is associated not only with severe back or leg pain but also with complicated surgical outcomes. The pathogenesis of DS is still unknown. Therefore, it is very important to ascertain the etiology of degenerative scoliosis and establish related molecular markers predicting and controlling the scoliosis. For the first time, we used two-dimensional fluorescence DIGE to compare the serum proteome profiles of 12 DS patients and controls. Proteins found to be differentially expressed were identified by MALDI-TOF mass spectrometric analysis, coupled with database interrogation. Eleven spots that were differentially expressed in the sera of DS patients were found, and eight gene products were identified among these spots. Clusterin, CLU cDNA FLJ57622, ALB cDNA FLJ50830, Hypothetical short protein, HLA-A MHC class 1 antigen. (Fragment), ALB 23 kDa protein, Isoform 1 of G protein-regulated inducer of neurite outgrowth 1 (GPRIN I)and Ficolin-3 were down-regulated in the sera of DS patients. The decreased levels of Clusterin and Ficolin-3 were confirmed by Western blot. The information obtained with this proteomic analysis will be very useful in understanding the pathophysiology of DS as well as in finding candidates as drug targets of DS. These results may provide a novel approach for the pathogenesis study of DS.

A possible association of responsiveness to adrenocorticotropic hormone with specific GRIN1 haplotypes in infantile spasms

Adrenocorticotropic hormone (ACTH) has been used as the major therapy for infantile spasms since 1958 because it effectively suppresses seizures; it also normalizes the electroencephalogram in the short-term treatment of infantile spasms. G protein-regulated inducer of neurite outgrowth 1 (GRIN1, also known as N-methyl-D-aspartate receptor 1, NMDAR1), a glutamate receptor, is the main component of functional N-methyl-D-aspartic acid receptors that are involved in the glucocorticoid-induced neuronal damage. Thus, it may be a candidate gene to be tested for responsiveness to ACTH in infantile spasms. In the present study, polymorphisms in the GRIN1 gene in infantile spasms were investigated using a case-control design. Twelve single nucleotide polymorphisms in the GRIN1 gene were genotyped in a Chinese case-control set consisting of 97 unrelated patients with infantile spasms (60 males, 37 females; mean age 6.4 mo, SD 2.7) and 96 healthy individuals (63 males, 33 females; mean age 7.3 mo, SD 3.8). Association analysis was performed on the genotyped data. Five estimated haplotypes with a frequency of more than 3% were detected. Results of the study showed that responsiveness to treatment with ACTH in homozygous carriers of the CTA haplotype was higher than that in heterozygous carriers and non-carriers (p=0.022). Furthermore, CTG, a rare haplotype, was strongly associated with infantile spasms (p=0.013). The results suggest that haplotypes of GRIN1 may influence responsiveness to ACTH. The findings necessitate further study for confirmation.

GRIN1 Regulates μ-Opioid Receptor Activities by Tethering the Receptor and G Protein in the Lipid Raft

The lipid raft location of mu-opioid receptor (MOR) determines the receptor activities. However, the manner in which MOR is anchored within the lipid rafts is undetermined. Using the targeted proteomic approach and mass spectrometry analyses, we have identified GRIN1 (G protein-regulated inducer of neurite outgrowth 1) can tether MOR with the G protein alpha-subunit and subsequently regulate the receptor distribution within the lipid rafts. Glutathione S-transferase fusion pulldown and receptor mutational analyses indicate that GRIN1-MOR interaction involves a receptor sequence (267)GSKEK(271) within the MOR third intracellular loop that is not involved in Galpha interaction. The GRIN1 domains involved in MOR interaction are also distinct from those involved in Galpha interaction. Pertussis toxin pretreatment reduced the amount of GRIN1 co-immunoprecipitated with MOR but not the amount with Galpha. Furthermore, overexpression of GRIN1 significantly enhanced the amount of MOR in lipid raft and the receptor signaling magnitude as measured by Src kinase activation. Such increase in MOR signaling was demonstrated further by determining the GRIN1-dependent pertussis toxin-sensitive neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control neuroblastoma N2A cells, overexpression of GRIN1 resulted in the increase in etorphine- and non-morphine-induced neurite outgrowth in these cells. Knocking down endogenous GRIN1 by small interfering RNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by methyl-beta-cyclodextrin also blocked neurite outgrowth. Hence, by tethering Galpha with MOR, GRIN1 stabilizes the receptor within the lipid rafts and potentiates the receptor signaling in the neurite outgrowth processes.

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Nicotine acetylcholine receptors (nAChRs) comprise a family of ligand-gated channels widely expressed in the mammalian brain. The beta2 subunit is an abundant protein subunit critically involved in the cognitive and behavioral properties of nicotine as well as in the mechanisms of nicotine addiction. In this work, we used matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS) to uncover protein interactions of the intracellular loop of the beta2 subunit and components of immunoprecipitated beta2-nAChR complexes from mouse brain. Using the beta2-knockout mouse to exclude nonspecific binding to the beta2 antibody, we identify 21 nAChR-interacting proteins (NIPs) expressed in brain. Western blot analysis confirmed the association between the beta2 subunit and candidate NIPs. Based on their functional profiles, the hypothesis is suggested that the identified NIPs can regulate the trafficking and signaling of the beta2-nAChR. Interactions of the beta2 subunit with NIPs such as G protein alpha, G protein-regulated inducer of neurite outgrowth 1, and G protein-activated K(+) channel 1 suggest a link between nAChRs and cellular G protein pathways. These findings reveal intracellular interactions of the beta2 subunit and may contribute to the understanding of the mechanisms of nAChR signaling and trafficking in neurons.

Functional Characterization of Gαo Signaling through G Protein-Regulated Inducer of Neurite Outgrowth 1

G protein-regulated inducer of neurite outgrowth 1 (GRIN1) was initially identified as a binding protein for guanosine 5'-3-O-(thio)triphosphate-bound Galphaz. GRIN1 is specifically expressed in brain and interacts selectively with activated alpha subunits of the Gi subfamily. GRIN1 colocalizes with Galphao at the growth cone of neuronal cells and promotes neurite extension in Neuro2a cells when coexpressed with constitutively active mutant GalphaoQ205L. These results suggest that GRIN1 functions as a downstream target for Galphao. However, GRIN1 does not contain domains that are homologous to known signaling motifs. To understand the mechanisms of Galphao-GRIN1 pathway, we analyzed functional domains of GRIN1 that are involved in binding with Galphao or with its targeting to the plasma membrane. Using pull-down assays with glutathione S-transferase-fused GRIN1 deletion mutants, Galphao binding regions were localized to amino acid residues 716 to 746 and 797 to 827 of GRIN1. The Galphao binding region of GRIN1 did not demonstrate GTPase accelerating activity for Galphao. GRIN1 localized in the cell periphery in Neuro2a cells, and two cysteine residues at C-terminal region of GRIN1 (Cys818 and Cys819) were shown to be critical for its membrane targeting. Coexpression of GRIN1 with GalphaoQ205L or GRIN1Delta(717-827), which lacks Galphao binding region, promoted microspike formation in Swiss 3T3 cells or neurite extension in Neuro2a cells. The dominant-negative mutant of Cdc42 blocked these morphological changes. Coexpression of GRIN1 and GalphaoQ205L stimulated the formation of GTP-bound Cdc42 in Swiss 3T3 cells. These results suggest that the binding of activated Galphao to GRIN1 induces activation of Cdc42, which leads to morphological changes in neuronal cells.