Parvovirus b19 infection associated with acute hepatitis, arthralgias, and rash.

Human parvovirus B19 is responsible for a wide variety of clinical syndromes, including erythema infectiosum, or fifth disease, polyarthritis, aplastic crisis in patients with hemolytic anemia, and chronic anemia in immunocompromised persons. Liver enzyme abnormalities are an infrequently reported association of parvovirus B19 infection in adults. We present a case of an acute transient hepatitis in the setting of parvovirus B19 infection, associated with arthralgias and an erythematous, edematous rash on the hands and leg.

Molecular dynamics of insulin/IGF-I receptor transmembrane signaling.

To examine the molecular basis of ligand-stimulated intramolecular beta-subunit autophosphorylation, hybrid receptors composed of wild-type and mutant insulin and insulin-like growth factor-1 (IGF-I) half-receptor precursors were characterized. Previous studies have demonstrated that assembly of the IGF-I wild-type half-receptor (alpha beta WT) with a kinase-defective half-receptor (alpha beta A/K) produced a substrate kinase-inactive holoreceptor in vitro [Treadway et al. (1991): Proc Natl Acad Sci USA 88:214-218]. To extend these studies, the vaccinia virus/bacteriophage T7 expression system was used to generate various hybrid receptor complexes in cultured cells. As was observed for hybrid receptors assembled in vitro, the wild-type/mutant hybrid receptors formed in situ were also incapable of phosphorylating several peptide substrates. However, ligand-stimulated beta-subunit autophosphorylation was still observed. To determine the molecular basis for this discrepancy, hybrid receptors were assembled from a truncated beta-subunit insulin half-receptor (alpha beta delta 43) and a kinase-defective half-receptor (alpha beta A/K). Under these conditions, insulin-stimulated autophosphorylation primarily occurred on the full-length kinase-inactive beta-subunit (alpha beta A/K) without significant labeling of the kinase-active truncated beta-subunit (alpha beta delta 43). A similar IGF-I hybrid receptor species was characterized, and the same pattern of autophosphorylation was observed in response to IGF-I. These data demonstrate that both insulin and IGF-I stimulate an intramolecular trans-autophosphorylation reaction between two adjacent beta-subunits within the holoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between alpha subunit ligand occupancy and beta subunit autophosphorylation in insulin/insulin-like growth factor-1 hybrid receptors.

Insulin receptor beta subunit autophosphorylation occurs in an intramolecular trans-reaction in which one beta subunit phosphorylates the adjacent beta subunit within an alpha 2 beta 2 holoreceptor complex (Frattali, A. L., Treadway, J. L., and Pessin, J. E. (1992) J. Biol. Chem. 267, 19521-19528). To determine the spatial relationship between alpha subunit occupancy and beta subunit autophosphorylation, the vaccinia virus/bacteriophage T7 transient expression system was used to generate insulin/insulin-like growth factor (IGF)-1 hybrid receptors. The extent of hybrid receptor formation was proportional to the molar ratio of the insulin and IGF-1 receptor expression plasmids used for transfection of cultured fibroblasts. Insulin/IGF-1 hybrid receptors displayed high affinity binding for insulin and IGF-1 similar to that observed for homotypic insulin and IGF-1 receptors, respectively. As expected, insulin poorly competed for 125I-IGF-1 binding to the insulin/IGF-1 hybrid receptors compared with IGF-1. IGF-1, however, competed more efficiently than insulin for 125I-insulin binding, indicating interactions between the alpha subunit binding sites. Furthermore, insulin or IGF-1 stimulated the autophosphorylation of both beta subunits within wild type insulin/IGF-1 hybrid receptors. Ligand-stimulated autophosphorylation of two different mutant/wild type insulin/IGF-1 hybrid receptors also resulted in the labeling of each beta subunit independent of which alpha subunit was occupied with ligand. These data demonstrate that insulin/IGF-1 hybrid receptors bind both ligands with high affinity and that occupancy of either alpha subunit results in a series of intramolecular trans-autophosphorylation reactions between beta subunits.

