Biomedical subjects

M F Shannon

Publications and source records attributed to M F Shannon.

At least 37 records · Page 2Linked to original sources

PubMed1996

High mobility group protein I(Y) is required for function and for c-Rel binding to CD28 response elements within the GM-CSF and IL-2 promoters.

CD28 response elements (CD28REs) within cytokine promoters are variant NF-kappaB-binding sites and are essential for transcription in response to CD28 receptor activation in T cells. We show that the CK-1 element (CD28RE) within the GM-CSF promoter binds the RelA and c-Rel transcription factors in response to CD28 activation. We further show that the high mobility group protein HMG I(Y) can bind to the CD28REs of both GM-CSF and IL-2 and that this binding is critical for c-Rel, but not RelA, binding. A second NF-kappaB site in the GM-CSF promoter that binds p50 and RelA, but neither c-Rel nor HMG I(Y), failed to respond to CD28 activation. Expression of HMG I or c-Rel antisense RNA inhibited CD28 activation of the IL-2 and GM-CSF promoters, implying that HMG I(Y) enhancement of c-Rel binding plays an important role in the activity of the CD28REs.

CD28 Antigens↗

PubMed1996

Costimulation of cytokine gene expression in T cells by the human T leukemia/lymphotropic virus type 1 trans activator Tax.

Many cell signals such as CD28 and CD4 binding can costimulate cytokine gene expression in activated T cells. We have found that the human T leukemia/lymphotropic virus type 1 viral protein Tax can also strongly costimulate expression of interleukin-2 (IL-2), IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA in T cells activated with the phorbol ester phorbol myristate acetate (PMA) and calcium ionophore, which can mimic activation through the antigen specific T-cell receptor. Reporter constructs also showed strong synergy between both stimuli and showed that Tax and the PMA-Ca2+ ionophore act through different regions of the IL-2 and GM-CSF genes. Furthermore, the Tax-responsive regions (TxRR) from both GM-CSF and IL-2 respond to costimulation through the CD28 surface receptor. The GM-CSF and IL-2 TxRRs showed significantly higher levels of NF-kappaB/rel binding, following induction by Tax, compared with that of the PMA-Ca2+ ionophore with only Tax capable of inducing c-Rel binding to a Consensus kappaB element within the GM-CSF TxRR. Tax protein mutants, however, showed that a pathway(s) other than NF-kappaB/rel induction could also cooperate with the PMA-Ca2+ ionophore to activate the GM-CSF and IL-2 genes. This high-level costimulation by Tax, through multiple pathways, may be important in the early stages of leukemia and in the nervous system disorder tropical spastic paraparesis.

Base Sequence↗

PubMed1995

ETS1 transactivates the human GM-CSF promoter in Jurkat T cells stimulated with PMA and ionomycin.

Activation of T helper cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one such cytokine whose increased expression results partly from increases in transcription. Cis-acting elements with NF kappa B, AP-1 and ETS-like motifs have been identified in the promoter region of the GM-CSF gene, which are important for transcriptional activity following PMA and ionomycin stimulation. A number of the ETS family of transcription factors are expressed in T cells, including ETS1 and ELF1. Here we describe the ability of these factors to interact with a site (GM5), located within the CLE0 element, -47 to -40 upstream of the GM-CSF transcription initiation site. Exogenous ETS1, but not ELF1, can transactivate GM-CSF, through the GM5 site, in a PMA/ionomycin dependent manner. Other unidentified ETS-like factors present in Jurkat cells are also capable of binding GM5. Mutation of the core ETS binding site from -GGAA- to -GGAT- prevents the binding of ETS-like factors with the exception of ETS1. The GM-CSF promoter, modified in this way to be ETS1 specific, is fully responsive to PMA/ionomycin induction, in addition to ETS1 transactivation in the presence of PMA and ionomycin. Together these data suggest that ETS1 may be involved in mediating the increased GM-CSF production associated with T cell activation.

