Biomedical subjects

W J Rutter

Publications and source records attributed to W J Rutter.

At least 109 records · Page 6Linked to original sources

PubMed1987

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning.

We have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase [Van den Bosch, H., Aarsman, A. J., DeJong, G. N., & Van Deenen, L. M. (1973) Biochim. Biophys. Acta 296, 94-104].

Amino Acid Sequence↗

PubMed1987

Use of directed mutagenesis to probe the role of tyrosine 198 in the catalytic mechanism of carboxypeptidase A.

Derivatization of Tyr198 in carboxypeptidase A (CPA) results in lowered catalytic activity toward peptide substrates (Cueni, L., and Riordan, J.F. (1978) Biochemistry 17, 1834-1842). We have synthesized via directed mutagenesis a rat CPA variant [Phe198] CPA containing a Tyr198-to-Phe substitution in order to test whether the phenolic hydroxyl plays a critical role in catalysis. A double mutant [Phe193, Phe248]CPA in which both Tyr198 and Tyr248 have been replaced by phenylalanine has also been engineered. Enzymatic characterization of [Phe198]CPA indicates that the Tyr198 hydroxyl is not obligatory for the hydrolysis of peptide and ester substrates. Furthermore, parallel studies with [Phe198, Phe248]CPA show that simultaneous removal of both the Tyr198 and Tyr248 hydroxyls does not abolish catalytic activity. Analysis of the acetylated derivatives of [Phe198]CPA, [Phe248]CPA, and [Phe198, Phe248]CPA establishes that Tyr198 and Tyr248 are the active site tyrosines which are modified by N-acetylimidazole. In addition, the perturbations of enzymatic activity which accompany acetylation of native CPA can be largely assigned to derivatization of Tyr248. The changes in the kinetic constants of substrate hydrolysis due to the Tyr198-to-Phe substitution are manifested as small decreases in the kcat values, but the Km values are essentially unaffected. This exclusive effect on the kcat values suggests that the Tyr198 hydroxyl participates in catalysis by stabilizing the rate-determining transition-state complex.

Amino Acid Sequence↗

PubMed1987

Cobra nerve growth factor: structure and evolutionary comparison.

We have cloned and sequenced an NGF cDNA from the cobra venom gland. The structure of the predicted cobra precursor resembles that of the mouse prohormone, with a highly conserved mature NGF protein at the C-terminus. In the non-NGF moiety, a putative signal peptide and two sets of basic residues presumably reflecting cleavage sites that are more conserved, while large interstitial regions are poorly conserved. This moiety may be involved in defining the folding of the precursor for subsequent proteolytic maturation and/or encode a bioactive polypeptide(s). Primer extension analysis indicates a single NGF transcript in the cobra venom gland.

Amino Acid Sequence↗

PubMed1987

Convenient vectors for cloning and sequencing EcoRI and HindIII fragments.

The polylinker regions of plasmid pUC and bacteriophage M13mp vectors have been specifically modified to provide alternative positions for cloning and reexcising EcoRI and HindIII fragments; the EcoRI and HindIII sites have been moved internal to BamHI and Bg/II sites. The location of EcoRI and HindIII sites in these HinEco vectors allows either selective linearization or excision of the cloned fragments at unique flanking sites.

Base Sequence↗

PubMed1987

Heterologous transmembrane signaling by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells.

A hybrid receptor molecule composed of the extracellular ligand-binding domain of the human insulin receptor and the transmembrane and cytoplasmic (protein-tyrosine kinase) domains of the chicken sarcoma virus UR2 transforming protein p68gag-ros has been constructed and expressed in Chinese hamster ovary (CHO) cells. The hybrid is processed normally into alpha and hybrid beta subunits, is expressed on the cell surface at high levels, and binds insulin with near-wild-type affinity. Furthermore, insulin stimulates the phosphorylation on tyrosine residues of the hybrid beta subunit in vivo and the phosphorylation of an exogenous substrate [poly(Glu,Tyr)] in vitro. Thus the hybrid is capable of heterologous transmembrane signaling. However, the hybrid mediates neither the insulin-activated uptake of 2-deoxyglucose nor the incorporation of [3H]thymidine into DNA, suggesting that the physiological response(s) mediated by ligand-activated protein-tyrosine kinases may utilize distinct intracellular mechanisms for postreceptor signaling.

