Localization of the gene encoding the human interleukin-2 receptor on chromosome 10

The human interleukin-2 receptor is an inducible growth factor receptor present on the surface of activated T lymphocytes. The receptor is required for a normal T-cell immune response. High-resolution fluorescence-activated chromosome sorting and DNA spot-blot analysis with complementary DNA's for the interleukin-2 receptor indicated that the receptor gene was located on chromosome 9, 10, 11, or 12. In situ hybridization studies showed that the interleukin-2 receptor gene is on the short arm of chromosome 10, p14----15.     

Structure and functional expression of a human interleukin-8 receptor

Interleukin-8 (IL-8) is a member of a family of pro-inflammatory cytokines. Although the best characterized activities of IL-8 include the chemoattraction and activation of neutrophils, other members of this family have a wide range of specific actions including the chemotaxis and activation of monocytes, the selective chemotaxis of memory T cells, the inhibition of hematopoietic stem cell proliferation, and the induction of neutrophil infiltration in vivo. A complementary DNA encoding the IL-8 receptor from human neutrophils has now been isolated. The amino acid sequence shows that the receptor is a member of the superfamily of receptors that couple to guanine nucleotide binding proteins (G proteins). The sequence is 29% identical to that of receptors for the other neutrophil chemoattractants, fMet-Leu-Phe and C5a. Mammalian cells transfected with the IL-8 receptor cDNA clone bind IL-8 with high affinity and respond specifically to IL-8 by transiently mobilizing calcium. The IL-8 receptor may be part of a subfamily of related G protein-coupled receptors that transduce signals for the IL-8 family of pro-inflammatory cytokines.     

Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues

The gene encoding human interleukin-2 (IL-2) has been cloned from human spleen cells, peripheral blood lymphocytes, and the Jurkat cell line. Nucleotide sequence analysis of the gene revealed that the encoded IL-2 protein has three cysteines located at amino acid residues 58, 105, and 125 of the mature protein. Site-specific mutagenesis procedures were used to modify the IL-2 gene by changing each of the cysteine codons individually to serine codons. Substitution of serine for cysteine residues at either position 58 or 105 of the IL-2 protein substantially reduced biological activity, indicating that the cysteines at these positions are necessary for maintenance of the biologically active conformation and may therefore be linked by a disulfide bridge. The modified IL-2 protein containing a substitution at position 125 retained full biological activity, suggesting that the cysteine at this position is not involved in a disulfide bond and that a free sulfhydryl group at that position is not necessary for receptor binding.     

Cloning and expression of the human interleukin-6 (BSF-2/IFN beta 2) receptor

Interleukin-6 (IL-6/BSF-2/IFN beta 2) is a multifunctional cytokine that regulates the growth and differentiation of various tissues, and is known particularly for its role in the immune response and acute phase reactions. A complementary DNA encoding the human IL-6 receptor (IL-6-R) has now been isolated. The IL-6-R consists of 468 amino acids, including a signal peptide of approximately 19 amino acids and a domain of approximately 90 amino acids that is similar to a domain in the immunoglobulin (Ig) superfamily. The cytoplasmic domain of approximately 82 amino acids lacks a tyrosine/kinase domain, unlike other growth factor receptors.     

Structure of an agonist-bound human A2A adenosine receptor

Activation of G protein-coupled receptors upon agonist binding is a critical step in the signaling cascade for this family of cell surface proteins. We report the crystal structure of the A(2A) adenosine receptor (A(2A)AR) bound to an agonist UK-432097 at 2.7 angstrom resolution. Relative to inactive, antagonist-bound A(2A)AR, the agonist-bound structure displays an outward tilt and rotation of the cytoplasmic half of helix VI, a movement of helix V, and an axial shift of helix III, resembling the changes associated with the active-state opsin structure. Additionally, a seesaw movement of helix VII and a shift of extracellular loop 3 are likely specific to A(2A)AR and its ligand. The results define the molecule UK-432097 as a "conformationally selective agonist" capable of receptor stabilization in a specific active-state configuration.     

Cloning of an interleukin-3 receptor gene: a member of a distinct receptor gene family

Interleukin-3 (IL-3) binds to its receptor with high and low affinities, induces tyrosine phosphorylation, and promotes the proliferation and differentiation of hematopoietic cells. A binding component of the IL-3 receptor was cloned. Fibroblasts transfected with the complementary DNA bound IL-3 with a low affinity [dissociation constant (Kd) of 17.9 +/- 3.6 nM]. No consensus sequence for a tyrosine kinase was present in the cytoplasmic domain. Thus, additional components are required for a functional high affinity IL-3 receptor. A sequence comparison of the IL-3 receptor with other cytokine receptors (erythropoietin, IL-4, IL-6, and the beta chain IL-2 receptor) revealed a common motif of a distinct receptor gene family.     

