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PubMed1988

Purification and NMR studies of [methyl-13C]methionine-labeled truncated methionyl-tRNA synthetase.

A procedure for the rapid purification of a truncated form of the Escherichia coli methionyl-tRNA synthetase has been developed. With this procedure, final yields of approximately 3 mg of truncated methionyl-tRNA synthetase per gram of cells, carrying the plasmid encoding the gene for the truncated synthetase [Barker, D.G., Ebel, J.-P., Jakes, R., & Bruton, C.J. (1982) Eur. J. Biochem. 127, 449], can be obtained. The catalytic properties of the purified truncated synthetase were found to be identical with those of the native dimeric and trypsin-modified methionyl-tRNA synthetases. A rapid procedure for obtaining milligram quantities of the enzyme is necessary before the efficient incorporation of stable isotopes into the synthetase becomes practical for physical studies. With this procedure, truncated methionyl-tRNA synthetase labeled with [methyl-13C]methionine was purified from an Escherichia coli strain auxotrophic for methionine and containing the plasmid encoding the gene for the truncated methionyl-tRNA synthetase. Both carbon-13 and proton observe-heteronuclear detect NMR experiments were used to observe the 13C-enriched methyl resonances of the 17 methionine residues in the truncated synthetase. In the absence of ligands, 13 of the 17 methionine residues could be resolved by carbon-13 NMR. Titration of the synthetase, monitoring the chemical shifts of resonances B and M (Figure 3), with a number of amino acid ligands and ATP yielded dissociation constants consistent with those derived from binding and kinetic data, indicating active site binding of the ligands under the conditions of the NMR experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed2001

Effects of carboxyl-terminal truncations on the activity and solubility of human monoamine oxidase B.

Monoamine oxidase is an outer mitochondrial membrane protein that catalyzes the deamination of a number of neurotransmitters and dietary amines. To determine the roles of the carboxyl-terminal amino acids on the activity and solubility of human monoamine oxidase (MAO B), 10 sequential mutants were made with stop codons at amino acid positions 511, 504, 498, 492, 486, 481, 476, 467, 417, and 397, respectively. All truncated mutants were expressed in Sf21 insect cells using baculovirus, and the enzyme kinetic parameters were determined. Truncations at amino acid positions 511, 504, and 498 slightly decreased MAO B catalytic activity and had no significant changes on deprenyl inhibition. Further deletions up to amino acid 417 decreased the specific activity 10--100-fold without significant changes of the K(m) for phenylethylamine or dopamine or the IC(50) for deprenyl and clorgyline. The truncation mutant C397, which lacks covalently attached FAD, was inactive. Progressive carboxyl-terminal truncations up to position 481 were correlated with increased solubility of MAO B mutants. 47% of the activity of the truncated C481 was found in the 105,000 x g supernatant in the absence of detergent. However, further truncated mutants, C476, C467, and C417, remained associated with the membrane fraction. In contrast to crude homogenate, the water-soluble C481 mutant was rapidly inactivated at 4 degrees C and 37 degrees C, which indicates that the membrane environment is required for the stability of MAO B. Expression of the green fluorescent protein-MAO B C481 fusion protein revealed that this mutant was located in the cytoplasm, whereas its counterpart in MAO A, truncated mutant C490, was located on the mitochondria. These results suggest that the carboxyl-terminal amino acid residues 417--520 of MAO B are not directly involved in the active site but are required for maintaining the appropriate conformation and interaction with the outer mitochondrial membrane. The different solubilities of the various carboxyl-terminal truncation mutants indicate that the interaction of MAO B with mitochondrial membrane is not simply anchoring through the carboxyl-terminal hydrophobic tail. Further, our results suggest that the carboxyl-terminal of MAO A and B plays different roles in mitochondrial attachment.

Amino Acid Sequence↗

PubMed1988

Expression and characterization of a truncated murine Fc gamma receptor.

