Homology model of human interferon-alpha 8 and its receptor complex.

Human interferon-alpha 8 (HuIFN alpha 8), a type I interferon (IFN), is a cytokine belonging to the hematopoietic super-family that includes human growth hormone (HGH). Recent data identified two human type I IFN receptor components. One component (p40) was purified from human urine by its ability to bind to immobilized type I IFN. A second receptor component (IFNAR), consisting of two cytokine receptor-like domains (D200 and D200'), was identified by expression cloning. Murine cells transfected with a gene encoding this protein were able to produce an antiviral response to human IFN alpha 8. Both of these receptor proteins have been identified as members of the immunoglobulin superfamily of which HGH receptor is a member. The cytokine receptor-like structural motifs present in p40 and IFNAR were modeled based on the HGH receptor X-ray structure. Models of the complexes of HuIFN alpha 8 with the receptor subunits were built by superpositioning the conserved C alpha backbone of the HuIFN alpha 8 and receptor subunit models with HGH and its receptor complex. The HuIFN alpha 8 model was constructed from the C alpha coordinates of murine interferon-beta crystal structure. Electrostatic potentials and hydrophobic interactions appear to favor the model of HuIFN alpha 8 interacting with p40 at site 1 and the D200' domain of IFNAR at site 2 because there are regions of complementary electrostatic potential and hydrophobic interactions at both of the proposed binding interfaces. Some of the predicted receptor binding residues within HuIFN alpha 8 correspond to functionally important residues determined previously for human IFN alpha 1, IFN alpha 2, and IFN alpha 4 subtypes by site-directed mutagenesis studies. The models predict regions of interaction between HuIFN alpha 8 and each of the receptor proteins, and provide insights into interactions between other type I IFNs (IFN-alpha subtypes and IFN-beta) and their respective receptor components.     

Human leukocyte interferon subtypes have different antiproliferative and antiviral activities on human cells

The antigrowth effects of 5 different cloned human leukocyte IFN subtypes (IFN-alpha A, B, C, D, F) and 2 molecular hybrids between them (IFN-alpha AD(Bg1II) and IFN-alpha DA(Bg1II)) were examined on 6 different human cell lines. The results indicate that the interferons sort into two distinct groups: IFN-alpha B, C and F showed comparable antiproliferative activity which was greater than that of IFN-alpha A, D, AD(Bg1II) and DA(Bg1II). The interferons could also be assigned to one of two groups on the basis of their antiviral activity. IFN-alpha A, D and AD(Bg1II) were observed to be more protective than IFN-alpha B, C and F against HSV-2 and EMCV infections, i.e. the relative antiviral efficacies of the cloned IFN subtypes were the reverse of their antiproliferative activities.     

Characterization of human beta-interferon-binding sites on human cells

Radioiodinated recombinant human beta-interferon (rHuIFN beta Ser), with almost full (greater than 90%) biological activity, was used to study the binding of human beta-interferon to Daudi cells. Specific binding was not observed with less biologically active (less than or equal to 10%) radioiodinated interferon. The bound radioiodinated interferon was shown to compete with human beta-interferon (HuIFN beta), rHuIFN beta Ser, human alpha-interferon (HuIFN alpha) and with human gamma-interferon (HuIFN gamma). Scatchard plot analyses suggest the presence of about 10,000 binding sites for HuIFN beta/Daudi cell. About 6,600 of these sites can be blocked by HuIFN alpha and 3,700 sites can be blocked by HuIFN gamma. The apparent Kd for HuIFN beta is 2.7 nM. The apparent Kd values for HuIFN alpha and HuIFN gamma are 3.7 and 1.1 nM, respectively. It was possible to demonstrate the cross-linking of HuIFN beta to two macromolecular components of Mr = 128,000 and 103,000. We propose the existence of at least two binding sites for HuIFN beta in Daudi cells, one site recognizing both HuIFN beta and HuIFN gamma, the other site recognizing both HuIFN beta and HuIFN alpha. Each site is capable of recognizing only HuIFN gamma or HuIFN alpha.     

Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells

We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells.     