Intramolecular subunit interactions between insulin and insulin-like growth factor 1 alpha beta half-receptors induced by ligand and Mn/MgATP binding.

We have previously demonstrated that isolated insulin and IGF-1 alpha beta half-receptors can be reconstituted into a functional alpha 2 beta 2 hybrid receptor complex [Treadway et al. (1989) J. Biol. Chem. 264, 21450-21453]. In the present study, we have examined this assembly process by determining the effect of ligand occupancy and Mn/MgATP binding on the dimerization of mutant and wild-type insulin and IGF-1 alpha beta half-receptors. IGF-1 or Mn/MgAMPPCP binding to wild-type IGF-1 alpha beta half-receptors resulted in the specific assembly of the alpha beta half-receptors into an alpha 2 beta 2 heterotetrameric IGF-1 holoreceptor complex. Similarly, insulin binding to the kinase-deficient mutant (A/K1018) insulin alpha beta half-receptor also resulted in the specific assembly into an alpha 2 beta 2 holoreceptor complex. In contrast, Mn/MgAMPPCP treatment of A/K1018 mutant insulin alpha beta half-receptors did not induce heterotetramer assembly, consistent with the inability of this mutant receptor to bind ATP. The ability of the insulin alpha beta receptors to assemble with the IGF-1 alpha beta half-receptors was used to examine the intermolecular subunit interactions responsible for dimerization. In the presence of Mn/MgAMPPCP, the wild-type insulin and wild-type IGF-1 alpha beta half-receptors were observed to assemble into an insulin/IGF-1 alpha 2 beta 2 hybrid receptor complex. Similarly, a combination of insulin and IGF-1 induced hybrid receptor formation between wild-type IGF-1 and A/K1018 mutant insulin alpha beta half-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmembrane signaling by the human insulin receptor kinase. Relationship between intramolecular beta subunit trans- and cis-autophosphorylation and substrate kinase activation.

To examine the role of intramolecular beta subunit trans- and cis-autophosphorylation in signal transduction, the vaccinia virus/bacteriophage T7 expression system was used to generate insulin holoreceptors composed of a kinase-defective half-receptor precursor (alpha beta A/K or alpha beta A/K.delta CT) and a kinase-active half-receptor precursor (alpha beta delta CT or alpha beta WT). In the alpha beta A/K-alpha beta delta CT hybrid insulin receptor, insulin stimulated a 20-fold increase in intramolecular beta subunit trans-phosphorylation, whereas cis-phosphorylation increased only 3-fold over the basal state. Similarly, in the alpha beta WT-alpha beta A/K.delta CT hybrid insulin receptor, insulin stimulated trans-phosphorylation approximately 30-fold and cis-phosphorylation only 3-fold over the basal state. Although cis-phosphorylation of the kinase-functional alpha beta half-receptor was observed within these hybrid receptor species, this was not sufficient to stimulate exogenous substrate kinase activity. These data demonstrate that insulin primarily activates an intramolecular beta subunit trans-phosphorylation reaction within the insulin holoreceptor and suggest that this reaction is necessary for activation of the holoreceptor. Furthermore, our results suggest a molecular basis for the dominant-negative phenotype observed in insulin-resistant patients possessing one kinase-defective insulin receptor allele.

Insulin/IGF-1 hybrid receptors: implications for the dominant-negative phenotype in syndromes of insulin resistance.