We have isolated a recombinant secreted Fc gamma R beta molecule by deletion of the transmembrane and cytoplasmic domains encoding sequence from a Fc gamma R beta 1 cDNA clone, and insertion of the truncated cDNA into a eukaryotic expression vector, pcEXV-3. To express and amplify the production of the truncated Fc gamma R beta molecule, we transfected the truncated cDNA plasmid into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and amplified the gene products with methotrexate. The resulting cell line secretes 2-3 micrograms/ml/24 h of truncated Fc gamma R beta, which can be readily purified by affinity chromatography on IgG-Sepharose. The truncated Fc gamma R beta has a Mr of 31-33,000 on SDS-PAGE and is glycosylated. N-glycosidase F cleavage reduces the Mr to 19,000, consistent with the size of the truncated product, 176 amino acid residues. There are two disulfide bonds in the protein. Binding of immune complexes formed between DNP20BSA and anti-DNP mAbs reveals better binding of IgG1 aggregates than that of IgG2b and IgG2a aggregates. The binding of the immune complexes was somewhat better at more acidic pH, in contrast to previous experiments with binding of purified Fc gamma R to immune complex-coated beads. We were surprised to observe that the truncated Fc gamma R beta did not react with the anti-Fc gamma R mAb 6B7C. Previous work had shown that 6B7C reacts with Fc gamma R on immunoblots, fails to bind to the surface of resting B cells and peritoneal macrophages, but does bind to macrophage cell lines and LPS-stimulated B cells. We show, by binding of mAb 6B7C to a peptide conjugate, that the 6B7C epitope lies within residues 169-183 of the intact Fc gamma R beta, which is just outside the plasma membrane. The availability of the truncated Fc gamma R beta in microgram quantities should facilitate further analysis of structure and function of these receptors.

Amino Acid Sequence↗

PubMed1995

Truncation artifact reduction in transmission CT for improved SPECT attenuation compensation.

Transmission computed tomography (TCT) has been shown to be an accurate method of acquiring non-uniform attenuation maps for single-photon emission computed tomography (SPECT) attenuation compensation. One commonly encountered problem, especially for convergent beam geometries, is image truncation. We describe two methods for reducing associated truncation artifacts, with the goal of improving SPECT attenuation compensation without unduly increasing imaging or reconstruction times: (i) the two-scan method, which reduces the degree of truncation by combining two short-duration, patient-shifted scans, and (ii) a quantitative extrapolation method, which fills in truncated projections accounting for the correct amount of attenuating medium in the slice. The methods are evaluated by imaging two phantoms on a fan beam TCT system. Projection sets are truncated to various degrees with software, and reconstructed images are compared to both untruncated filtered backprojection and iterative reconstructions of truncated data. A fundamental analysis of SPECT attenuation factors is performed, and attenuation compensation of a cardiac insert is analysed. Results indicate the two-scan method can effectively reduce the degree of truncation in many cases, and the quantitative extrapolation method greatly improves SPECT attenuation compensation over using truncated maps.

Artifacts↗

PubMed2026

C-Terminal Truncation and Fusion Partner Determine Oncogenicity of FGFR3.

UNLABELLED: Genomic alterations affecting components of the fibroblast growth factor (FGF) signaling axis can trigger aberrant pathway activation and tumor development. Genomic truncation of the FGF receptor 2 (FGFR2) exon 18 (E18) disrupts the FGFR2 carboxy (C)-terminal tail, acting as a potent driver alteration across multiple tumor types. In this study, we analyzed human oncogenomic datasets to reveal that E18 truncations are similarly prevalent in FGFR3, an FGFR2 paralog. FGFR3 E18 truncations primarily occur due to rearrangements (RE) that involve transforming acidic coiled-coil-containing protein 3 (TACC3), resulting in FGFR3ΔE18-TACC3 gene fusions. In contrast to E18-truncated FGFR2, functional in vitro and in vivo examination of Fgfr3 variants demonstrated that the truncation of Fgfr3 E18 is insufficient to promote oncogenic activity in cell lines or in the lungs and mammary glands of mice. Only the combination of an Fgfr3 E18 truncation with a RE partner gene that encodes a receptor-dimerizing domain resulted in the development of tumors, which were sensitive to FGFR inhibition. Overall, these findings suggest that patients with cancers that are positive for rearranged FGFR3, resulting in E18 truncation and a fusion to dimerizing partners, should be considered for FGFR-targeted therapies. SIGNIFICANCE: FGFR3, unlike its paralog FGFR2, requires both a C-terminal truncation and fusion to a partner gene that retains the expression of a dimerizing domain to effectively drive oncogenic signaling and tumorigenesis.