Subtype-selective noncompetitive or competitive inhibition of human alpha1-adrenergic receptors by rho-TIA

The 19-amino acid conopeptide (rho-TIA) was shown previously to antagonize noncompetitively alpha(1B)-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human alpha(1)-AR subtypes (alpha(1A), alpha(1B), and alpha(1D)). Radioligand binding assays showed that rho-TIA was 10-fold selective for human alpha(1B)-over alpha(1A)- and alpha(1D)-ARs. As observed with hamster alpha(1B)-ARs, rho-TIA decreased the number of binding sites (B(max)) for human alpha(1B)-ARs without changing affinity (K(D)), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, rho-TIA had opposite effects at human alpha(1A)-ARs and alpha(1D)-ARs, decreasing K(D) without changing B(max), suggesting it acts competitively at these subtypes. rho-TIA reduced maximal NE-stimulated [(3)H]inositol phosphate formation in HEK293 cells expressing human alpha(1B)-ARs but competitively inhibited responses in cells expressing alpha(1A)- or alpha(1D)-ARs. Truncation mutants showed that the amino-terminal domains of alpha(1B)- or alpha(1D)-ARs are not involved in interaction with rho-TIA. Alanine-scanning mutagenesis of rho-TIA showed F18A had an increased selectivity for alpha(1B)-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at alpha(1B)-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus rho-TIA noncompetitively inhibits alpha(1B)-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at alpha(1B)-ARs.     

Detection of functional interferon alpha receptors in human neuroendocrine tumor cell lines using a new monoclonal antibody.

While first described as antiviral agents, interferons (IFNs) exhibit significant antiproliferative and antitumor effects as well. IFN alpha has been successfully used in clinical trials to treat several malignancies, including leukemias and certain solid tumors. While many cell types have been studied for IFN alpha receptor expression, very little is known about receptor expression on human neuroendocrine cells. Using a novel anti-IFN alpha receptor monoclonal antibody, we examined IFN alpha receptor expression in 10 human cell lines derived from tumors of neuroendocrine origin, including neuroblastoma, neuroepithelioma and small cell lung carcinoma. All cell lines studied displayed a similar pattern of IFN alpha receptor expression and 5 of 8 cell lines demonstrated reduced thymidine incorporation following IFN alpha treatment. Addition of exogenous IFN alpha caused a decrease in IFN alpha receptor expression, while differentiating agents, such as phorbol esters and retinoic acid, induced an increase in receptor number without altering receptor affinity.     

Epitopes of human interferon-alpha defined by the reaction of monoclonal antibodies with alpha interferons and interferon analogues

Five monoclonal antibodies reactive with human interferon (HuIFN)-alpha 2, but not with HuIFN-alpha 1, have been analyzed for their reaction with a series of IFN analogues and hybrid IFN molecules. Using analogues containing alpha 1 or gamma substitutions, it was shown that amino acids in the 107 to 113 region are implicated in the epitopes recognized by four of the five antibodies tested. Surprisingly, two of the antibodies that did not react with [alpha 1(113 to 114)112 to 113]alpha 2 also did not react with a truncated IFN-alpha 2(4 to 155). The presence of an epitope determined by amino acids at 112 and 113 and by the amino and carboxyl ends of the molecule supports a model for IFN where the carboxyl- and amino-terminals are adjacent to the proposed reverse turn around amino acid 110 to 115. The fifth alpha 2 specific antibody whose reaction with HuIFN-alpha 2 is not affected by the above substitutions and truncations recognizes IFN or IFN hybrids that, like alpha 2, have arginine at position 120, but does not react with IFN that, like alpha 1, have lysine at position 120. Amino acids 107 to 113 and 120 lie in regions of the molecule that have high hydrophilicity and are probably structurally involved in the epitopes recognized by the antibodies. Under conditions of antibody excess, the antibodies described here inhibit binding of HuIFN-alpha 2 to both human and bovine cells.     

激动剂作用下α1-肾上腺素受体亚型在HEK293A细胞中的分布变化

为了明确α1-肾上腺素受体（α1-adrenergic receptor,α1-AR）三种亚型在人胚胎肾（human embryonic kidney,HEK）293A细胞株中的分布特点,及其在激动剂作用下在细胞内的定位改变,本研究采用放射配体结合实验、实时荧光共聚焦成像和Western blot方法检测α1-AR三种亚型在细胞中的定位及蛋白质表达的变化。结果发现：（1）α1-AR三种亚型在HEK293A细胞株转染效率相同,均达90％以上。三株细胞的粗制膜上α1B-AR表达量最高,α1D-AR最低,α1A-AR居中,但三者的解离常数（配）相等;（2）在无激动剂作用时,α1A-AR均匀地分布在HEK293A细胞的胞膜和胞浆,α1B-AR主要位于胞膜,而α1D-AR则主要分布在胞浆中：（3）用α1-AR激动剂苯‘肾上腺素（phenylephrine,PE）刺激细胞1h后,α1A-和α1B-AR在胞膜上分布明显减少,而在胞浆中分布增加,其中α1B-AR变化更为显著,α1D-AR的分布在PE作用下无明显变化。以上结果提示,在激动剂作用下,α1-AR二种亚型在HEK293A细胞中的定位特点和分布变化各有不同。     

Interferon alpha induces rapid tyrosine phosphorylation of the alpha subunit of its receptor.

