Biomedical subjects

O R Colamonici

Publications and source records attributed to O R Colamonici.

At least 19 recordsLinked to original sources

PubMed2000

Role of the cytoplasmic domains of the type I interferon receptor subunits in signaling.

Type I interferons are imperative in maintaining a defense against viral infection. These cytokines also play an important role in the control of cell proliferation. These effects are triggered by ligand binding to a specific cell surface receptor. In the present article, we attempt to analyze the advances made in the last four years on type I interferon signaling. This review will focus on the contribution of the cytoplasmic domain of the alpha and betaL chains of the receptor to the activation of the Jak-Stat pathway. We also analyze the possible role of other pathways in interferon signaling.

Animals↗

PubMed2000

Structure-function study of the extracellular domain of the human type I interferon receptor (IFNAR)-1 subunit.

Despite accumulating information about the different effector molecules and signaling cascades that are invoked on interferon-alpha (IFN-alpha) binding to the type 1 IFN receptor, little is known about the specifics of the binding interactions between the ligand and the receptor complex. The IFN-alpha/beta receptor (IFNAR)-2 subunit of the IFN receptor is considered the primary binding chain of the receptor, yet it is clear that both receptor subunits, IFNAR-1 and IFNAR-2, cooperate in the high-affinity binding of IFN to the receptor complex. Earlier results from our laboratory suggested that an association of IFNAR-1 with membrane Galalpha1-4Gal-containing glycolipids facilitates receptor-mediated signaling. The data implicated amino acid residues in the SD100 domain of IFNAR-1 in the glycosphingolipid (GSL) modification of the type 1 IFN receptor. Interestingly, the human and murine counterparts of IFNAR-1 exhibit remarkable species specificity despite their considerable amino acid sequence identity. Certainly, those amino acid residues that effect GSL modification of IFNAR-1 are conserved between species, yet specific regions of IFNAR-1 that confer species specificity have not been defined. To delineate further the role of the IFNAR-1 SD100A domain in receptor function, a chimeric cDNA was assembled, in which the SD100A domain of the murine IFNAR-1 chain was replaced with the human sequence. This construct was expressed in IFNAR-1-/- mouse embryonic fibroblasts, and stable transfectants were established. Transfectants are fully sensitive to murine IFN-alpha4 treatment with respect to the induction of IFN-stimulated gene factor 3 (ISGF3) and sis-inducing factor (SIF) signal transducer and activator of transcription factor (Stat) complexes, exhibiting comparable levels of Stat activation to those observed in IFNAR-1-/- cells reconstituted with intact MuIFNAR-1. Similar results were obtained with IFN-induced antiviral and growth inhibitory responses. Viewed together, these data suggest that the SD100A domain of IFNAR-1 does not contribute to species-specific IFN binding.

Amino Acid Sequence↗

PubMed1999

The proximal tyrosines of the cytoplasmic domain of the beta chain of the type I interferon receptor are essential for signal transducer and activator of transcription (Stat) 2 activation. Evidence that two Stat2 sites are required to reach a threshold of interferon alpha-induced Stat2 tyrosine phosphorylation that allows normal formation of interferon-stimulated gene factor 3.

The precise role of the different subunits (alpha/IFNAR1 and betaL/IFNAR2) of the type I interferon receptor (IFN-R) in the activation of signal transducer and activator of transcription (Stat) 1, Stat2, and Stat3 has not yet been established. In this report we demonstrate that there are functionally redundant phosphotyrosine-dependent and -independent binding sites for Stat2 in the alpha and beta subunits of the type I IFN-R. Expression of a type I IFN-R containing only the constitutive Stat2 site or the proximal tyrosines of betaL, but not the docking site on the alpha chain (Tyr466 and Tyr481), supported low levels of Stat2 activation. However, the presence of only one intact Stat2 site did not lead to induction of interferon-stimulated gene factor 3 (ISGF3) or an antiviral state. Normal levels of Stat2 tyrosine phosphorylation, induction of ISGF3, and an antiviral effect always required the proximal tyrosines of betaL and at least one of the other Stat2 sites (Tyralpha466, 481 or betaL404-462). These data suggest that a threshold of Stat2 tyrosine phosphorylation is required for complete activation of ISGF3. Interestingly, a receptor in which all tyrosines were mutated to phenylalanine shows normal Stat3 phosphorylation and low levels of activation of Stat1.