Receptor, Fibroblast Growth Factor, Type 3↗

PubMed2003

Water permeability of C-terminally truncated aquaporin 0 (AQP0 1-243) observed in the aging human lens.

PURPOSE: To first assess the distribution of posttranslationally truncated products of aquaporin 0 (AQP0) in dissected sections of a normal human lens and to determine the effect of backbone cleavage on the water permeability of AQP0. METHODS: A 27-year-old lens was concentrically dissected into six sections. Membrane protein was isolated from each section and cleaved with cyanogen bromide, and the peptides were separated and analyzed by reverse-phase (RP)-HPLC-mass spectrometry (MS). The sites of posttranslational AQP0 C-terminal truncation were determined by mass spectrometry. Truncated forms of AQP0 were expressed in a Xenopus laevis oocyte system, and the effect of truncation on AQP0 water permeability was assessed in an oocyte osmotic swelling assay. RESULTS: The extent of truncation at many sites within the C terminus increased with fiber cell age, and the effects of truncations after residues 234, 238, and 243 on AQP0 water permeability were examined. Truncation after residue 243 resulted in an approximate 15% decrease in permeability compared with the full-length protein, AQP0 1-263. However, rather than a direct effect on water transport, analysis of surface protein expression indicated that the decrease in permeability was a result of less efficient protein trafficking to the oocyte surface and that the permeabilities of full-length and 1-243 AQP0 were indistinguishable. Further, C-terminal truncation of AQP0 to 1-234 and 1-238, completely impaired trafficking into the plasma membrane, precluding the measurement of permeability. CONCLUSIONS: These data provide evidence that loss of 20 amino acids from the C terminus may not directly affect the ability of AQP0 to transport water.

Adult↗

PubMed1995

Selective binding and internalisation by truncated receptors restrict the availability of BDNF during development.

The tyrosine kinase receptor trkB is thought to mediate the biological actions of brain-derived neurotrophic factor. This receptor is expressed by a large variety of neurons during development. Truncated trkB molecules lacking the tyrosine kinase domain have also been described, but their functions remain elusive. In order to gain insight into their role, we studied the pattern of expression and properties of these truncated receptors in the chick embryo. mRNA coding for truncated trkB was detected already early during neurogenesis and in situ hybridisation experiments indicated that the expression was in non-neuronal cells, as previously observed in the brain of adult rodents. Ependymal and leptomeningeal cells expressing high levels of truncated trkB were found to completely surround the developing brain and the spinal cord throughout development. In the otic vesicle, mesenchymal cells expressing truncated trkB surround cells producing brain-derived neurotrophic factor, as well as neurons expressing trkB with its tyrosine kinase domain. Non-neuronal cells were found not to express trkB mRNA coding for the tyrosine kinase domain. Studies with radioiodinated brain-derived neurotrophic factor performed on frozen sections of the chick embryo revealed that non-neuronal cells expressing truncated trkB bind brain-derived neurotrophic factor with high affinity and selectivity. In addition, experiments with dissociated leptomeningeal cells revealed that binding is rapidly followed by selective internalisation of the ligand. These results suggest that truncated trkB molecules form an efficient and selective barrier preventing the diffusion of brain-derived neurotrophic factor and eliminating it by internalisation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative Splicing↗

PubMed2003

SsrA-mediated trans-translation plays a role in mRNA quality control by facilitating degradation of truncated mRNAs.

An important unsolved question regarding the bacterial SsrA system is the fate of target mRNAs replaced by SsrA RNA during trans-translation. The aim of the present study is to address the potential role of SsrA system in mRNA quality control, focusing on truncated mRNAs that are expected to arise from 3'-to-5' exonucleolytic attack. We found that significant amounts of truncated mRNAs and polypeptides were produced from genes lacking a rho-independent terminator in SsrA-deficient cells. These truncated mRNAs, hence truncated polypeptides, were no longer observed in the presence of SsrA RNA. The data indicate that the SsrA system facilitates degradation of "nonstop" mRNAs by presumably removing the stalled ribosomes. Furthermore, analysis of affinity-purified proteins indicated that truncated polypeptides could be produced even from a gene with an intact rho-independent terminator, although less efficiently, implying that C-terminally truncated proteins and 3'-truncated mRNA may be produced from virtually all protein-coding genes. We conclude that the SsrA system not only promotes the degradation of incomplete polypeptides but also minimizes the synthesis of incomplete polypeptides by facilitating the degradation of truncated mRNAs that are produced in cells.