Base Sequence↗

PubMed1995

Multiple signals are required for function of the human granulocyte-macrophage colony-stimulating factor gene promoter in T cells.

The human granulocyte-macrophage CSF (GM-CSF) gene is expressed in T cells in response to TCR activation that can be mimicked by treatment of the cells with PMA and Ca2+ ionophore. The gene contains a proximal functional promoter region (-620 to +34), as well as a powerful enhancer located 3 kb upstream, both of which are involved in the response of the gene to TCR activation. The proximal promoter contains a region termed CLEO (-54 to -31) that consists of a purine-rich element abutting an activator protein-1 (AP-1)-like site, as well as an upstream nuclear factor-kappa B (NF-kappa B) site (-85 to -76) and a CK-1 element (-101 to -92). We show in this work that mutations in either the purine-rich region of the CLEO element or the NF-kappa B site result in reduced PMA/Ca2+ activation of a 620-bp human GM-CSF promoter-luciferase reporter construct in Jurkat T cells by 65% and 50%, respectively. The major inducible protein complex that binds to the human CLEO (hCLEO) element is an AP-1-like complex that is inducible by PMA alone, but shows increased binding in response to PMA together with Ca2+ ionophore. Although the binding of this complex is not cyclosporin-sensitive, promoter induction is inhibited by cyclosporin treatment. A second weak inducible complex resembling nuclear factor of activated T cells (NF-AT) was also observed binding to the hCLEO region. By using recombinant proteins, we confirmed that AP-1, NF-ATp, and a higher order NF-ATp/AP-1 complex could all form with the hCLEO element, and we have also defined the sequence requirements for binding of each of these complexes. We found that expression of a constitutively active form of calcineurin could substitute for Ca2+ ionophore and synergize with PMA to activate the GM-CSF promoter, and conversely that mutant-activated Ras could substitute for PMA and cooperate with Ca2+ ionophore. Co-expression of Ras and calcineurin, however, did not activate the GM-CSF promoter, but required the additional expression of NF-kappa B p65. These results imply that at least three signals are required to activate the GM-CSF proximal promoter, and that the signals impinge on distinct transcription factors that bind to the hCLEO and NF-kappa B regions of the promoter.

Base Sequence↗

PubMed1995

Interaction of GM-CSF and IL-3 with the common beta-chain of their receptors.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL-3) are cytokines active in both normal and abnormal hemopoiesis, inflammation, and immunity. Their biological activity is mediated via receptors that comprise a ligand-specific alpha chain and a beta chain that is common to the GM-CSF, IL-3, and IL-5 receptors. To understand the mechanism of action of GM-CSF and IL-3 in both normal and pathological conditions, we are seeking to define the structural elements required for ligand/receptor and receptor/receptor contact and their role in cellular activation. To this end we have identified a conserved motif in the first helix of GM-CSF, Glu21 that is critical for high affinity binding and biological activity. Charge-reversal mutagenesis of this residue generates a GM-CSF analogue that is devoid of biological activity and can antagonize the activity of wild-type GM-CSF. This probably results from the selective deficiency in interaction with the beta chain of the receptor and suggests that similar antagonists for IL-3 and IL-5 are also feasible. Complementary mutagenesis studies on the receptor beta chain have identified the putative B'-C' loop in the membrane-proximal domain as being critical for the high affinity binding of GM-CSF but not IL-3. Characterization of the specificity of sites of interaction between the ligands and receptors may permit the design of specific or genetic antagonists that may have important therapeutic implications.

Amino Acid Sequence↗

PubMed1995

GM-CSF and IL-2 share common control mechanisms in response to costimulatory signals in T cells.