Animals↗

PubMed1999

Activation of the Jak-Stat pathway in cells that exhibit selective sensitivity to the antiviral effects of IFN-beta compared with IFN-alpha.

We determined whether selective activation of components of the Jak-Stat pathway by different type I interferons (IFN) occurs in human myocardial fibroblasts that exhibit much higher sensitivity to the antiviral effects of IFN-beta than of IFN-alpha. Similar levels of activation of the Tyk2 kinase and the Stat3 transcription factor were induced in response to either IFN-beta or IFN-alpha treatment. However, activation of the Jak1 tyrosine kinase was detectable only in IFN-beta-treated but not IFN-alpha-treated cells. Consistent with this, tyrosine phosphorylation of Stat1 and Stat2 and formation of the IFN-stimulated gene factor 3 (ISGF3) complex occurred to a much higher degree in response to IFN-beta stimulation. These findings demonstrate that differential activation of distinct components of the Jak-Stat pathway by different type I IFN can occur. Furthermore, they strongly suggest that such selective activation accounts for the occurrence of differences in the antiviral properties of distinct type I IFN in certain cell types.

Antiviral Agents↗

PubMed1998

Jak2 is essential for signaling through a variety of cytokine receptors.

A variety of cytokines activate receptor-associated members of the Janus family of protein tyrosine kinases (Jaks). To assess the role of Jak2, we have derived Jak2-deficient mice. The mutation causes an embryonic lethality due to the absence of definitive erythropoiesis. Fetal liver myeloid progenitors, although present based on the expression of lineage specific markers, fail to respond to erythropoietin, thrombopoietin, interleukin-3 (IL-3), or granulocyte/macrophage colony-stimulating factor. In contrast, the response to granulocyte specific colony-stimulating factor is unaffected. Jak2-deficient fibroblasts failed to respond to interferon gamma (IFNgamma), although the responses to IFNalpha/beta and IL-6 were unaffected. Lastly, reconstitution experiments demonstrate that Jak2 is not required for the generation of lymphoid progenitors, their amplification, or functional differentiation. Therefore, Jak2 plays a critical, nonredundant role in the function of a specific group of cytokines receptors.

Animals↗

PubMed1998

Identification of a domain in the beta subunit of the type I interferon (IFN) receptor that exhibits a negative regulatory effect in the growth inhibitory action of type I IFNs.

Expression of human alpha and long form of the beta (betaL) subunits of type I interferon receptor (IFN-R) in mouse cells is sufficient to activate the Jak-Stat pathway and to elicit an antiviral state in response to human IFNalpha2 and IFNbeta. We demonstrate herein, however, that these cells respond to the antiproliferative effects of murine IFNalphabeta but not human type I IFNs. These results suggest that an unknown species-specific component is required for the antiproliferative effect of human type I IFNs. The absence of this component can be complemented by expressing the human betaL chain truncated at amino acid 346. Thus, the distal region of betaL appears to function as a negative regulator of the growth inhibitory effects of type I IFNs. Further studies looking for possible targets of the betaL regulatory domain demonstrated that this region associates with a tyrosine phosphatase. These results suggest that a protein associated with the negative regulatory domain of betaL, likely a tyrosine phosphatase, plays a role in regulating the growth inhibitory effects of human type I IFNs.

Animals↗

PubMed1998

Differential use of the betaL subunit of the type I interferon (IFN) receptor determines signaling specificity for IFNalpha2 and IFNbeta.

The signaling specificity for cytokines that have common receptor subunits is achieved by the presence of additional cytokine-specific receptor components. In the type I interferon (IFN) family, all 14 subtypes of IFNalpha, IFNbeta, and IFNomega bind to the same alpha and betaL subunits of the type I IFN-R, yet differences in signaling and biological effects exist among them. Our data demonstrate that IFNalpha2 and IFNbeta utilize different regions of the betaL subunit for signaling. Thus, in contrast to other cytokine systems, signal diversity in the type I IFN system can be accomplished within the same receptor complex by utilizing different regions of the same receptor subunits.

