Structural and functional analyses of glycosylation on the distinct molecules of human GM-CSF receptors.

We have previously demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors are composed of at least two molecules of 80 and 135 kDa, which were denoted alpha- and beta-chains, respectively [Chiba, S., Shibuya, K., Piao, Y.-F., Tojo, A., Sasaki, N., Matsuki, S., Miyagawa, K., Miyazono, K. & Takaku, F. (1990) Cell Regul. 1, 327-335]. In this paper, we describe an investigation of the biochemical disparity noted between the alpha- and beta-chains of GM-CSF receptors using proteolytic and deglycosidic enzymes, and further demonstrate the potential importance of carbohydrate structures of the GM-CSF receptors using different lectins and glycoprotein synthesis inhibitors. Cross-linked alpha- and beta-chains with 125I-GM-CSF were digested by Staphylococcus aureus V8 protease and gave a different pattern. Furthermore, the size of the alpha-chain was reduced by 25 kDa by the removal of the N-linked oligosaccharides with peptidase: N-glycosidase F treatment, whereas that of the beta-chain remained unmodified by the enzyme. These results suggest that the alpha-chain of GM-CSF receptors agrees with the recently cloned low-affinity GM-CSF receptor [Gearing, D.P., King, J.A., Gough, N. M. & Nicola, N.A. (1989) EMBO J. 8, 3667-3676] having approximately 30% N-linked oligosaccharides and is biochemically different from the alpha beta-chain. By analyses using lectins, some of the oligosaccharides in the alpha-chain seem to be the complex-type and/or hybrid-type, because wheat germ agglutinin and leukoagglutinating phytohemagglutinin inhibited both GM-CSF-induced proliferation and GM-CSF binding to its receptors. Further analyses using glycoprotein synthesis inhibitors showed that N-linked processing of the alpha-chain, especially glucose removal by glucosidase I and II (whose activities are inhibited by deoxynojirimycin), appeared to be required for the expression onto the cell surface although the beta-chain expression was little affected by their inhibitors. Thus the beta-chain, probably located near the alpha-chain on the cell surface, was associated with a high-affinity class of GM-CSF receptors.     

Kinetic analysis of ligand binding to interleukin-2 receptor complexes created on an optical biosensor surface.

The interleukin-2 receptor (IL-2R) is composed of at least three cell surface subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma c. On activated T-cells, the alpha- and beta-subunits exist as a preformed heterodimer that simultaneously captures the IL-2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL-2 to biosensor surfaces containing either the alpha-subunit, the beta-subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent-exposed thiols. Equilibrium analysis of the binding data established IL-2 dissociation constants for the individual alpha- and beta-subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the alpha- and beta-subunits, similar to that reported for the pseudo-high-affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher-affinity binding sites detected on surfaces containing both alpha- and beta-subunits resulted from capture of IL-2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure-function relationships of this and other multiple subunit receptor systems.     

Mutational study on the roles of disulfide bonds in the beta-subunit of gastric H+,K+-ATPase

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the non-catalytic beta-subunit. Correct assembly between the alpha- and beta-subunits is essential for the functional expression of H(+),K(+)-ATPase. The beta-subunit contains nine conserved cysteine residues; two are in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain. The six cysteine residues in the ectodomain form three disulfide bonds. In this study, we replaced each of the cysteine residues of the beta-subunit with serine individually and in several combinations. The mutant beta-subunits were co-expressed with the alpha-subunit in human embryonic kidney 293 cells, and the role of each cysteine residue or disulfide bond in the alpha/beta assembly, stability, and cell surface delivery of the alpha- and beta-subunits and H(+),K(+)-ATPase activity was studied. Mutant beta-subunits with a replacement of the cytoplasmic and transmembrane cysteines preserved H(+),K(+)-ATPase activity. All the mutant beta-subunits with replacement(s) of the extracellular cysteines did not assemble with the alpha-subunit, resulting in loss of H(+),K(+)-ATPase activity. These mutants did not permit delivery of the alpha-subunit to the cell surface. Therefore, each of these disulfide bonds of the beta-subunit is essential for assembly with the alpha-subunit and expression of H(+),K(+)-ATPase activity as well as for cell surface delivery of the alpha-subunit.     