Animals↗

PubMed1987

Activation of transforming potential of the human insulin receptor gene.

A retrovirus containing part of the human insulin receptor (hIR) gene was constructed by replacing ros sequences in the avian sarcoma virus UR2 with hIR cDNA sequences coding for 46 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains of the beta subunit of hIR. The resulting virus, named UIR, contains the hIR sequence fused to the 5' portion of the UR2 gag gene coding for p19. UIR is capable of transforming chicken embryo fibroblasts and promoting formation of colonies in soft agar; however, it does not form tumors in vivo. A variant that arose from the parental UIR is capable of efficiently inducing sarcomas in vivo. UIR-transformed cells exhibit higher rates of glucose uptake and growth than normal cells. The 4-kilobase UIR genome codes for a membrane-associated, glycosylated gag-hIR fusion protein of 75 kDa designated P75gag-hir. P75gag-hir contains a protein tyrosine kinase activity that is capable of undergoing autophosphorylation and of phosphorylating foreign substrates in vitro; it is phosphorylated at both serine and tyrosine residues in vivo.

Animals↗

PubMed1987

A mutational analysis of the insulin gene transcription control region: expression in beta cells is dependent on two related sequences within the enhancer.

Cell-specific expression of the insulin gene is controlled by cis-acting DNA sequences located within approximately equal to 350 base pairs of the 5' flanking DNA immediately upstream from the transcription start site. Using synthetic oligonucleotides, we have constructed a systematic series of block replacement mutants spanning this region. No single sequence appears to be absolutely required for expression. However, three of the mutants exhibit 5-10 times less activity and several others show 2-3 times less. Simultaneous mutation of two of the most mutationally sensitive regions leads to virtual abolition of activity. These two elements are structurally related and presumably represent key components of the machinery determining the cell-specific expression of the insulin gene.

Animals↗

PubMed1987

Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity.

To test whether the tyrosine kinase activity of the insulin receptor is crucial for insulin action, we have constructed mutations of the human insulin receptor at Lys-1030, which is in the presumed ATP-binding region. By using oligonucleotide-directed mutagenesis, this lysine residue was replaced with either methionine, arginine, or alanine. Chinese hamster ovary cells were transfected by mutant cDNAs and the expressed insulin receptors were characterized. We show here that none of these mutants exhibited insulin-activated autophosphorylation and kinase activity in vitro. They also do not mediate insulin- and antibody-stimulated uptake of 2-deoxyglucose. The tyrosine kinase activity is thus required for a key physiological response of insulin.

Adenosine Triphosphate↗

PubMed1987

Regulation of secretion of the hepatitis B virus major surface antigen by the preS-1 protein.

P24, P30, and P39, the three major surface antigens of the envelope of hepatitis B virus, are co-carboxy-terminal proteins with different amino-terminal extensions. We prompted expression of these proteins in Chinese hamster ovary (CHO) cells by placing the appropriate coding sequence(s) under the control of the simian virus 40 early promoter. P24 and P30 formed 22-nm particles which were efficiently secreted. In contrast, P39 accumulated in a perinuclear structure, presumably the Golgi complex, and was not secreted. Coexpressing P39 and P24 resulted in the localization of both in the perinuclear region and restricted the secretion of P24. We found that P39 must be expressed at a relatively low level to allow efficient secretion of P24 in typical spherical particles. We hypothesize that P39, by inhibiting the formation of spherical particles, helps to induce formation of filamentous particles and mature Hepatitis B virus.

Animals↗

PubMed1987

Mouse nerve growth factor gene: structure and expression.

The organization and biologically significant sequences of the entire mouse nerve growth factor (NGF) gene have been determined. The gene spans 45 kilobases and contains several small 5' exons. Transcription of the gene results in four different mRNA species, which can be accounted for by alternative splicing and independent initiation from two promoters. These transcripts encode proteins which have divergent N termini and the NGF moiety at their C termini. The levels of the various NGF transcripts have been determined in different tissues and throughout postnatal development. We have also examined the expression of these transcripts in the brain in response to specific early sensory deprivation. The results suggest that the expression of NGF mRNA during postnatal development is regulated independently of the formation of complex neural networks.