The mechanisms of generation of second messengers after binding of interferon alpha (IFN alpha) to its receptor remain unknown. We have studied the phosphorylation of the alpha subunit of the IFN alpha receptor, which is recognized by the monoclonal antibody IFNa receptor 3. Immunoblotting experiments showed that IFN alpha induced rapid tyrosine phosphorylation of the alpha subunit in the IFN alpha-sensitive H-929, U-266, and Daudi cell lines. Immunoprecipitation experiments performed with 32P-labeled cells showed that the alpha subunit is phosphorylated before IFN alpha treatment and that the level of phosphorylation increases after IFN alpha stimulation. Phosphoamino acid analysis confirmed the IFN alpha-induced tyrosine phosphorylation and demonstrated that the base-line phosphorylation corresponded to serine phosphorylation that increased 50% upon IFN alpha treatment. Tyrosine phosphorylation of the alpha subunit was time- and dose-dependent, further demonstrating the specificity of the process. Phosphorylation of the alpha subunit of the receptor occurred rapidly after IFN alpha binding, both at 37 and 4 degrees C. Exposure of the cells to the tyrosine kinase inhibitor genistein blocked the IFN alpha-induced tyrosine phosphorylation of this subunit of the IFN alpha receptor. In contrast H7, a specific protein kinase C inhibitor, and acute and chronic exposure to phorbol esters had no effect on tyrosine phosphorylation, suggesting that protein kinase C does not regulate the tyrosine phosphorylation of the alpha subunit of the IFN alpha receptor. No IFN alpha-induced tyrosine phosphorylation was observed in the IFN alpha-resistant U-937 cell line that expresses a variant IFN alpha receptor. Altogether these data suggest that tyrosine phosphorylation of the alpha subunit may play a role in the signal transduction pathway of IFN alpha.     

Effects of human interferon on cellular response to UV in UV-sensitive human cell strains

Cells of a human RSa cell line, with high sensitivity to UV killing and low capacity for DNA repair, when pretreated with 1-100 units/ml of human interferon (HuIFN) preparations for more than 12 h before irradiation, acquired an enhancement of UV-induced DNA-repair replication synthesis in association with recovery from inhibition of total cellular DNA synthesis and UV survival. Prompt and transient induction of plasminogen activator activities was also found within 5 min after UV irradiation in the cells pretreated with HuIFN but not in the cells non-pretreated with HuIFN. The enhancement and induction effects of HuIFN were observed, irrespective of the kind of HuIFN preparation used (alpha, beta or gamma, and natural or recombinant) and in other UV-sensitive fibroblast cells which were derived from Cockayne syndrome and xeroderma pigmentosum fibroblasts (XP1KY). However, all of the enhancement of DNA-repair synthesis and the induction of plasminogen activator activities by HuIFN was suppressed by treatment with cycloheximide immediately after UV irradiation.     

Arthropod D2 receptors positively couple with cAMP through the Gi/o protein family

The pyloric network is an important model system for understanding neuromodulation of rhythmic motor behaviors like breathing or walking. Dopamine (DA) differentially modulates neurons within the pyloric network. However, while the electrophysiological actions of DA have been well characterized, nothing is known about the signaling events that mediate its effects. We have begun a molecular characterization of DA receptors (DARs) in this invertebrate system. Here, we describe the cloning and characterization of the lobster D(2) receptor, D(2 alpha Pan). We found that when expressed in HEK cells, the D(2 alpha Pan) receptor is activated by DA, but not other monoamines endogenous to the lobster nervous system. This receptor positively couples with cAMP through multiple Gi/o proteins via two discrete pathways: 1) a G alpha mediated inhibition of adenylyl cyclase (AC), leading to a decrease in cAMP and 2) a G beta gamma-mediated activation of phospholipase C beta (PLC beta), leading to an increase in cAMP. Alternate splicing alters the potency and efficacy of the receptor, but does not affect monoamine specificity. Finally, we show that arthropod D(2) receptor coupling with cAMP varies with the cellular milieu.     