Base Sequence↗

PubMed2002

Truncated amplification: a method for high-fidelity template-driven nucleic acid amplification.

The error rate of conventional PCR is problematic when amplifying from single cells or amplifying segments for protein functional analysis by in vitro translation. We describe truncated amplification, a method for high-fidelity amplification in which DNA polymerase errors are not propagated efficiently and original DNA templates exert greater influence on the amplification process. Truncated amplification utilizes pairs of oligonucleotides and thermal cycling, but it differs from PCR. Truncated amplification amplifies non-exponentially with one or two chimeric oligonucleotides and produces truncated terminal products that are no more than three rounds of replication from the original template. Exon 6 of the p53 gene was utilized as a model system to demonstrate proof of principle. Chimeric oligonucleotides containing three 3'-->5' reversed-deoxynucleotides or 2'-OMe-ribonucleotides at 6-8 nucleotides from the 3 'terminus retained sequence specificity and primer extension activity. With PfuTurbo but not with Taq or Vent (exo-) DNA polymerases, the modified nucleotides completely truncated the DNA polymerase elongation. The resulting truncated terminal products are not templates for further amplification because of the short length of the 3' complementary region. Truncated amplific ation can amplify quadratically or geometrically depending on whether two or one chimeric oligonucleotides are used. Truncated amplification is a promising approach when template-driven amplification is desired to increase thefrequency of error-free products.

Base Sequence↗

PubMed2000

Performance of ordered-subset reconstruction algorithms under conditions of extreme attenuation and truncation in myocardial SPECT.

UNLABELLED: We studied the bias and variance characteristics of the ordered-subset expectation maximization (OSEM) and rescaled block-iterative EM (RBIEM) iterative reconstruction algorithms in myocardial SPECT under extreme, but realistic, conditions. METHOD: We used the 2-dimensional mathematic cardiac torso phantom to simulate 2 patient anatomies: a large male with a raised diaphragm and a female with large breast size, approximating extreme cases of attenuation conditions found in the clinic. For each anatomy, realistic 201Tl projection data were simulated for a 180 degrees acquisition arc. Three cases of truncation for a 90 degrees-configured dual detector system were simulated: no truncation, moderate truncation, and extreme truncation. For each case, an ensemble of 250 noise simulations was generated, and each noisy dataset was reconstructed with the OSEM and RBIEM algorithms. The reconstructions modeled only the effects of nonuniform attenuation and used a range of subset configurations. Over the ensemble, we computed means and variances of activity in 8 regions of interest (ROIs) in the heart as a function of iteration. RESULTS: Under conditions of no truncation and moderate truncation, the results from OSEM and RBIEM were very close to those from maximum-likelihood EM (MLEM); in all cases, the difference in ROI means was <2.5%. For extreme truncation, the errors increased to as much as 11% with OSEM, but these were no greater than the errors for MLEM under the same conditions. The OSEM algorithm with 2 views per subset was found to result in much higher variance of ROI estimates for the same bias as compared with RBIEM or OSEM with 4 or more views per subset. CONCLUSION: The OSEM and RBIEM algorithms are at least as robust to highly attenuating patients and truncation as MLEM algorithm and can be adequate substitutes for MLEM, even in extreme cases. Clinical users should apply the smallest number of subsets that can be accommodated by allowable processing time to reduce image noise and variance in quantitative estimates.

Algorithms↗

PubMed2004

[Construction and functional study of truncated beta catenin genes].