Regulation of expression of the interleukin-2 receptor on hematopoietic cells by interleukin-3

Remarkable similarities in the intracellular and genetic events occur when lymphoid and hematopoietic cells are exposed to their specific growth factors. The interleukin-2 (IL-2) receptor, whose cell-surface expression is an absolute requirement for the growth and differentiation of lymphoid cells, was detected on various nonlymphoid hematopoietic cell types in this study. Cell lines consisting either of granulocyte-macrophage precursors or mast cells, which are dependent on interleukin-3 (IL-3) for their growth, expressed high levels of the IL-2 receptor on their surface. Analysis of the binding characteristics of these receptors with 125I-labeled recombinant IL-2 revealed that only receptors with low affinity for IL-2 were present on these cells. Addition of purified recombinant IL-3 to these cell lines led to an increase in IL-2 receptor gene expression within 1 hour in isolated nuclei. This IL-3--induced increase in the number of IL-2 receptors on the cell surface is maximal within 24 hours. Addition of 10,000 units of IL-2 to these cells had no apparent effect on their growth or differentiation. The presence of the receptor with only low affinity for IL-2 on hematopoietic cells and the regulation by IL-3 suggest that this receptor is involved in some important metabolic event in hematopoiesis.     

Cloning of complementary DNA encoding a functional human interleukin-8 receptor

Interleukin-8 (IL-8) is an inflammatory cytokine that activates neutrophil chemotaxis, degranulation, and the respiratory burst. Neutrophils express receptors for IL-8 that are coupled to guanine nucleotide-binding proteins (G proteins); binding of IL-8 to its receptor induces the mobilization of intracellular calcium stores. A cDNA clone from HL-60 neutrophils, designated p2, has now been isolated that encodes a human IL-8 receptor. When p2 is expressed in oocytes from Xenopus laevis, the oocytes bind 125I-labeled IL-8 specifically and respond to IL-8 by mobilizing calcium stores with an EC50 of 20 nM. This IL-8 receptor has 77% amino acid identity with a second human neutrophil receptor isotype that binds IL-8 with higher affinity. It also exhibits 69% amino acid identity with a protein reported to be an N-formyl peptide receptor from rabbit neutrophils, but less than 30% identity with all other known G protein-coupled receptors, including the human N-formyl peptide receptor.     

鸡白细胞介素-2基因在Pichia methanolica酵母中的表达

鸡白细胞介素-2(chIL-2)是近年来新发现的一种禽类细胞因子.为建立用甲醇型酵母Pichia methanolica (P. methanolica)表达鸡白细胞介素-2基因(chIL-2)的新途径,将chIL-2基因插入到P. methanolica分泌型表达载体pMETαA构建了重组PMAD11表达菌株(pMETαA/rchIL-2).经SDS-PAGE、Western印迹测定,证实chIL-2基因在P. methanolica中表达成功,rchIL-2的分子量约为16 kDa,其表达量占分泌总蛋白的35%.     

The structure of interleukin-2 complexed with its alpha receptor

Interleukin-2 (IL-2) is an immunoregulatory cytokine that binds sequentially to the alpha (IL-2Ralpha), beta (IL-2Rbeta), and common gamma chain (gammac) receptor subunits. Here we present the 2.8 angstrom crystal structure of a complex between human IL-2 and IL-2Ralpha, which interact in a docking mode distinct from that of other cytokine receptor complexes. IL-2Ralpha is composed of strand-swapped "sushi-like" domains, unlike the classical cytokine receptor fold. As a result of this domain swap, IL-2Ralpha uses a composite surface to dock into a groove on IL-2 that also serves as a binding site for antagonist drugs. With this complex, we now have representative structures for each class of hematopoietic cytokine receptor-docking modules.     

