Evidence for a subtype of insulin-like growth factor I receptor in brain

We examined the structure of receptors for insulin-like growth factor I (IGF-I), insulin, and epidermal growth factor (EGF) in human brain and human placenta using affinity cross-linking procedures and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In human brain, proteins specifically cross-linked to 125I-IGF-I, 125I-insulin, and 125I-EGF had apparent molecular weights of 120,000, 115,000 and 170,000, respectively. In human placenta, proteins cross-linked to 125I-IGF-I and 125I-insulin were 10 kDa larger than the corresponding subunits in brain. The receptor labeled by 125I-EGF in placenta was indistinguishable from the EGF receptor in brain. The size discrepancy of IGF-I receptors in brain and placenta was no longer apparent after removing the carbohydrate moieties of the proteins with endo-beta-N-acetylglucosaminidase F (EndoF). Furthermore, the brain IGF-I receptor was not cleaved by neuraminidase, whereas, the placental IGF-I receptor had increased mobility on SDS gels following neuraminidase treatment. The results indicate that receptors for IGF-I and insulin in human brain are structurally distinct from the corresponding receptors in human placenta, the structural heterogeneity of the receptors involves differences in N-linked glycosylation, particularly the terminal processing steps, and EGF receptors are present in human brain and human placenta but are structurally similar in these tissues. We conclude that there is a selective modification in the glycosylation of receptors for IGF-I and insulin in brain.     

Anti-formyl peptide antibodies

Antibodies that selectively bind to N-formylmethionyl leucyl phenylalanine (fMLF, also known as fMLP) have been generated. These antibodies bound to fMLF with higher affinity than to non-formylated peptide MLF: the differences in the binding energies between fMLF and MLF were 1.4->2.1 kcal/mol.     

Insulin receptors in the mammalian central nervous system: binding characteristics and subunit structure

Insulin receptors in rat and human central nervous system have been identified by binding of 125I-insulin on purified synaptic plasma membranes; affinity labelling of receptors by chemical cross-linking 125I-insulin; or phosphorylation of receptors with [gamma-32P]ATP. Brain insulin receptors showed significant differences in their binding characteristics and subunit structure when compared with receptors in other tissues like adipose and liver cells: absence of negatively cooperative interactions; a distinct binding specificity i.e. porcine proinsulin, coypu insulin and insulin-like growth factor I and II showed 2-5 times higher binding affinity in brain than in other cell types; a smaller molecular size of the brain receptor alpha-subunit than in other tissues (Mr approximately 115,000 instead of 130,000). In contrast, the size (Mr approximately 94,000) and function of the insulin receptor beta-subunit kinase was identical with that described in other cells. We conclude, that insulin receptors in mammalian brain represent a receptor subtype which may mediate growth rather than metabolic activity of insulin.     

Subcellular localization of full-length human myeloid leukemia factor 1 (MLF1) is independent of 14-3-3 proteins

Myeloid leukemia factor 1 (MLF1) is associated with the development of leukemic diseases such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, information on the physiological function of MLF1 is limited and mostly derived from studies identifying MLF1 interaction partners like CSN3, MLF1IP, MADM, Manp and the 14-3-3 proteins. The 14-3-3-binding site surrounding S34 is one of the only known functional features of the MLF1 sequence, along with one nuclear export sequence (NES) and two nuclear localization sequences (NLS). It was recently shown that the subcellular localization of mouse MLF1 is dependent on 14-3-3 proteins. Based on these findings, we investigated whether the subcellular localization of human MLF1 was also directly 14-3-3-dependent. Live cell imaging with GFP-fused human MLF1 was used to study the effects of mutations and deletions on its subcellular localization. Surprisingly, we found that the subcellular localization of full-length human MLF1 is 14-3-3-independent, and is probably regulated by other as-yet-unknown proteins.     

mCPP but not TFMPP is an antagonist at cardiac 5HT3 receptors.