Antigen complexed with major histocompatibility complex class I or II molecules on the surface of antigen presenting cells interacts with the T cell receptor (TCR) on the surface of T cells and initiates an activation cascade. So called costimulatory signals, mediated by other cell surface interactions or soluble cytokines produced by antigen presenting cells, are also required for complete T cell activation. High levels of cytokine gene expression in T cells also required both TCR and costimulatory signals. The granulocyte-macrophage colony-stimulating factor requires sequences in the promoter as well as a powerful enhancer located 3kb upstream to respond to TCR-like signals. These promoter and enhancer regions are mainly activated by the transcription factor nuclear factor of activated T cells (NFAT). The activation of NFAT by TCR signals has been well described for interleukin-2 (IL-2) and IL-4 gene transcription in T cells. Costimulatory signals, such as activation of the CD28 cell surface molecule on T cells, lead to activation through a distinct region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter. This region is termed the CK-1 or CD28RE and appears to bind specific members of the NF-kappa B family of transcription factors. Human T leukemia virus type 1 (HTLV-1) infects T cells and can lead to increase GM-CSF expression. We have found that the HTLV-1 transactivator protein, tax, acts as a costimulatory signal for GM-CSF and IL-2 gene transcription, in that it can cooperate with TCR signals to mediate high level gene expression. Tax activates the GM-CSF promoter through the CK-1/CD28RE region and also activates nuclear factor-kappa B binding to this region. However, other transcription factors or coactivators of NF-kappa B are required for tax activation but these remain to be identified. The CK-1/CD28RE of GM-CSF shows a high degree of similarity to the IL-2 CD28RE and the IL-3 gene also contains a related region. This observation, together with the fact that both GM-CSF and IL-2 respond to TCR signals via NFAT, implies a high degree of conservation in the regulation of cytokine gene expression in T cells.

Base Sequence↗

PubMed1995

Human granulocyte-macrophage colony-stimulating factor enhancer function is associated with cooperative interactions between AP-1 and NFATp/c.

The promoter of the human granulocyte-macrophage colony-stimulating factor gene is regulated by an inducible upstream enhancer. The enhancer encompasses three previously defined binding sites for the transcription factor NFAT (GM170, GM330, and GM550) and a novel NFAT site defined here as the GM420 element. While there was considerable redundancy within the enhancer, the GM330, GM420, and GM550 motifs each functioned efficiently in isolation as enhancer elements and bound NFATp and AP-1 in a highly cooperative fashion. These three NFAT sites closely resembled the distal interleukin-2 NFAT site, and methylation interference assays further defined GGA(N)9TCA as a minimum consensus sequence for this family of NFAT sites. By contrast, the GM170 site, which also had conserved GGA and TCA motifs but in which these motifs were separated by 15 bases, supported strong independent but no cooperative binding of AP-1 and NFATp, and this site functioned poorly as an enhancer element. While both the GM330 and GM420 elements were closely associated with the inducible DNase I-hypersensitive site within the enhancer, the GM420 element was the only NFAT site located within a 160-bp HincII-BalI fragment defined by deletion analysis as the essential core of the enhancer. The GM420 element was unusual, however, in containing a high-affinity NFATp/c-binding sequence (TGGAAAGA) immediately upstream of the sequence TGACATCA which more closely resembled a cyclic AMP response-like element than an AP-1 site. We suggest that the cooperative binding of NFATp/c and AP-1 requires a particular spacing of sites and that their cooperativity and induction via independent pathways ensure very tight regulation of the granulocyte-macrophage colony-stimulating factor enhancer.

Base Sequence↗

PubMed1994

A sequence-specific single-strand DNA binding protein that contacts repressor sequences in the human GM-CSF promoter.