Cooperativity of alpha- and beta-subunits of group II chaperonin from the hyperthermophilic archaeum Aeropyrum pernix K1

alpha- and beta-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of alpha- and beta-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant alpha- and beta- subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified alpha- and beta-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both alpha- and beta-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at 43 degreesC and 50 degreesC, respectively, and MDH from thermal inactivation at 80 degreesC and 85 degreesC. Moreover, in the presence of ATP, the protective effects of alpha- and beta-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at 85 degreesC) from thermal inactivation was not observed. Specifically, the presence of both alpha- and beta- subunits could effectively protect MDH from thermal inactivation at 80 degreesC in an ATP-dependent manner.     

Comparative quantum-chemical analysis of the electronic structure and M?ssbauer parameters of the active site models for deoxymyoglobin and alpha- and beta-subunits of tetrameric deoxyhemoglobin

The results of iterative extended Huckel calculations of the electronic structure of the penta-coordinated Fe(II)-porphyn-imidazole complexes as models for deoxymyoglobin and alpha- and beta-subunits of tetrameric deoxyhemoglobin are presented. Temperature dependences of the Fe-57 nuclei quadrupole splitting and isomer shift for deoxymyoglobin and alpha- and beta-subunits of tetrameric deoxyhemoglobin models were calculated taking into account the spin-orbit coupling between ground and low-lying Fe(II) high spin terms. The results show that the electronic structure and M?ssbauer parameters are sensitive to the stereochemical differences of the active sites in deoxymyoglobin and alpha- and beta-subunits of tetrameric deoxyhemoglobin.     

Insulin and IGF-1 receptors contain covalently bound palmitic acid

We have studied the biosynthesis of the insulin receptor in a human hepatoma cell line, HepG2. As previously reported, these cells synthesize a disulphide-bonded alpha 2 beta 2 tetrameric insulin receptor. Labelling of HepG2 cells with [3H]palmitate or [3H]myristate followed by immunoprecipitation with a polyclonal antireceptor antibody revealed the incorporation of palmitate, but not myristate, into the beta-subunit and alpha beta-precursor of the receptor in a hydroxylamine-sensitive linkage. The extracellular alpha-subunit was not labelled, demonstrating the specificity of incorporation. Acylation of the insulin receptor was an early event as judged by fatty acid incorporation into the alpha beta-precursor and prevention by protein synthesis inhibitors. Pulse-chase studies demonstrated the expected processing of the alpha beta-precursor to mature alpha- and beta-subunits, but no evidence for preferential turnover of the fatty acid moiety was found. The site of acylation appears to be in the transmembrane or cytoplasmic domain since proteolytic treatment of intact cells produced a truncated beta-subunit still containing label. Binding studies showed that HepG2 cells contain approximately half as many insulin-like growth factor-1 receptors as insulin receptors, raising the possibility that this receptor may also be acylated. Indeed, immunoprecipitation with the antiinsulin receptor serum of MDCK cells expressing IGF-1 receptors, but not insulin receptors, revealed bands corresponding to the alpha beta-precursor, alpha- and beta-subunits, of which the alpha beta-precursor and beta-subunits incorporated [3H]palmitate but the alpha-subunit did not.     