The prototypic arylpiperazines, meta-chlorophenylpiperazine (mCPP), meta-trifluoromethylphenylpiperazine (TFMPP) and quipazine are widely studied serotonergic ligands with nonselective effects at 5HT1 and 5HT2 receptor subtypes. The present study was designed to compare the affinities of these arylipiperazines at 5HT3 receptors, and to determine agonist or antagonist activity at 5HT3 receptors. Quipazine showed high affinity at brain 5HT3 receptors (IC50 = 4.4 nM) and was a potent agonist of the von Bezold-Jarisch reflex in anesthetized rats, a response mediated by cardiac 5HT3 receptors. In concentrations that activated 5HT3 receptors, quipazine also antagonized serotonin-induced bradycardia in anesthetized rats. Taken together, these data suggest that quipazine is an agonist/antagonist with high affinity at 5HT3 receptors in both brain and cardiac tissue. Although mCPP also showed relatively high affinity at brain 5HT3 receptors (IC50 = 61.4 nM), it did not activate the von Bezold-Jarisch reflex; instead, mCPP potently antagonized serotonin-induced bradycardia. Thus, mCPP acts as an antagonist at 5HT3 receptors in the periphery. Although both quipazine and mCPP possessed relatively high affinity at brain 5HT3 receptors, TFMPP did not bind appreciably to 5HT3 receptors in brain (IC50 = 2373 nM) and neither activated nor inhibited cardiac 5HT3 receptors. That TFMPP did not interact with 5HT3 receptors, whereas quipazine and mCPP did, is in marked contrast to the similar effects of all three arylpiperazines at other serotonin receptors. The selectivity of TFMPP for 5HT1 and 5HT2 receptors (i.e., its minimal affinity for 5HT3 receptors) suggests that this arylpiperazine may be a preferred ligand relative to mCPP when studying 5HT1 or 5HT2 receptor mediated responses.     

Structural and Functional Characteristics of Insulin Receptors in Rat Neuroblastoma Cells

Insulin receptors were detected in a variety of rat neuroblastoma and glioma cell lines. The binding of 125I-insulin to B103 neuroblastoma cells had characteristics typical of insulin receptors in other tissues, including high affinity for insulin, low affinity for insulin-like growth factor I (IGF-I), and curvilinear Scatchard plots. Using photoaffinity labeling procedures and sodium dodecyl sulfate (SDS) gel electrophoresis to analyze the subunit structure of insulin receptors in B103 cells, the predominantly labeled protein had an apparent molecular weight of 125K and the mobility of this protein was shifted after removal of sialic acid residues. On the basis of size and susceptibility to neuraminidase, the insulin binding subunit in neuroblastoma cells was identical to the alpha-subunit of insulin receptors in adipocytes and different from the 115K subunit found in brain. The presence of an "adipocyte" form of the insulin receptor in clonal cells derived from brain is probably a consequence of transformation and results from more extensive oligosaccharide processing of the 115K receptor expressed in normal brain cells. The fully glycosylated receptors in neuroblastoma cells were capable of exerting functions typical of insulin receptors in adipocytes such as internalization of insulin and stimulation of glucose transport.     

Identification and characterization of receptors for tumor necrosis factor-alpha in the brain.

Specific receptors for murine TNF have been identified in homogenates of rodent brain. These receptors are saturable and bind TNF with sufficient affinity to ensure occupancy by cytokine elaborated during infection. 125I-mTNF was detected in four specific complexes of Mr 130,000, 90,000, 66,000 and 60,000 after affinity labeling. Solubilization of brain membranes into detergent increased binding capacity 4-fold which indicates the presence of latent receptors for mTNF in the brain. Specific binding was greatest in the brainstem, least in the cerebellum and was also detected in the cortex, thalamus and basal ganglia.     

Shuttling imbalance of MLF1 results in p53 instability and increases susceptibility to oncogenic transformation

Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cell's susceptibility to oncogenic transformation.     

Clozapine: selective labeling of sites resembling 5HT6 serotonin receptors may reflect psychoactive profile.

BACKGROUND: Clozapine, the classic atypical neuroleptic, exerts therapeutic actions in schizophrenic patients unresponsive to most neuroleptics. Clozapine interacts with numerous neurotransmitter receptors, and selective actions at novel subtypes of dopamine and serotonin receptors have been proposed to explain clozapine''s unique psychotropic effects. To identify sites with which clozapine preferentially interacts in a therapeutic setting, we have characterized clozapine binding to brain membranes. MATERIALS AND METHODS: [3H]Clozapine binding was examined in rat brain membranes as well as cloned-expressed 5-HT6 serotonin receptors. RESULTS: [3H]Clozapine binds with low nanomolar affinity to two distinct sites. One reflects muscarinic receptors consistent with the drug''s anticholinergic actions. The drug competition profile of the second site most closely resembles 5HT6 serotonin receptors, though serotonin itself displays low affinity. [3H]Clozapine binding levels are similar in all brain regions examined with no concentration in the corpus striatum. CONCLUSIONS: Besides muscarinic receptors, clozapine primarily labels sites with properties resembling 5HT6 serotonin receptors. If this is also the site with which clozapine principally interacts in intact human brain, it may account for the unique beneficial actions of clozapine and other atypical neuroleptics, and provide a molecular target for developing new, safer, and more effective agents.     

Correlating Function and Imaging Measures of the Medial Longitudinal Fasciculus

Objective

To test the validity of diffusion tensor imaging (DTI) measures of tissue injury by examining such measures in a white matter structure with well-defined function, the medial longitudinal fasciculus (MLF). Injury to the MLF underlies internuclear ophthalmoparesis (INO).