D1-D2 dopamine receptor interaction within the nucleus accumbens mediates long-loop negative feedback to the ventral tegmental area (VTA)

The objective of the present study was to examine the effects of perfusion of dopamine (DA) D1- and D2-like receptor agonists in the nucleus accumbens (ACB) on the long-loop negative feedback regulation of mesolimbic somatodendritic DA release in the ventral tegmental area (VTA) of Wistar rats employing ipsilateral dual probe in vivo microdialysis. Perfusion of the ACB for 60 min with the D1-like receptor agonist SKF 38393 (SKF, 1-100 microM) dose-dependently reduced the extracellular levels of DA in the ACB, whereas the extracellular levels of DA in the VTA were not changed. Similarly, application of the D2-like receptor agonist quinpirole (Quin, 1-100 microM) through the microdialysis probe in the ACB reduced the extracellular levels of DA in the ACB in a concentration-dependent manner, whereas extracellular levels of DA in the VTA were not altered. Co-application of SKF (100 microM) and Quin (100 microM) produced concomitant reductions in the extracellular levels of DA in the ACB and VTA. The reduction in extracellular levels of DA in the ACB and VTA produced by co-infusion of SKF and Quin was reversed in the presence of either 100 microM SCH 23390 (D1-like antagonist) or 100 microM sulpiride (D2-like antagonist). Overall, the results suggest that (a) activation of dopamine D1- or D2-like receptors can independently regulate local terminal DA release in the ACB, whereas stimulation of both subtypes is required for activation of the negative feedback pathway to the VTA.     

The effect of selective sst1, sst2, sst5 somatostatin receptors agonists, a somatostatin/dopamine (SST/DA) chimera and bromocriptine on the "clinically non-functioning" pituitary adenomas in vitro

The aim of the work was to investigate the effects of somatostatin analogs acting selectively on sst1 (BIM-23926), sst2 (BIM-23120) and sst5 (BIM-23206) receptor subtypes on the viability of "clinically non-functioning" pituitary adenomas in vitro. The effects of native SST (SST-14), a SST/DA chimera (BIM-23A387) and a D(2)-dopamine receptor agonist bromocriptine (BC) were also examined. The study was performed on 10 surgically removed pituitary macroadenomas, diagnosed before surgery as "non-functioning". A part of each tumor was mechanically dispersed and digested with collagenase to isolate the tumoral cells. Another part of each tumor was fixed, embedded in paraffin and immunostained to reveal the pituitary hormones and SST receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, sst5). The tumoral cell suspensions were incubated for 24 h with the substances mentioned above. The quantity of viable cells was estimated using the EZ4U system. The results were compared with the immunohistochemical evaluation of the hormonal profile of adenoma and the sst receptor subtype immunoreactivities present. The findings indicate that selective sst1, sst2 and sst5 receptors agonists, SST/DA chimera and D(2)-dopamine receptor agonist bromocriptine affect the viability of some, but not all, "clinically non-functioning" pituitary adenomas in vitro. The most effective was bromocriptine. The investigated somatostatin analogs including SST/DA chimera exerted roughly similar inhibitory effects. Further studies are needed to fully evaluate the potential usefulness of these compounds in the pharmacological treatment of "non-functioning" pituitary tumors.     

Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.     

Two interferons alpha influence each other during their interaction with the extracellular domain of human type interferon receptor subunit 2

The interaction between two human interferons alpha (IFN-alphas) and the extracellular (EC) domain of human type I IFN receptor subunit 2 (IFNAR2) was analyzed. Previous experiments using Daudi cells showed that IFN-alpha21b and some IFN-alpha hybrids (made from IFN-alpha2c and 21b) competed poorly for the IFN-alpha2b binding site. This study examined the causes of the poor competition between these IFN-alphas. IFN-alpha2c and the IFN hybrid CM3 {IFN-alpha21b(1-75)(81-95)/IFN-alpha2c(76-80) (96-166), Y86K} were selected for this study based on their cell binding and biological properties. Competitive binding ELISA, native electrophoresis followed by Western blot, electrospray ionization mass spectrometry (ESI-MS), surface plasmon resonance biosensor (SPR) analysis, as well as neutralization of antiproliferative activities on Daudi cells in the presence of soluble IFNAR2-EC show evidence that each of the described IFN-alpha subtypes affected the binding of the other IFN-alpha to IFNAR2-EC by affecting the stability of the complex, i.e., dissociation of the complex. Moreover, native electrophoresis with different IFNAR2-EC mutants showed that IFN-alpha2c and CM3 utilize different amino acids in the binding domain of IFNAR2-EC. In addition to that, analytical ultracentrifugation (AUC) revealed differences in the oligomeric state of the two studied interferons. Our results demonstrated that two individual IFN-alphas interact differentially with IFNAR2-EC and influence each other during this interaction. This study contributes to the understanding of the mutual interaction between multiple IFN-alpha subtypes during the competition for binding to the receptor.     