猪白细胞介素-2基因的克隆与表达

根据GenBank上发表的猪白细胞介素-2（Interleukin．2,IL-2）基因序列,设计1对引物。采用RT-PCR技术,以ConA刺激的猪外周血淋巴细胞为材料,从总RNA中扩增出484bp的特异性片段,将其克隆入pGEM-Teasy载体。序列测定表明;扩增片段为猪IL-2基因,该基因与GenBank上发表的猪IL-2基因的核苷酸序列同源性达99．8％。将其定向克隆至原核表达载体pET32a,构建了表达重组猪IL-2的基因工程菌株,经IPTG诱导表达和层析纯化,获得了纯化的重组猪IL-2蛋白。     

Cloning, sequencing, and expression of the gene coding for the human platelet alpha 2-adrenergic receptor

The gene for the human platelet alpha 2-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of alpha 2-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human beta 2- and beta 1-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional alpha 2-adrenergic receptor subtypes.     

Chromosomal location of human T-cell receptor gene Ti beta

A complementary DNA probe corresponding to the beta-chain gene of Ti, the human T lymphocyte receptor, has been molecularly cloned. The chromosomal origin of the Ti beta gene was determined with the complementary DNA by screening a series of 12 cell hybrid (mouse X human) DNA's containing overlapping subsets of human chromosomes. DNA hybridization (Southern) experiments showed that the human Ti beta gene resides on chromosome 7 and is thus not linked to the immunoglobulin loci or to the major histocompatibility locus in humans.     

Structure and diversity of the human T-cell receptor beta-chain variable region genes

In order to characterize the variability of the expressed human T-cell receptor (TCR) beta-chain repertoire and contrast this variability to the known murine beta-chain repertoire, 15 independent complementary DNA (cDNA) clones containing TCR beta-chain variable region (V beta) genes were isolated from a human tonsil cDNA library. The nucleotide and derived amino acid sequences of these 15 V beta genes were analyzed together with 7 previously defined sequences. Fifteen different human V beta genes could be identified from 22 independent sequences. By means of DNA hybridization and sequence homology comparisons, it was possible to group these 15 genes into ten distinct V beta subfamilies, each containing from one to seven members. Minimal polymorphism was noted between individuals, except in multimember subfamilies. The amino acid sequences of these genes contain conserved amino acids that are also shared by murine TCR V beta genes and immunoglobulins; no features were found that distinguish human V beta genes from their murine counterparts. Evaluation of secondary structure showed that maximum variability coincides with generally hydrophilic portions of the amino acid sequence, while specific hydrophobic regions were conserved in all V beta genes examined.     

Interleukin-2 receptor beta chain gene: generation of three receptor forms by cloned human alpha and beta chain cDNA's

Interleukin-2 (IL-2) binds to two distinct receptor molecules, the IL-2 receptor alpha (IL-2R alpha, p55) chain and the newly identified IL-2 receptor beta (IL-2R beta, p70-75) chain. The cDNA encoding the human IL-2R beta chain has now been isolated. The overall primary structure of the IL-2R beta chain shows no apparent homology to other known receptors. Unlike the IL-2R alpha chain, the IL-2R beta chain has a large cytoplasmic region in which a functional domain (or domains) mediating an intracellular signal transduction pathway (or pathways) may be embodied. The cDNA-encoded beta chain binds and internalizes IL-2 when expressed on T lymphoid cells but not fibroblast cells. Furthermore, the cDNA gives rise to the generation of high-affinity IL-2 receptor when co-expressed with the IL-2R alpha chain cDNA.     

Novel interleukin-2 receptor subunit detected by cross-linking under high-affinity conditions

Interleukin-2 (IL-2) binds to both high- and low-affinity classes of IL-2 receptors on activated T lymphocytes. Only the high-affinity receptors are involved in receptor-mediated endocytosis and normally transduce the mitogenic signals of IL-2; however, the structural features distinguishing the high- and low-affinity receptors are unknown. When 125I-labeled IL-2 was chemically cross-linked to activated human T lymphocytes, two major bands were identified. First, as predicted, a 68- to 72-kilodalton band, consisting of IL-2 (15.5 kilodaltons) cross-linked to the IL-2 receptor (55 kilodaltons), was observed. Second, an unpredicted 85- to 92-kilodalton moiety was detected. This band was not present when IL-2 was cross-linked to transfected C127 cells, which exclusively express low-affinity receptors. The data presented are most consistent with the existence of a 70- to 77-kilodalton glycoprotein subunit (p70) which, upon associating with the 55-kilodalton low-affinity receptor (p55), transforms it into a high-affinity site. It is proposed that p55 and p70 be referred to as the alpha and beta subunits, respectively, of the high-affinity IL-2 receptor.     

Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist

Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.