Methods

40 MS patients with chronic INO and 15 healthy controls were examined under an IRB-approved protocol. Tissue integrity of the MLF was characterized by DTI parameters: longitudinal diffusivity (LD), transverse diffusivity (TD), mean diffusivity (MD) and fractional anisotropy (FA). Severity of INO was quantified by infrared oculography to measure versional disconjugacy index (VDI).

Results

LD was significantly lower in patients than in controls in the medulla-pons region of the MLF (p < 0.03). FA was also lower in patients in the same region (p < 0.0004). LD of the medulla-pons region correlated with VDI (R = -0.28, p < 0.05) as did FA in the midbrain section (R = 0.31, p < 0.02).

Conclusions

This study demonstrates that DTI measures of brain tissue injury can detect injury to a functionally relevant white matter pathway, and that such measures correlate with clinically accepted evaluation indices for INO. The results validate DTI as a useful imaging measure of tissue integrity.     

Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3

Myeloid leukemia factor 1 (MLF1) was first identified as the leukemic fusion protein NPM-MLF1 generated by the t(3;5)(q25.1;q34) chromosomal translocation. Although MLF1 expresses normally in a variety of tissues including hematopoietic stem cells and the overexpression of MLF1 correlates with malignant transformation in human cancer, little is known about how MLF1 is involved in the regulation of cell growth. Here we show that MLF1 is a negative regulator of cell cycle progression functioning upstream of the tumor suppressor p53. MLF1 induces p53-dependent cell cycle arrest in murine embryonic fibroblasts. This action requires a novel binding partner, subunit 3 of the COP9 signalosome (CSN3). A reduction in the level of CSN3 protein with small interfering RNA abrogated MLF1-induced G1 arrest and impaired the activation of p53 by genotoxic stress. Furthermore, ectopic MLF1 expression and CSN3 knockdown inversely affect the endogenous level of COP1, a ubiquitin ligase for p53. Exogenous expression of COP1 overcomes MLF1-induced growth arrest. These results indicate that MLF1 is a critical regulator of p53 and suggest its involvement in leukemogenesis through a novel CSN3-COP1 pathway.     

An endogenous morphine-like factor in mammalian brain.

An endogenous morphine-like substance (MLF) found in rat and calf brains has a regional distribution correlating with that of opiate receptors, with the highest levels in the caudate and negligible amounts in the cerebellum. In binding assays MLF behaves like an opiate agonist. Sodium ion and enzyme and reagent treatment of membranes decrease its potency and manganese ion enhances it. MLF is localized in synaptosomal fractions, stored in an osmotically labile compartment, and can be degraded by carboxypeptidase A and leucine amino peptidase, implying a peptide structure. Its molecular weight is about 1000 as determined by gel chromatography.     

An oxidation resistant radioligand for corticotropin-releasing factor receptors.

The methionine residues in Tyr-corticotropin-releasing factor (CRF) and Tyr-sauvagine radioligands are subject to oxidation, which renders them biologically inactive. Therefore [Tyr(0,) Gln(1,) Leu(17)]sauvagine (YQLS), in which the methionine was replaced with leucine was synthesized and labeled with (125)Iodine using chloramine-T. Mass spectroscopy revealed that chloramine-T-treatment did not oxidize YQLS. (125)I-YQLS bound with high affinity to cells expressing the murine CRF receptor 1 (CRFR1), CRF receptor 2 (CRFR2), and the mouse brain regions known to express both CRF receptors. (125)I-YQLS chemically cross-linked to CRFR1. In conclusion, (125)I-YQLS is oxidation-resistant, high affinity radioligand that can be chemically cross-linked to the CRF receptors.     

Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R(3) -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were performed to distinguish [(125) I]IGF-I binding to IGF-I receptors and IGFBPs. Specific [(125) I]IGF-I binding in brain regions and the posterior pituitary was completely displaced by DES(1-3)IGF-I and R(3) -IGF-I, indicating binding to IGF-I receptors. In contrast, [(125) I]IGF-I binding in the anterior pituitary was not displaced by DES(1-3)IGF-I and R(3) -IGF-I, suggesting binding to an IGF-binding site that is different from the IGF-I receptor. Binding affinity of IGF-I to this site was about 10-fold lower than for the IGF-I receptor. Using western immunoblotting we were also unable to detect IGF-I receptors in human anterior pituitary. Instead, western immunoblotting and immunoprecipitation experiments showed a 150-kDa IGFBP-3-acid labile subunit (ALS) complex in the anterior pituitary and not in the posterior pituitary and other brain regions. RT-PCR experiments showed the expression of ALS mRNA in human anterior pituitary indicating that the anterior pituitary synthesizes ALS. In the brain regions and posterior pituitary, IGFBP-3 was easily washed away during pre-incubation procedures as used in the [(125) I]IGF-I binding experiments. In contrast, the IGFBP-3 complex in the anterior pituitary could not be removed by these washing procedures. Our results indicate that the human anterior pituitary contains a not previously described tightly cell membrane-bound 150-kDa IGFBP-3-ALS complex that is absent in brain and posterior pituitary.     