Modifying effects of interferon and puromycin on cytogenetic damage induced by alkylating drug phopurine in human lymphocytes.

The induction of sister chromatid exchanges (SCEs) by the bifunctional alkylating antineoplastic drug phopurine (2-dimethyl-amino-6-diethyleneiminophosphamido-7-methylpurine) and its modification by human recombinant interferons alpha 2, beta and gamma (HuIFN alpha 2, HuIFN beta and HuIFN gamma) and puromycin (PM) were studied in human lymphocytes. Results demonstrated a striking similarity in the modifying action of HuIFN alpha 2 and PM: 1) both modifiers reduced SCE values induced by phopurine, 2) at high and low doses of phopurine the effect of both modifiers was minimal, and 3) both agents were able to convert DNA lesions from short-term to long-term.     

Physiological significance of alpha(2)-adrenergic receptor subtype diversity: one receptor is not enough

Alpha(2)-adrenergic receptors mediate part of the diverse biological effects of the endogenous catecholamines epinephrine and norepinephrine. Three distinct subtypes of alpha(2)-adrenergic receptors, alpha(2A), alpha(2B), alpha(2C), have been identified from multiple species. Because of the lack of sufficiently subtype-selective ligands, the specific biological functions of these receptor subtypes were largely unknown until recently. Gene-targeted mice carrying deletions in the genes encoding for individual alpha(2)-receptor subtypes have added important new insight into the physiological significance of adrenergic receptor diversity. Two different strategies have emerged to regulate adrenergic signal transduction. Some biological functions are controlled by two counteracting alpha(2)-receptor subtypes, e.g., alpha(2A)-receptors decrease sympathetic outflow and blood pressure, whereas the alpha(2B)-subtype increases blood pressure. Other biological functions are regulated by synergistic alpha(2)-receptor subtypes. The inhibitory presynaptic feedback loop that tightly regulates neurotransmitter release from adrenergic nerves also requires two receptor subtypes, alpha(2A) and alpha(2C). Similarly, nociception is controlled at several levels by one of the three alpha(2)-receptor subtypes. Further investigation of the specific function of alpha(2)-subtypes will greatly enhance our understanding of the relevance of closely related receptor proteins and point out novel therapeutic strategies for subtype-selective drug development.     

Characterization of the murine alpha interferon gene family

Mouse and human genomes carry more than a dozen genes coding for closely related alpha interferon (IFN-alpha) subtypes. IFN-alpha, as well as IFN-beta, IFN-kappa, IFN-epsilon, and limitin, are thought to bind the same receptor, raising the question of whether different IFN subtypes possess specific functions. As some confusion existed in the identity and characteristics of mouse IFN-alpha subtypes, the availability of data from the mouse genome sequence prompted us to characterize the murine IFN-alpha family. A total of 14 IFN-alpha genes were detected in the mouse genome, in addition to three IFN-alpha pseudogenes. Four IFN-alpha genes (IFN-alpha1, IFN-alpha7/10, IFN-alpha8/6, and IFN-alpha11) exhibited surprising allelic divergence between 129/Sv and C57BL/6 mice. All IFN-alpha subtypes were found to be stable at pH 2 and to exhibit antiviral activity. Interestingly, some IFN subtypes (IFN-alpha4, IFN-alpha11, IFN-alpha12, IFN-beta, and limitin) showed higher biological activity levels than others, whereas IFN-alpha7/10 exhibited lower activity. Most murine IFN-alpha turned out to be N-glycosylated. However, no correlation was found between N-glycosylation and activity. The various IFN-alpha subtypes displayed a good correlation between their antiviral and antiproliferative potencies, suggesting that IFN-alpha subtypes did not diverge primarily to acquire specific biological activities but probably evolved to acquire specific expression patterns. In L929 cells, IFN genes activated in response to poly(I*C) transfection or to viral infection were, however, similar.