Age Factors↗

PubMed1987

Mapping polypeptide hormone genes in the mouse: somatostatin, glucagon, calcitonin, and parathyroid hormone.

Mouse-Chinese hamster hybrids segregating mouse chromosomes were analyzed by Southern hybridization techniques to map the genes for somatostatin (Smst), glucagon (Gcg), calcitonin (Calc), and parathyroid hormone (Pth). The mouse gene for somatostatin, detected on a 20-kb EcoRI fragment, is located on mouse chromosome 16. Glucagon cDNA hybridized to a 14-kb EcoRI fragment residing on chromosome 2. Calcitonin and parathyroid hormone genes, detected on 7.8-kb HindIII and 6.0-kb BamHI fragments, respectively, were on mouse chromosome 7. The calcitonin and parathyroid hormone genes appear to be part of a larger linkage group which has been conserved in mouse and man.

Animals↗

PubMed1987

A membrane-anchored cytoplasmic domain of the human insulin receptor mediates a constitutively elevated insulin-independent uptake of 2-deoxyglucose.

Insulin stimulates the autophosphorylation of the beta-subunit of the insulin receptor (IR) on tyrosine residues. Mutations which compromise IR autophosphorylation in vivo result in a decrease of the insulin-activated uptake of 2-deoxyglucose. These results are consistent with previous results which implicate IR autophosphorylation in the generation of the insulin response by cells. To further explore the specificity of the IR tyrosine phosphokinase (TPK) domain in IR function, we have altered the human IR (hIR) cDNA to encode truncated insulin-independent TPKs, which are expressed in chinese hamster ovary (CHO) cells as either membrane-anchored or cytosolic proteins. Both mutant hIRs exhibit TPK activity in vitro, although the cytosolic form is approximately 20 times more active. The carbohydrate moiety of the membrane-anchored form is of the high mannose type, consistent with an intracellular localization for this mutant hIR. The two mutant hIRs mediate very different physiological responses in transfected cells: the membrane-anchored, but not the cytosolic, hIR TPK mediates a constitutively elevated (135% the maximum insulin-stimulated response in CHO cells) insulin-independent uptake of 2-deoxyglucose. These results thus suggest that the hIR TPK is in fact specific for this aspect of IR function and, when membrane-associated, can mediate the insulin-independent uptake of 2-deoxyglucose. Neither of these mutant hIRs appears to transform CHO cells.

Animals↗

PubMed1987

The human insulin receptor cDNA: a new tool to study the function of this receptor.

The human insulin receptor (hIR) is an integral transmembrane glycoprotein comprised of two alpha and two beta subunits. An immediate consequence of insulin binding to the extracellular alpha subunit is the autophosphorylation of tyrosine residues on the intracellular domain of the beta subunit. The placental hIR cDNA has been cloned and sequenced, providing the primary structural features of the protein. In order to investigate the functions of the beta subunit and particularly the role of autophosphorylation and tyrosine phosphokinase (TPK) activity (a feature shared by other receptors and oncogene proteins) in transmembrane signalling, we designed an expression system of the hIR cDNA in eucaryotic cells. Superexpressing CHO cell lines that contain about 10(6) functional hIR/cell have been developed. In these cells half maximum stimulation of glucose uptake occurs at 5 X 10(-10)M insulin, whereas normal CHO cells require 5 X 10(-12)M insulin. In this expression system we have carried out site-directed mutagenesis experiments in which domains of the molecule have been deleted or particular amino acids have been replaced by others. The replacement of either or both the tyrosine residues 1162 and 1163 compromise an autophosphorylated site that is important for kinase function and the insulin response. Expression of an isolated membrane-bound form of the beta-subunit produces a 6 fold increase in glucose uptake. This insulin-independent effect disappears if the twin tyrosines are mutated or if the beta subunit is expressed in the cytoplasm. These studies also show that the C terminal 112 amino acid portion of the beta subunit is important for the stability of this protein.

Amino Acid Sequence↗

PubMed1986

Cloning of a Drosophila cDNA encoding a polypeptide similar to the human insulin receptor precursor.