Classical insulin and IGF-1 receptors are alpha 2 beta 2 heterotetrameric complexes synthesized from two identical alpha beta half-receptor precursors. Recent data strongly suggests, however, that nonidentical alpha beta half-receptor precursors can assemble to generate hybrid holoreceptor species both in vivo and in vitro. This review focuses primarily on two types of hybrid receptors. The first type is an insulin/IGF-1 hybrid receptor generated by the association of an alpha beta insulin half-receptor with an alpha beta IGF-1 half-receptor. The second type is one formed from a wildtype (kinase-active) insulin or IGF-1 alpha beta half-receptor and a mutant (kinase-inactive) insulin alpha beta half-receptor. Although the functional properties of insulin/IGF-1 hybrid receptors have not yet been completely defined, wildtype/mutant hybrid receptors are essentially substrate kinase inactive. These data indicate that the mutant alpha beta half-receptor exerts a transdominant inhibition upon the wildtype alpha beta half-receptor within the alpha 2 beta 2 holoreceptor complex. This defect in substrate kinase activity may contribute to the molecular defect underlying some syndromes of severe insulin resistance and diabetes. Heterozygous individuals expressing both wildtype and mutant tyrosine kinase-defective insulin receptor precursors demonstrate varying degrees of insulin resistance and diabetes. In addition, cell lines which express both endogenous wildtype and transfected kinase-defective insulin receptors display markedly decreased insulin and IGF-1 sensitivity and responsiveness. Formation of hybrid receptors which results in premature termination of insulin signal transduction may be one mechanism underlying the observation that kinase-inactive receptors inhibit the function of native receptors.

Evidence supporting a passive role for the insulin receptor transmembrane domain in insulin-dependent signal transduction.

We previously have demonstrated that intramolecular interactions between alpha beta-alpha beta subunits are necessary for insulin-dependent activation of the protein kinase domain within a single alpha 2 beta 2 heterotetrameric insulin-receptor complex (Wilden, P. A., Morrison, B. D., and Pessin, J. E. (1989) Biochemistry 28, 785-792). To evaluate the role of the beta subunit transmembrane domain in the insulin-dependent signalling mechanism, mutant human insulin receptors containing a series of nested transmembrane domain deletions (amino acids 941-945) were generated and stable Chinese hamster ovary-transfected cell lines were obtained. In addition, a substitution of Val-938 for Glu (E/V938) similar to the oncogenic mutation found in the neu transmembrane domain was also introduced into the insulin receptor. Scatchard analysis of insulin binding to the stable Chinese hamster ovary cell lines expressing either wild type or mutant insulin receptors indicated equivalent receptor number (2-4 x 10(6)/cell) and similar high affinity binding constants (Kd 0.1-0.3 nM). 125I-Insulin affinity cross-linking demonstrated that all of the expressed insulin receptors were assembled and processed into alpha 2 beta 2 heterotetrameric complexes. Surprisingly, all the mutant insulin receptors retained insulin-stimulated autophosphorylation both in vivo and in vitro. Furthermore, endogenous substrate phosphorylation in vivo as well as insulin-stimulated thymidine incorporation into DNA were unaffected by the transmembrane domain mutations. These data demonstrate that marked structural alterations in the insulin receptor transmembrane domain do not interfere with insulin-dependent signal transduction.

Effects of mutagenesis of C912 in the streptomycin binding region of Escherichia coli 16S ribosomal RNA.

Four different mutations were produced at position 912 of Escherichia coli 16S rRNA in the multicopy plasmid pKK3535. Cells transformed with the mutant plasmids were assayed for growth in steptomycin. The U912 mutant conferred low level streptomycin resistance as reported originally by Montandon and co-workers (EMBO J 1986; 5:3705-3708). The G912 mutant also gave low level resistance but, unlike U912, caused significant retardation in growth rate and tended to select for fast-growing revertants. The A912 mutant was without effect on growth rate or streptomycin sensitivity, while deletion of C912 was lethal. Cells with U912 were selected for increased streptomycin resistance (MIC up to 160 micrograms/ml) and then cured of the plasmid. The cured cells retained a higher level of streptomycin resistance (MIC: 80 micrograms/ml) than the original wild type strain (MIC: 10 micrograms/ml), but sequencing by reverse transcriptase showed no evidence of U912 in the cellular 16S rRNA. Thus, recombination of the plasmid-coded U912 mutation into host rrn operons was not the mechanism by which increased streptomycin resistance occurred. The plasmid with U912 was transformed into three different streptomycin-dependent strains to determine whether the rRNA mutation, which presumably alters streptomycin binding, was compatible with S12 mutations which require bound streptomycin in order to function properly. In one strain, no transformants could be isolated, indicating that the plasmid was lethal. The two other streptomycin-dependent strains were transformed, but ribosomes containing the mutant rRNA were non-functional.