AIM: To observe the subcellular location and the change of the transcriptional activity of truncated beta catenins in mammalian cells. METHODS: The full length wild type beta catenin gene was cloned by RT-PCR. The truncated beta catenins were constructed by deleting the N terminal, the C terminal or the Armadillo domain sequences, and were inserted into the EGFP expression vector. After they were transfected into 293 cells, the transcriptional activity of truncated beta catenins in 293 cells was detected by dual luciferase reporter system. The subcellular location of truncated beta catenin in transfected COS7 cells was observed under fluorescence microscope. RESULTS: The eukaryotic expression vector containing truncated beta catenin gene and expression vector of truncated beta catenin gene fused with EGFP gene were constructed successfully. C terminus-deleted beta catenin localized mainly in the cytoplasm of transfected COS7 cells, and the transcriptional activity of the N terminus- and the C terminus- deleted beta catenins was lower than that of the wild type beta catenin in transfected 293 cells. CONCLUSION: The different locations of truncated beta catenins in transfected COS7 cells suggest that the truncated ones play different roles in the transcriptional activity.

Animals↗

PubMed1992

Thymus-independent expression of a truncated T cell receptor-alpha mRNA in murine kidney.

During studies on gene expression in the kidney, we unexpectedly observed that murine kidney expresses a truncated form of TCR-alpha mRNA (1.3-1.4 kb). This transcript was not associated with the presence of complete TCR-alpha mRNA (1.7 kb) or detectable TCR-beta or -delta transcripts, thus indicating that the truncated TCR-alpha mRNA could not be attributed to blood contamination of the kidney RNA preparation. The truncated TCR-alpha message appeared to contain at least the C alpha region, as suggested by hybridization with an intra-C alpha 24 oligonucleotide probe, by amplification of the C alpha region with the polymerase chain reaction from total kidney mRNA, and by sequencing of, and hybridization with, the amplified products. In situ hybridization of kidney sections indicated that the transcript was expressed in interstitial cells. Northern blots of cortex and medulla RNA showed that the cells expressing the truncated TCR-alpha mRNA were predominantly located in the medulla. To investigate the possibility that the transcript was not produced by T cells or NK cells, fractionation of renal cell suspensions were performed. The truncated TCR-alpha mRNA was detected in a fraction containing large (low buoyant density) cells in which no expression of CD3, Thy 1, or NK-1.1 was detected, indicating that these cells are not mature T cells, do not express a functional TCR, and are not NK cells. The cells expressing the truncated TCR-alpha mRNA were radiosensitive, and were not thymus dependent, because this transcript was as abundant in nude mice as in normal mice. The transcript was not detected in bone marrow. Expression of the truncated TCR-alpha mRNA was not dependent on an intact recombinase activity as its expression was not affected by the severe combined immunodeficiency mutation. Our results show that murine kidney contains a population of radiosensitive thymus-independent large interstitial cells that express a truncated TCR-alpha mRNA that is not associated with surface expression of functional TCR. These cells may have attempted to rearrange TCR-alpha genes, suggesting that they may be related to the lymphoid lineage.

Animals↗

PubMed2006

Whole-body 18F-FDG PET/CT in the presence of truncation artifacts.

UNLABELLED: We investigated the effect of CT truncation in whole-body (WB) PET/CT imaging of large patients, and we evaluated the efficacy of an extended field-of-view (eFOV) correction technique. METHODS: Two uniform phantoms simulating a "torso" and an "arms-up" setup were filled with (18)F-FDG/water. A third, nonuniform "body phantom was prepared with hot and cold lesions. All 3 phantoms were positioned in the center of the PET/CT gantry with >or=10% of their volume extending beyond the maximum CT FOV. An eFOV algorithm was used to estimate complete CT projections from nonlinear extrapolation of the truncated projections. CT-based attenuation correction (CT AC) of the phantom data was performed using CT images reconstructed from truncated and extended projections. For clinical validation, we processed truncated datasets from 10 PET/CT patients with and without eFOV correction. RESULTS: When using truncated CT images for CT AC, PET tracer distribution was suppressed outside the transverse CT FOV in phantom and patient studies. PET activity concentration in the truncated regions was only 10%-32% of the true value but increased to 84%-100% when using the extended CT images for CT AC. At the same time, the contour of phantoms and patients was recovered to the anatomically correct shape from the uncorrected emission images, and the apparent distortion of lesions near the maximum CT FOV was reduced. CONCLUSION: Truncation artifacts in WB PET/CT led to visual and quantitative distortions of the CT and attenuation-corrected PET images in the area of truncation. These artifacts can be corrected to improve the accuracy of PET/CT for diagnosis and therapy response evaluation.