Isolation of male germ-line stem cells; influence of GDNF

The identification and physical isolation of testis stem cells, a subset of type A spermatogonia, is critical to our understanding of their growth regulation during the first steps of spermatogenesis. These stem cells remain poorly characterized because of the paucity of specific molecular markers that permit us to distinguish them from other germ cells. Thus, the molecular mechanisms driving the first steps of spermatogenesis are still unknown. We show in the present study that GFR alpha-1, the receptor for GDNF (glial cell line-derived neurotrophic factor), is strongly expressed by a subset of type A spermatogonia in the basal part of the seminiferous epithelium. Using this characteristic, we devised a method to specifically isolate these GFR alpha-1-positive cells from immature mouse testes. The isolated cells express Ret, a tyrosine kinase transmembrane receptor that mediates the intracellular response to GDNF via GFR alpha-1. After stimulation with rGDNF, the isolated cells proliferated in culture and underwent the first steps of germ cell differentiation. Microarray analysis revealed that GDNF induces the differential expression of a total of 1124 genes. Among the genes upregulated by GDNF were many genes involved in early mammalian development, differentiation, and the cell cycle. This report describes the first isolation of a pure population of GFR alpha-1-positive cells in the testis and identifies signaling pathways that may play a crucial role in maintaining germ-line stem cell proliferation and/or renewal.     

Assembly of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase with its pre-existing beta-subunit in Xenopus oocytes

The alpha- and beta-subunits of Torpedo californica Na+/K(+)-ATPase were expressed in turn in single oocytes by alternately microinjecting the specific mRNAs for the alpha- and beta-subunits. The mRNA first injected was degraded prior to the injection of the second mRNA by injecting the antisense oligonucleotide specific for the first mRNA. The pre-existing beta-subunit, which had been synthesized by injecting mRNA for the beta-subunit, could assemble with the alpha-subunit expressed later in the single oocytes and the resulting alpha beta complex acquired both ouabain-binding and Na+/K(+)-ATPase activities. On the other hand, formation of the alpha beta complex was not detected when the alpha-subunit was expressed first, followed by the beta-subunit. These data suggest that the beta-subunit acts as a receptor or a stabilizer for the alpha-subunit upon the biogenesis of Na+/K(+)-ATPase.     

Beta 1- and beta 3-subunits can associate with presynthesized alpha-subunits of Xenopus oocyte Na,K-ATPase.

Oligomerization of newly synthesized alpha- and beta-subunits is a prerequisite for the structural and functional maturation of Na,K-ATPase. In this study, we have tested the competence of presynthesized alpha- and beta-subunits to assemble into functional enzyme complexes. Antisense oligonucleotides complementary to alpha-mRNA were used to inhibit alpha-subunit synthesis in Xenopus oocytes leaving a presynthesized trypsin-sensitive alpha-subunit pool. beta-Subunits expressed in these oocytes from injected cRNA assembled with the preexisting alpha-subunits, rendered them trypsin-resistant, and permitted the expression of more ouabain binding sites at the plasma membrane. Similarly, presynthesized beta 1- or beta 3-subunits produced in Xenopus oocytes by injection of beta-cRNA and later of specific antisense oligonucleotides were stabilized and transported out of the endoplasmic reticulum when alpha-cRNA was injected into oocytes. These data indicate that alpha- and beta-subunits can insert into endoplasmic reticulum membranes independent of each other in an assembly-competent form and retain their ability for oligomerization after synthesis.     

IL-3 and GM-CSF induce the expression of the inositol trisphosphate receptor in K562 myeloblast cells.

When treated with IL-3 plus GM-CSF, K562 myeloblast cells acquired the ability to mobilize nonmitochondrial stores of intracellular Ca2+ in response to added Ins (1, 4, 5) P3. Untreated K562 cells are capable of sequestering intracellular Ca2+ but released none of this Ca2+ in response to Ins (1, 4, 5) P3. Untreated K562 cells were shown to have no detectable specific [3H] Ins (1, 4, 5) P3 binding sites and no InsP3 receptor mRNA as assayed by Northern blot and PCR. However, following IL-3 and GM-CSF treatment, both a single class of low nM KD Ins (1, 4, 5) P3 binding site and a 10 kb InsP3 receptor mRNA were detectable. The results suggest that IL-3 and GM-CSF regulate the expression of the Ins (1, 4, 5) P3 receptor gene.     