Animals↗

PubMed1998

Interferon alpha activates the tyrosine kinase Lyn in haemopoietic cells.

We investigated whether the src-family tyrosine kinase Lyn is involved in the generation of interferon alpha (IFN alpha) signals in haemopoietic cells. In vitro kinase assays using IFN alpha-sensitive cells of B-cell origin demonstrated the presence of IFN alpha-dependent kinase activity in anti-Lyn immunoprecipitates. Further studies demonstrated that Lyn associates via its src homology 2 (SH2) domain with the Janus family tyrosine kinase Tyk-2. This interaction was IFN alpha-dependent and involved direct binding of the SH2 domain of Lyn to the IFN alpha-activated form of Tyk-2. Thus, during binding of IFN alpha to its receptor in malignant haemopoietic cells, Lyn is engaged in an IFN alpha-signalling pathway, probably downstream of Tyk-2.

Blotting, Western↗

PubMed1997

A region of the beta subunit of the interferon alpha receptor different from box 1 interacts with Jak1 and is sufficient to activate the Jak-Stat pathway and induce an antiviral state.

Coexpression of the alpha and betaL subunits of the human interferon alpha (IFNalpha) receptor is required for the induction of an antiviral state by human IFNalpha. To explore the role of the different domains of the betaL subunit in IFNalpha signaling, we coexpressed wild-type alpha subunit and truncated forms of the betaL chain in L-929 cells. Our results demonstrated that the first 82 amino acids (AAs) (AAs 265-346) of the cytoplasmic domain of the betaL chain are sufficient to activate the Jak-Stat pathway and trigger an antiviral state after IFNalpha2 binding to the receptor. This region of the betaL chain, required for Jak1 binding and activation, contains the Box 1 motif that is important for the interaction of some cytokine receptors with Jak kinases. However, using glutathione S-transferase fusion proteins containing amino- and carboxyl-terminal deletions of the betaL cytoplasmic domain, we demonstrate that the main Jak1-binding region (corresponding to AAs 300-346 on the beta subunit) is distinct from the Box 1 domain (AAs 287-295).

Animals↗

PubMed1997

The IRS-pathway operates distinctively from the Stat-pathway in hematopoietic cells and transduces common and distinct signals during engagement of the insulin or interferon-alpha receptors.

Binding of interferon-alpha (IFN-alpha) to its receptor on hematopoietic cells activates the signal transducers and activators of transcription (Stat)- and insulin receptor substrate (IRS)-pathways, and regulates expression of antiproliferative and antiviral activities. However, it remains unknown whether these two pathways cooperate in the generation of IFN-alpha responses or function independently, and whether IRS-proteins transduce distinct downstream signals in response to IFNs or insulin/insulin-like growth factor (IGF)-1-mediated activation. Our data show that in response to IFN-alpha treatment, IRS-1 functions selectively as a docking protein for the SH2 domains of the p85 subunit of the PI 3'-kinase, but not the SH2 domain of Grb-2 which is engaged during insulin/IGF-1 signaling. In studies with THP-1 human myelomonocytic cells and 32D mouse myeloid cells, which are IRS-defective, we found that the IFN-alpha-regulated activation of Stat-1, Stat-2, and Stat-3 does not require the function of the IRS-system. Furthermore, THP-1 cells are responsive to the protective effect of IFN-alpha against vesicular stomatitis virus. Both 32D and THP-1 cells were resistant to the growth inhibitory effect of IFN-alpha, but this effect was not reversible by expression of IRS-1 or IRS-2 alone in 32D cells. Taken altogether these data show that: (1) The IRS-system transduces common and distinct signals in response to IFN-alpha or insulin/lGF-1 stimulation of hematopoietic cells. (2) The IRS-pathway operates separately from the Stat-pathway, and its function is not essential for the generation of the antiviral effect of IFN-alpha. (3) Neither the IRS- nor the Stat-pathways alone are sufficient to mediate the antiproliferative effects of IFN-alpha in hematopoietic cells, and additional signaling elements are required.