Algorithms↗

PubMed1995

Induction of B cell lymphomas by overexpression of a Myb oncogene truncated at either terminus.

The c-myb oncogene encodes a nuclear transcriptional transactivator that is often terminally truncated in hematopoietic tumors. To directly assess the tumorigenic activity of full length and terminally-truncated variants of c-myb, we have overexpressed several structurally-altered forms of myb within an avian retroviral vector and have shown that overexpression of truncated (but not full length) myb transforms both myeloid cells in vitro and mesenchymal cells in vivo. In vivo infection with these truncated myb viruses is now shown to induce metastatic B cell lymphomas in a significant minority of animals. Evaluation of the lymphomas revealed two distinct mechanisms of myb-induced tumorigenesis. In most of the lymphomas, proviral DNA inserted into the endogenous chicken c-myb gene and promoted the expression of a 5'-truncated myb transcript encoding an amino terminal truncated protein. In comparison, some animals infected with a virus encoding a carboxyl (C) terminal truncated myb (T-myb) developed non-insertional B cell lymphomas that directly expressed the provirally-encoded T-myb gene. The lymphomagenic T-myb protein lacks 214 C terminal amino acids including all of the myb transcription inhibition domain. This novel lymphomagenic activity for a C terminal truncated myb suggests that a loss of regulatory sequences at either end of c-myb is sufficient to create a B cell-specific transforming gene.

Animals↗

PubMed1996

Positive linear correlation between the length of truncated apolipoprotein B and its secretion rate: in vivo studies in human apoB-89, apoB-75, apoB-54.8, and apoB-31 heterozygotes.

Apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins secreted from the liver, plays crucial roles in the secretion, transport, and receptor-mediated clearance of lipoproteins. A minority of cases of familial hypobetalipoproteinemia is due to genetically determined truncations of apoB-100 that range in size from apoB-9 to apoB-89, but truncated apoBs smaller than apoB-27.6 were not detected in plasma. To ascertain the physiologic bases of the hypobetalipoproteinemia, we studied in vivo metabolic parameters of the products of both the normal and mutant apoB alleles in human apoB truncation/apoB-100 heterozygotes (apoB-89/apoB-100, n = 2, apoB-75/apoB-100, n = 2; apoB-54.8/apoB-100, n = 6; apoB-31/apoB-100, n = 1) using endogenous labeling with [13C]leucine, mass spectrometry, and multicompartmental modeling. All truncated forms of apoB were secreted at reduced rates. The secretion rates of apoB-89, apoB-75, apoB-54.8, and apoB-31 were 92%, 64%, 37%, and 12%, respectively, of the respective apoB-100s on a molar basis. Additionally, particles containing apoB-89, apoB-75, and apoB-54.8 had increased fractional catabolic rates (FCR), while apoB-31-containing particles had a decreased FCR. On regression analysis, the secretion rate was linearly linked to the length of the truncated apoB (r2 = 0.86, P < 0.0001), with secretion being reduced by 1.4% for each 1% of apoB truncated. The linear regression line of apoB size versus apoB secretion rate has a zero intercept for apoB secretion at apoB-28, which is consonant with the apparent absence in plasma of truncations smaller than apoB-25. We conclude that secretion of apoB in vivo is dependent on the length of the truncation of apoB, possibly because the smaller the truncated apoB, the less it is protected from intracellular degradation.

Apolipoprotein B-100↗

PubMed1996

A semiparametric extension of the Mann-Whitney test for randomly truncated data.

In many applications, statistical data are frequently observed subject to a retrospective sampling criterion resulting in pure right-truncated data. In classical testing problems, the Mann-Whitney test is used for testing the equality of two distributions. A semiparametric extension of this test is developed for the case when truncation is present. We consider a model in which the truncation distribution is parameterized, while the lifetime distribution is left as a nonparametric component. The method is seen to be applicable to many patterns of truncation including left truncation, right truncation, and doubly truncated data for which no other nonparametric or semiparametric test is currently available. Applications of the semiparametric method are given. Simulation results indicate that for pure right-truncated data the semiparametric test is more powerful than a recent nonparametric test.

Acquired Immunodeficiency Syndrome↗

PubMed1995

Dissociation of mitogenesis and transforming activity by C-terminal truncation of the insulin-like growth factor-I receptor.