NF-GMb is a nuclear factor that binds to the proximal promoter of the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene. NF-GMb has a subunit molecular weight of 22 kDa, is constitutively expressed in embryonic fibroblasts and binds to sequences within the adjacent CK-1 and CK-2 elements (CK-1/CK-2 region), located at approximately -100 in the GM-CSF gene promoter. These elements are conserved in haemopoietic growth factor (HGF) genes. NF-GMb binding requires the presence of repeated 5'CAGG3' sequences that overlap the binding sites for positive activators. Surprisingly, NF-GMb was found to bind solely to single-strand DNA, namely the non-coding strand of the GM-CSF CK-1/CK-2 region. NF-GMb may belong to a family of single-strand DNA binding (ssdb) proteins that have 5'CAGG3' sequences within their binding sites. Functional analysis of the proximal GM-CSF promoter revealed that sequences in the -114 to -79 region of the promoter containing the NF-GMb binding sites had no intrinsic activity in fibroblasts but could, however, repress tumour necrosis factor-alpha (TNF-alpha) inducible expression directed by downstream promoter sequences (-65 to -31). Subsequent mutation analysis showed that sequences involved in repression correlated with those required for NF-GMb binding.

Base Sequence↗

PubMed1994

Synergistic transcriptional activation of the IL-8 gene by NF-kappa B p65 (RelA) and NF-IL-6.

Transcriptional activation of the IL-8 gene by several inflammatory mediators, including the cytokines IL-1 and TNF-alpha, is mediated through sequences located between nucleotide -94 and -71 of the IL-8 promoter. Because adjacent binding sites for the inducible transcription factors NF-kappa B and NF-IL-6 are located within this region, we examined the functional interaction of these two transcription factor families in IL-8 gene regulation. Maximal transcriptional activation by PMA in Jurkat T lymphocytes was shown to require intact binding sites for both NF-kappa B and NF-IL-6. Electrophoretic mobility shift analysis indicates that NF-IL-6, as well as other related members of this family, bind specifically to the NF-IL-6 site in the IL-8 promoter. In addition, NF-kappa B p65 (RelA), but not NF-kappa B p50 (NFKB1), binds specifically to the NF-kappa B site. When incubated together, RelA and NF-IL-6/C/EBP form a ternary complex with this region of the IL-8 promoter; this binding is dependent on intact binding sites for both NF-IL-6 and RelA. Transient cotransfection analyses indicate that the cooperative association of NF-IL-6 and RelA with the IL-8 promoter results in synergistic transcriptional activation. Mutational analyses of RelA demonstrate that the C-terminal transactivation domain and the DNA binding domain are required for synergistic activation with NF-IL-6. In addition, overexpression of the NF-kappa B inhibitor molecule, I kappa B, abolished the RelA- and RelA/NF-IL-6-dependent synergistic activation. These data demonstrate that RelA and members of the C/EBP/NF-IL-6 family can functionally cooperate in transcriptional activation of the IL-8 gene and suggest a common mechanism for inducible regulation of cytokine gene expression.

Base Sequence↗

PubMed1994

Specific human granulocyte-macrophage colony-stimulating factor antagonists.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hemopoietic growth factor and activator of mature myeloid cell function. We have previously shown that residue 21 in the first helix of GM-CSF plays a critical role in both biological activity and high-affinity receptor binding. We have now generated analogues of GM-CSF mutated at residue 21, expressed them in Escherichia coli, and examined them for binding, agonistic, and antagonistic activities. Binding experiments showed that GM E21A, E21Q, E21F, E21H, E21R, and E21K bound to the GM-CSF receptor alpha chain with a similar affinity to wild-type GM-CSF and had lost high-affinity binding to the GM-CSF receptor alpha-chain-common beta-chain complex. From these mutants, only the charge reversal mutants E21R and E21K were completely devoid of agonistic activity. Significantly we found that E21R and E21K antagonized the proliferative effect of GM-CSF on the erythroleukemic cell line TF-1 and primary acute myeloid leukemias, as well as GM-CSF-mediated stimulation of neutrophil superoxide production. This antagonism was specific for GM-CSF in that no antagonism of interleukin 3-mediated TF-1 cell proliferation or tumor necrosis factor alpha-mediated stimulation of neutrophil superoxide production was observed. E. coli-derived GM E21R and E21K were effective antagonists of both nonglycosylated and glycosylated wild-type GM-CSF. These results show that low-affinity GM-CSF binding can be dissociated from receptor activation and have potential clinical significance for the management of inflammatory diseases and certain leukemias where GM-CSF plays a pathogenic role.