Structural insights of the MLF1/14-3-3 interaction

Myeloid leukaemia factor 1 (MLF1) binds to 14-3-3 adapter proteins by a sequence surrounding Ser34 with the functional consequences of this interaction largely unknown. We present here the high-resolution crystal structure of this binding motif [MLF1(29-42)pSer34] in complex with 14-3-3ε and analyse the interaction with isothermal titration calorimetry. Fragment-based ligand discovery employing crystals of the binary 14-3-3ε/MLF1(29-42)pSer34 complex was used to identify a molecule that binds to the interface rim of the two proteins, potentially representing the starting point for the development of a small molecule that stabilizes the MLF1/14-3-3 protein-protein interaction. Such a compound might be used as a chemical biology tool to further analyse the 14-3-3/MLF1 interaction without the use of genetic methods. Database Structural data are available in the Protein Data Bank under the accession number(s) 3UAL [14-3-3ε/MLF1(29-42)pSer34 complex] and 3UBW [14-3-3ε/MLF1(29-42)pSer34/3-pyrrolidinol complex] Structured digital abstract ? 14-3-3 epsilon?and?MLF1?bind?by?x-ray crystallography?(View interaction) ? 14-3-3 epsilon?and?MLF1?bind?by?isothermal titration calorimetry?(View Interaction:?1,?2).     

Physiological roles of urocortins, human homologues of fish urotensin I, and their receptors

Urocortin 1, a human homologue of fish urotensin I, together with its related-compounds (urocortins 2 and 3), comprises a distinct family of stress peptides. Urocortin 1 has a high affinity for both corticotropin-releasing factor (CRF) type 1 receptor (CRF1) and CRF type 2 receptor (CRF2), and urocortins 2 and 3 have a high affinity for CRF2, while CRF has a low affinity for CRF2 and a high affinity for CRF1. These differences of the binding affinity with receptors make the biological actions of these peptides. Besides the binding affinity with receptors, the limited overlap of the distribution of CRF and urocortins may also contribute to the differences of physiological roles of each peptide. Urocortins show 'stress-coping' responses such as anxiolysis and dearousal in the brain. In the periphery, recent studies show the potent effects of urocortins on the cardiovascular and immune systems. In this review article, we take a look over the series of peptides included in this family, especially in terms of the versatility of biological actions, along with the various characters of the receptors.     

Purification of basic FGF receptors from rat brain

Receptor molecules for basic fibroblast growth factor (bFGF) were isolated from rat brain by a novel and rapid procedure and characterized. Purification was performed by wheatgerm agglutinin (WGA) gel affinity chromatography in combination with bFGF gel affinity chromatography, utilizing a novel elution method involving heparin. The eluted proteins were active in binding bFGF and were separated as two bands with respective molecular masses of 140 kDa and 110 kDa on SDS-PAGE. More than half of this bFGF-binding activity was lost after 16 h at 4 degrees C. Thus, bFGF receptors were purified as labile glycoconjugates.     

Substance P receptors in mammalian central nervous system.

1. Multiple distinct affinity states or sites of substance P (SP) receptors exist in freshly-prepared rat brain membranes. 2. Substance P receptors may couple with islet-activating protein (pertussis toxin) sensitive GTP-binding protein(s). 3. Substance P receptors may be regulated Mg2+ and Na+ in an opposite manner. 4. Some important factor(s), in addition to GTP-binding protein, appear to be involved in SP binding activity. 5. An apparent molecular weight of the SP binding site is approximately 46,000 Da.     

Structural derivatives of pindolol: relationship between in vivo and in vitro potencies for their interaction with central beta-adrenergic receptors

Although (-)-125I-iodopindolol (IPIN) can be used to label beta-adrenergic receptors in the central nervous system (CNS) in vivo, use of this ligand for receptor imaging studies in humans may be limited due to its relatively poor penetration into the CNS. A series of derivatives related to pindolol was therefore studied in an effort to determine the factors that might influence the penetration and interaction of these compounds with central beta-adrenergic receptors in vivo. Evaluation of the ability of these derivatives to displace the binding of IPIN in the brain upon systemic administration provides an assessment of whether the derivatives penetrate and interact with central beta-adrenergic receptors in vivo. Multiple regression analyses showed that the most important factor which influences the ability of the pindolol derivatives to penetrate into the brain and interact with beta-adrenergic receptors in vivo is the affinity of the derivatives for binding to beta-adrenergic receptors in vitro. Both lipophilicity and the molecular weights of the derivatives are important secondary factors which influence their in vivo potency.