Animals↗

PubMed1997

Interaction of p59fyn with interferon-activated Jak kinases.

During IFN alpha stimulation, p59(fyn) associates with the Type I IFNR-associated Tyk-2 kinase in several human hematopoietic cell lines in vivo. This interaction is direct, and is mediated by the SH2 domain in p59(fyn), as shown by binding studies using glutathione-S-transferase fusion proteins and far western blots. Furthermore, in response to IFN alpha-treatment of cells, the SH2 domain of Fyn interacts with the Tyk-2-associated c-cbl proto-oncogene product. In a similar manner, during IFN gamma stimulation, p59(fyn) associates via its SH2 domain with the activated form of the IFN gamma-dependent Jak-2 kinase. These data suggest that p59(fyn) is a common element in IFN alpha and IFN gamma signaling, and is selectively engaged by the Type I or II IFN receptors via specific interactions with distinct Jak kinases.

Antibodies, Monoclonal↗

PubMed1997

The vav proto-oncogene product (p95vav) interacts with the Tyk-2 protein tyrosine kinase.

The vav proto-oncogene product participates in the signaling pathways activated by various cell-surface receptors, including the type I IFN receptor. During engagement of the type I IFN receptor, p95vav is phosphorylated on tyrosine residues, but the kinase regulating its phosphorylation has not been identified to date. Our studies demonstrate that p95vav forms a stable complex with the IFN-receptor-associated Tyk-2 kinase in vivo, and strongly suggest that this kinase regulates its phosphorylation on tyrosine. Thus, p95vav is engaged in IFN-signaling by a direct interaction with the functional type I IFN receptor complex to transduce downstream signals.

Benzoquinones↗

PubMed1997

Expression of type I interferon receptor in solid tumors of childhood.

Interferon alpha (IFN alpha) is used as an antineoplastic agent, both in hematopoietic malignancies and in solid tumors, because of its immunomodulatory action and direct antitumor activity. IFN alpha binds to specific cell-surface receptors that mediate its biologic activity. We studied the expression of IFN alpha receptors in pediatric solid tumors by use of the monoclonal antibody IFNaR3, which specifically recognizes the alpha subunit of the IFN Type I receptor. In three cell lines derived from those tumors, we determined the structure of the receptors by affinity cross-linking and immunoprecipitation techniques, and we determined their ability to mediate an antiproliferative effect. All of the tumor specimens studied by immunocytochemical analysis, including neuroblastomas, primitive neuroectodermal tumors, and rhabdomyosarcomas, stained positive for the IFN alpha receptor antibody, although in some cases immunoreactivity was weak. The three cell lines, derived from a neuroblastoma, a primitive neuroectodermal tumor, and a Ewing's sarcoma, respectively, showed the same pattern of IFN alpha receptor expression, both by affinity crosslinking and immunoprecipitation assays. Treatment with IFN alpha of those cell lines induces growth inhibition in vitro. These results suggest that IFN Type I receptor might be expressed in most solid tumors of childhood and that its structure is identical to the receptor expressed by the majority of hematologic malignancies.

Adolescent↗

PubMed1996

Differences in interferon alpha and beta signaling. Interferon beta selectively induces the interaction of the alpha and betaL subunits of the type I interferon receptor.

All Type I interferons (IFNalpha, IFNbeta, IFNomega) bind to the Type I IFN receptor (IFNR) and elicit a common set of signaling events, including activation of the Jak/Stat and IRS pathways. However, IFNbeta selectively induces the association of the alpha subunit of the Type I IFNR with p100, a tyrosyl phosphoprotein, to transduce IFNbeta-specific signals. Using antibodies raised against the different components of the Type I IFNR, we identified p100 as the long form of the beta subunit (betaL subunit) of the Type I IFNR. This was also confirmed in experiments with mouse L-929 cells transfected with truncated forms of betaL. Thus, IFNbeta stimulation of human cells or mouse L-929 transfectants expressing the human alpha and betaL subunits, selectively induces the formation of a signaling complex containing the alpha and betaL subunits of the receptor. The IFNbeta-regulated interaction of the alpha and betaL chains is rapid and transient and follows a similar time course with the tyrosine phosphorylation of these receptor components. These data demonstrate that the signaling specificity for different Type I IFNs is established early in the signaling cascade, at the receptor level, and results from distinct interactions between components of the Type I IFNR.