Amino Acid Sequence↗

PubMed1994

Identification of residues in the first and fourth helices of human granulocyte-macrophage colony-stimulating factor involved in biologic activity and in binding to the alpha- and beta-chains of its receptor.

Residues within the first and fourth helices of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) were analyzed for their role in biologic activity and interaction with the alpha- and beta-chains of the hGM-CSF receptor. Within the first helix substitution of the surface residues Glu14, Asn17, Gln20, Arg23, Arg24, and Asn27 or the buried residues Ala18, Leu25, and Leu28 did not significantly impair bioactivity or receptor binding. Substitutions at the buried residues Ala22 and Leu26 had intermediate bioactivity. However, substitutions of the surface residue Glu21 or the buried residue Ile19 reduced the relative bioactivity of the analogues to as little as 0.45% and 0.3%, respectively. Substitution of the charged surface residues of the fourth helix showed that substitution at Glu104, Lys107, and Lys111 had no significant effect on bioactivity, but substitution at Glu108 and Asp112 reduced the potency of the analogues to 34% and 7%, respectively. Receptor binding studies showed that, whereas Glu21 is the critical residue for binding to the hGM-CSF-receptor beta-chain, Asp112 is likely to be involved in binding to the GM-CSF-receptor alpha-chain. These results establish the relative contribution of residues in the first and fourth helices for GM-CSF bioactivity and receptor binding, and support a model where the fourth helix of GM-CSF interacts with the alpha-chain, and the first helix with the beta-chain of the GM-CSF receptor.

Amino Acid Sequence↗

PubMed1994

Requirement for nuclear factor (NF)-kappa B p65 and NF-interleukin-6 binding elements in the tumor necrosis factor response region of the granulocyte colony-stimulating factor promoter.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor produced by mesenchymal and myeloid cells following activation by inflammatory stimuli. It has previously been shown that a region of the G-CSF promoter, (-200 to -165) containing the decanucleotide CK-1 element and two repeated sequences that resemble nuclear factor (NF)-interleukin-6 (IL-6) binding sites, is required for activation of the G-CSF gene by tumor necrosis factor-alpha (TNF-alpha) and IL-1 beta. We now show that the NF-kappa B p65 protein can bind to and activate this TNF response region. There are several unusual features of this p65 interaction with the TNF response region. First, NF-kappa B p65 but not the related NF-kappa B p50 binds to the CK-1 element and a p50/65 hybrid protein that relies on the p50 rel homology domain for DNA binding does not transactivate the TNF response region. Second, p65 transactivation of this region is cell specific and requires not only its own binding site but also the NF-IL6 consensus sites. NF-IL6 also binds to the TNF response region of the G-CSF promoter. Electrophoretic mobility shift studies show that p65 and NF-IL6 can bind cooperatively to the TNF response region. The ability of this region to respond to TNF-alpha or p65 is correlated with the ability to form the p65/NF-IL6 ternary complex.

Animals↗

PubMed1994

Two contiguous residues in human interleukin-3, Asp21 and Glu22, selectively interact with the alpha- and beta-chains of its receptor and participate in function.