A Drosophila cDNA clone was obtained using the human insulin receptor cDNA sequence as a probe. The 3586 bp nucleotide sequence predicted a single polypeptide of 1095 amino acid residues which showed considerable homology (35.2%) with the human insulin receptor precursor. Although the cDNA was incomplete at its 5'-terminal region, it encodes a transmembrane glycoprotein as a single precursor of a two subunit molecule having a structural architecture similar to that of the human insulin receptor precursor. The presumptive beta subunit carries a well conserved Tyr kinase domain which showed 63.5% homology with that of human insulin receptor; however the protein of the alpha subunit is only weakly conserved (25%).

Amino Acid Sequence↗

PubMed1986

Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose.

Insulin stimulates the autophosphorylation of tyrosine residues of the beta subunit of the insulin receptor (IR); this modified insulin-independent kinase has increased activity toward exogenous substrates in vitro. We show here that replacement of one or both of the twin tyrosines (residues 1162 and 1163) with phenylalanine results in a dramatic reduction in or loss of insulin-activated autophosphorylation and kinase activity in vitro. In vivo, these mutations not only result in a substantial decrease in insulin-stimulated IR autophosphorylation but also in a parallel decrease in the insulin-activated uptake of 2-deoxyglucose. Furthermore, a truncated IR protein (lacking the last 112 amino acids) has an unstable beta subunit; this mutant has no kinase activity in vitro or in vivo and does not mediate insulin-stimulated uptake of 2-deoxyglucose. IR autophosphorylation is thus implicated in the regulation of IR activities, with tyrosines 1162 and 1163 as major sites of this regulation.

Amino Acid Sequence↗

PubMed1986

Assignment of the gene for carboxypeptidase A to human chromosome 7q22----qter and to mouse chromosome 6.

A rat cDNA probe for preprocarboxypeptidase A was used to follow the segregation of the human gene for carboxypeptidase A (CPA) in 49 human X mouse somatic cell hybrids using Southern filter hybridization techniques. CPA was assigned to human chromosome 7q22----qter. Similarly, the probe was used to follow the segregation of the mouse gene for carboxypeptidase A (Cpa) in 19 mouse X Chinese hamster somatic cell hybrids. Cpa was assigned to mouse chromosome 6. The gene for carboxypeptidase A forms part of a syntenic group that is conserved in man and mouse.

Animals↗

PubMed1986

Selective expression of rat pancreatic genes during embryonic development.

We present the developmental profiles of the mRNAs of 10 selectively expressed pancreatic exocrine genes and of insulin. The mRNA profiles fall into three related classes, but each profile is in some respect unique. The data on gene expression suggest there are four developmental states of the exocrine pancreas: early morphogenesis and low-level gene expression (the protodifferentiated state), the embryonic differentiated state, a modulated state in neonatal animals, and the adult differentiated state. Each state is characterized by distinct ratios of the exocrine mRNAs and presumably involves a distinct regulatory transition. This complex differentiative program must involve multiple regulatory molecules.

Amylases↗

PubMed1986

Regulation of rat insulin 1 gene expression: evidence for negative regulation in nonpancreatic cells.

Two cis-acting elements, the enhancer and the promoter, independently contribute to the cell-specific expression of the rat insulin 1 gene. The activities of these elements are presumably mediated by trans-acting factors. We have performed intracellular competition experiments that suggest the presence of a negative factor(s) that represses the enhancer activity in cells that do not express the insulin gene. In these experiments fibroblast cells (COS-7) were transfected with two plasmids: a test plasmid containing the gene for chloramphenicol acetyltransferase under the control of the thymidine kinase promoter and the insulin enhancer; and a competitor plasmid containing insulin enhancer sequences and the simian virus 40 origin of replication to permit its replication in the recipient cells. The presence of the competitor plasmid led to a 5- to 6-fold increase in chloramphenicol acetyltransferase activity as compared with the activity detected when insulin enhancer was absent from either the competitor or the test plasmid. A 5-fold increase in chloramphenicol acetyltransferase activity was also seen when the rat amylase enhancer was present on the competitor plasmid; in contrast the simian virus 40 enhancer exerted no effect. Efficient derepression required additional sequences downstream from those essential for enhancer activity. We propose that the activity of the rat insulin 1 enhancer is modulated by a negative trans-acting factor(s) that is active in cells not expressing insulin but is overridden by the dominant positive trans-acting factor(s) present in insulin-producing cells.

Animals↗