Animals↗

PubMed1996

The type-I interferon receptor. The long and short of it.

The type-I interferon receptor is a multisubunit receptor of the cytokine receptor superfamily. The production of specific monoclonal antibodies against the receptor and the cloning of different receptor subunits have contributed to understanding the type-I interferon receptor structure and function. The present article analyzes these new advances and the role of the different receptor subunits in type-I interferon signaling.

Binding Sites↗

PubMed1996

Immunohistochemical detection of the type I interferon receptor in human fetal, adult, and neoplastic tissues.

We have used the monoclonal antibody IFNaR3 that recognizes the alpha subunit of the type I interferon (IFN) receptor to study the expression of this receptor in a large series of normal human adult and fetal tissues, as well as in a large number of tumors of diverse origin. Among fetal tissues (8-20 weeks) the type I IFN receptor was expressed in liver, striated muscle, epidermis, renal tubules, choroid plexus of the CNS, and epithelia of different origins (bronchial, gastrointestinal, and pancreatic). Adult tissues showed a similar pattern that includes epithelia from salivary ducts, genital tract, bladder, breast, as well as germinal centers of lymph nodes, tonsils, and spleen. The study of a large series of tumors revealed that the type I IFN receptor is expressed in most, but not all, melanomas, bladder, kidney, small bowel, lung, and breast adenocarcinomas. The majority of lymphomas, sarcomas, and endocrine tumors proved negative. These results support the concept that the type I IFN receptor is rather ubiquitously expressed in normal and malignant epithelial tissues. More interestingly, the expression of the type I IFN receptor was not detected in all tumors, raising the question of whether some cases may fail IFN alpha therapy due to the lack of receptor expression. This report demonstrates that the IFNaR3 monoclonal antibody can be used for receptor detection in paraffin-embedded sections and it could represent a useful tool in the search for correlations between IFN alpha response and receptor expression in different diseases.

Adult↗

PubMed1995

Homodimerization and intermolecular tyrosine phosphorylation of the Tyk-2 tyrosine kinase.

The Jak kinases and Stat transcription factors play a major role in signaling of various cytokines including IFN alpha. In this report we show a ligand-independent interaction between Tyk-2 and Jak-1 kinases. We also demonstrate that the Tyk-2 kinase forms a homodimer that has the ability to undergo intermolecular tyrosine phosphorylation. The formation of the Tyk-2 homodimer is independent of both tyrosine phosphorylation and the presence of the tyrosine kinase domain.

Cell Line↗

PubMed1995

Cloning and expression of a long form of the beta subunit of the interferon alpha beta receptor that is required for signaling.

The interferon alpha beta receptor (IFN alpha R) or type I IFN-R is formed by a 110-kDa alpha subunit or IFNAR and by a beta subunit, which has short and long forms (molecular masses of 55 and 95-100 kDa, respectively). In this report, we demonstrate that the IFN alpha/beta R cDNA recently cloned corresponds to the 55-kDa or short form of the beta subunit, while the 95-100-kDa species reported here corresponds to a longer form of the IFN alpha/beta R cDNA that is probably produced by alternative splicing of the same gene. Stable transfection of the alpha subunit with either form of the beta subunit results in the expression of low and high affinity receptors, while expression of either form of the beta subunit alone only produces low affinity receptors. More important, only expression of the alpha and long form of the human beta subunits in mouse L-929 cells reconstitutes the activation of the Jak kinases and the Stat factors, as well as the antiviral response to human type I IFNs.

Amino Acid Sequence↗