We have previously reported that the predicted first helix of human interleukin (IL)-3 contains a hydrophilic region encompassing residues Asp21, Glu22, and Thr25 that is crucial for biological activity and IL-3 receptor binding. Using single amino acid substitution mutagenesis, we have now determined that Asp21 and Glu22, but not Thr25, were crucial for full IL-3 activity. Mutant D21R was 30-fold less potent than wild type IL-3 in the stimulation of biological activity. It also exhibited a similar reduction in its ability to bind to the cloned high affinity IL-3 receptor complex (alpha- and beta-chains) or to the receptor alpha-chain alone, indicating that residue 21 is involved in contacts with the alpha-chain. Mutant E22R was approximately 20,000-fold less potent than wild type IL-3 in the stimulation of biological activity and in binding to the IL-3 receptor high affinity complex. However, the binding of E22R to the IL-3 receptor alpha-chain alone was similar to that of wild type IL-3, suggesting that this mutant was defective in interactions with the receptor beta-chain. These results show that two contiguous residues in the N-terminal region of IL-3 mediate binding to the two different chains of the IL-3 receptor and emphasize the functional significance of the conserved Glu in the first helix of the IL-3, granulocyte-macrophage colony-stimulating factor, and IL-5 cytokine subfamily.

Amino Acid Sequence↗

PubMed1994

T-cell functional regions of the human IL-3 proximal promoter.

The human interleukin-3 (IL-3) gene is expressed almost exclusively in activated T cells. Its expression is regulated at both the transcriptional and post-transcriptional level. We have previously shown that treatment of Jurkat T cells with phytohemaglutinin (PHA) and the phorbol ester, PMA, activated transcription initiation from the IL-3 gene. To define the regions of the gene required for transcription activation, we generated a series of reporter constructs containing different regions of the IL-3 gene 5' and 3' flanking sequences. Both positive and negative regulatory elements were identified in the proximal 5' flanking region of the IL-3 gene. The promoter region between -173 and -60 contained the strongest activating elements. The transcription factor AP-1 could bind to this positive activator region of the promoter. We also examined the function of the IL-3 CK-1/CK-2 elements that are present in many cytokine genes and found that they acted as a repressor of basal level expression when cloned upstream of a heterologous promoter but were also inducible by PMA/PHA.

Base Sequence↗

PubMed1993

The granulocyte-macrophage colony-stimulating factor/interleukin 3 locus is regulated by an inducible cyclosporin A-sensitive enhancer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) are pleiotropic hemopoietic growth factors whose genes are closely linked and induced in T lymphocytes in a cyclosporin A (CsA)-sensitive fashion. Since we found that the human GM-CSF and IL-3 proximal promoters were not sufficient to account for the observed regulation of these genes, we mapped DNase I hypersensitive sites across the GM-CSF/IL-3 locus in the Jurkat human T-cell line to identify additional regulatory elements. We located an inducible DNase I hypersensitive site, 3 kb upstream of the GM-CSF gene, that functioned as a strong CsA-sensitive enhancer of both the GM-CSF and IL-3 promoters. Binding studies employing Jurkat cell nuclear extracts indicated that four sites within the enhancer associate with the inducible transcription factor AP1. Three of these AP1 elements lie within sequences that also associate with factors resembling the CsA-sensitive, T cell-specific transcription factor NFAT. We provide additional evidence suggesting that an AP1-like factor represents one of the components of NFAT. We propose that the intergenic enhancer described here is required for the correctly regulated activation of both GM-CSF and IL-3 gene expression in T cells and that it mediates the CsA sensitivity of the GM-CSF/IL-3 locus.

Base Sequence↗

PubMed1993

HTLV-1 tax activation of the GM-CSF and G-CSF promoters requires the interaction of NF-kB with other transcription factor families.

The trans-activator protein, tax, from the human T leukemia virus type 1 (HTLV-1) trans-activates both viral and cellular genes. It has previously been shown that granulocyte macrophage-colony stimulating factor (GM-CSF) is constitutively expressed in HTLV-1 infected cells and in cells artificially expressing tax. We show here that the GM-CSF promoter is tax responsive in fibroblasts and T cells, whereas the granulocyte (G)-CSF promoter is tax responsive only in fibroblasts. The tax protein can activate cellular genes through a least two families of transcription factors; the NF-kB/rel and CREB/ATF families. We have used mutant tax proteins to show that the activation of NF-kB proteins is essential for tax trans-activation of both the GM-CSF and G-CSF promoters. The ability of tax to activate CREB/ATF proteins is also essential for GM-CSF transactivation. We have identified a 44 bp region of the GM-CSF promoter that contains tax responsive elements. This region contains a classical NF-kB site, a CK-1 element that can bind the NF-kB p65 protein, as well as a putative ATF binding site. The tax response of the G-CSF promoter requires not only the conserved CK-1 sequence but also an adjacent NF-IL6 binding site that may explain the cell restricted function of the G-CSF promoter.