We have investigated the mitogenic and transforming ability of an IGF-I receptor with a 108-amino-acid C-terminal truncation in R- cells, which are 3T3-like cells derived from mouse embryos in which the IGF-I receptor genes have been disrupted by targeted homologous recombination. R- cells stably transfected with expression plasmids encoding either a wild-type or a truncated human IGF-I receptor were capable of growing in serum-free medium supplemented solely with IGF-I. This response was observed over a wide range of receptor levels. R- cells overexpressing the wild-type IGF-I receptor also formed colonies in soft agar, and colony formation was augmented by coexpression of the SV40 large T antigen. However, all the examined clones of R- cells expressing the truncated IGF-I receptor exhibited a dramatically impaired ability to grow in soft agar, even in the presence of the T antigen. The inability to form colonies in soft agar was not due to a quantitative impairment of signal transduction, because: (1) SV40-transformed cells with a physiological level of the wild-type IGF-I receptor did not respond to IGF-I with cell proliferation, but grew in soft agar; (2) R- cells stably transfected with both a truncated receptor and T antigen, on the contrary, responded with mitogenesis to IGF-I but could not form colonies in soft agar; (3) some clones with the truncated receptor expressed levels of receptor roughly 100-fold the level of wild-type cells; and (4) several parameters of IGF-I receptor signal transduction were not impaired in cells stably transfected with a truncated receptor. Furthermore, overexpression of an activated ras in cells with the truncated IGF-IR did not restore their ability to proliferate under anchorage-independent conditions. We conclude that the 108 amino acids of the IGF-I receptor are not essential for a mitogenic response to IGF-I, but are required for transformation (as assessed by the ability to grow in soft agar), indicating that these two functions can be dissociated at an intramolecular level. Moreover, although ras (activated) certainly plays a role in transformation, the transforming activity of the IGF-IR also requires signaling elements that are ras-dependent.

3T3 Cells↗

PubMed1999

COOH-terminal truncated cardiac myosin-binding protein C mutants resulting from familial hypertrophic cardiomyopathy mutations exhibit altered expression and/or incorporation in fetal rat cardiomyocytes.

Mutations in human cardiac myosin-binding protein C (cMyBP-C) gene are associated with familial hypertrophic cardiomyopathy (FHC), and most of them are predicted to produce COOH-truncated proteins. To understand the molecular mechanism(s) by which such mutations cause FHC, we analyzed (i) the accumulation of human cMyBP-C mutants in fetal rat cardiomyocytes, and (ii) the protein sequence of the human wild-type (wt) cMyBP-C by hydrophobic cluster analysis with the aim of identifying new putative myosin-binding site(s). Accumulation and sarcomeric localization of the wt protein and of four FHC-mutant cMyBP-Cs (E542Q and three COOH-truncated proteins) were studied in cardiomyocytes by immunostaining and confocal microscopy after transfection with myc-tagged constructs. We found that: (i) 10 % of the cells expressing COOH-truncated mutants exhibit an incorporation into the A-band of the sarcomere without any alteration of the myofibrillar architecture versus 76 % of those expressing the wt or E542Q mutant cMyBP-Cs (p<0.001); (ii) 90 % of the cells expressing the truncated mutants show a diffuse localization of these proteins in the cardiomyocytes, out of which 45 % exhibit a significant alteration of the sarcomeric structure (p<0.0001 versus wt); and (iii) the two shortest mutant cMyBP-Cs accumulate at very low levels in fetal rat cardiomyocytes as compared to the wt (p<0.008). Protein sequence analysis indicated that a 45-residue sequence in the NH2-terminal C0 domain of cMyBP-C exhibits a consistent homology (sequence similarity score of 42 %) with a segment of the NH2-terminal domain of myomesin, another myosin-binding protein. This result suggests that the C0 domain of human cMyBP-C contains a novel putative myosin-binding site that could account for the A-band incorporation of the truncated mutants. In addition, the faint accumulation and the diffuse localization of truncated mutants could probably be explained by a low affinity of the C0 domain for myosin. We conclude that COOH-truncated cMyBP-Cs may act as poison polypeptides that disrupt the myofibrillar architecture and result in the defects observed in FHC.

Amino Acid Motifs↗