Base Sequence↗

PubMed1992

A human interleukin 3 analog with increased biological and binding activities.

Human interleukin 3 (IL-3) variants generated by site-directed mutagenesis were analyzed in multiple biological and binding assays to identify residues critical for IL-3 activity. Two mutants carrying substitutions in the predicted hydrophilic region within the first alpha-helix, [Ala21,Leu22]IL-3 and [Ala21,Leu22,Ala25]IL-3 showed loss of biological activity and high-affinity binding. Mutants in a second predicted hydrophilic region, [Ala44,Leu45,Ala46]IL-3 and [Ala44,Ala46]IL-3, however, showed similar biological and binding activities to wild-type IL-3. Mutations in a C-terminal hydrophilic region that overlaps the fourth predicted alpha-helix led to either loss or gain of function. IL-3 analogs [Glu104,Asp105]-, [Leu108]-, [Asn108]-, [Thr108]-, and [Ala101,Leu108]IL-3 were less active than wild-type IL-3, whereas [Ala101]IL-3 and [Val116]IL-3 were 2- to 3-fold more potent. Significantly, the double mutant [Ala101,Val116]IL-3 exhibited a 15-fold greater potency than native IL-3. Receptor binding studies showed that [Ala101,Val116]IL-3 exhibited increased binding to the high- and low-affinity receptors of monocytes. These results show the generation of an IL-3 analog with increased biological and binding activities and support a model where the C terminus of IL-3 interacts with the alpha chain of the IL-3 receptor, making this region a useful focus for the development of more potent IL-3 agonists or antagonists.

Amino Acid Sequence↗

PubMed1992

Residue 21 of human granulocyte-macrophage colony-stimulating factor is critical for biological activity and for high but not low affinity binding.

The functional role of the predicted first alpha-helix of human granulocyte-macrophage colony-stimulating factor (GM-CSF) was analysed by site-directed mutagenesis and multiple biological and receptor binding assays. Initial deletion mutagenesis pointed to residues 20 and 21 being critical. Substitution mutagenesis showed that by altering Gln20 to Ala full GM-CSF activity was retained but that by altering Glu21 for Ala GM-CSF activity and high affinity receptor binding were decreased. Substitution of different amino acids for Glu21 showed that there was a hierarchy in the ability to stimulate the various biological activities of GM-CSF with the order of potency being Asp21 greater than Ser21 greater than Ala21 greater than Gln21 greater than Lys21 = Arg21. To distinguish whether position 21 was important for GM-CSF binding to high or low affinity receptors, GM-CSF (Arg21) was used as a competitor for [125I]GM-CSF binding to monocytes that express both types of receptor. GM-CSF (Arg21) exhibited a greatly reduced capacity to compete for binding to high affinity receptors, however, it competed fully for [125I]GM-CSF binding to low affinity receptors. Furthermore, GM-CSF (Arg21) was equipotent with wild-type GM-CSF in binding to the cloned low affinity alpha-chain of the GM-CSF receptor. These results show that (i) this position is critical for high affinity but not for low affinity GM-CSF receptor binding thus defining two functional parts of the GM-CSF molecule; (ii) position 21 of GM-CSF is critical for multiple functions of GM-CSF; and (iii) stimulation of proliferation and mature cell function by GM-CSF are mediated through high affinity receptors.

Amino Acids↗