Identification of ERβ1 and ERβ2 in human seminoma, in embryonal carcinoma and in their adjacent intratubular germ cell neoplasia

Background  

Estrogens exert a role on germ cell physiology of normal human testis through the mediation of the estrogen receptor (ER) beta subtypes. Epidemiological studies evidenced an increased incidence of testicular germ cell cancer after elevated pre-natal estrogen exposure but the expression of estrogen receptors in these testicular neoplasms has not been well elucidated.     

Tryptic activation of the insulin receptor. Proteolytic truncation of the alpha-subunit releases the beta-subunit from inhibitory control

Trypsin exerts insulin-like effects in intact cells and on partially purified preparations of insulin receptors. To elucidate the mechanism of these insulinomimetic effects, we compared the structures of insulin- and trypsin-activated receptor species with their functions, including insulin binding, autophosphorylation, and tyrosine kinase activity. In vitro treatment of wheat germ agglutinin-purified receptor preparations with trypsin resulted in proteolysis of both alpha- and beta-subunits. The activated form of the receptor had an apparent molecular mass of 110 kDa under nonreducing conditions, compared to the 400-kDa intact receptor, and was separated following reduction into an 85-kDa beta-subunit related fragment and a 25-kDa alpha-subunit related fragment. Treatment of whole cells with trypsin prior to isolation of the insulin receptor resulted in proteolytic modification of the alpha-subunit only. In this case, the total molecular mass of the activated species was 116 kDa, comprised of an intact 92-kDa beta-subunit and again a 25-kDa alpha-subunit related fragment. Values of Km for peptide substrate phosphorylation and Ki for inhibition of receptor autophosphorylation, and sites of autophosphorylation within the beta-subunits were similar for receptors activated either by insulin or trypsin. Insulin had no additional effect on the rate of autophosphorylation of the truncated receptor, and no binding of insulin by the truncated receptor was detected either by direct assay or cross-linking with bifunctional reagents. Based on the deduced amino acid sequence of the insulin receptor and the structural studies presented here we concluded that this activated form of the receptor resulted from tryptic cleavage at the dibasic site Arg576-Arg577. This was accompanied by loss of the insulin binding site and separation of alpha-beta heterodimers. As truncation of the alpha-subunit results in beta-subunit activation, it appears that the beta-subunit is a constitutively activated kinase and that the function of the alpha-subunit in the intact receptor is to inhibit the beta-subunit.     

Fractionation of protein components of plasma membranes from the electric organ of Torpedo marmorata

A procedure has been developed for the separation of intrinsic proteins of plasma membranes from the electric organ of Torpedo marmorata. (Na+ + K+)-ATPase, nicotinic acetylcholine receptor and acetylcholinesterase remained active after solubilization with the nonionic detergent dodecyl octaethylene glycol monoether (C12E8). These components could be separated by ion exchange chromatography on DEAE-Sephadex A-25. Fractions enriched in ouabain-sensitive K+-phosphatase or (Na+ + K+)-ATPase activity showed two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to the alpha- and beta-subunits. The (Na+ + K+)-ATPase was shown to have immunological determinants in common with a 93 kDa polypeptide which copurified with the nicotinic acetylcholine receptor, also after solubilization in Triton X-100 and chromatography on Naja naja siamensis alpha-toxin-Sepharose columns. The data suggest that the alpha-subunit of (Na+ + K+)-ATPase associates with the acetylcholine receptor in the membranes of the electric organ.     

The separation of subunits of phenylalanyl-tRNA-synthetase from Escherichia coli MRE-600 by means of affinity chromatography in dissociation conditions

The method of affinity chromatography on sepharose with immobilized tRNA in the presence of urea was developed for separating the subunits of phenylalanyl-tRNA synthetase from E. coli MRE-600 (subunit structure alpha 2 beta 2). Specific binding of large beta-subunits of the enzyme on immobilized tRNA testifies the localization of the tRNA-binding center on the beta-subunit of phenylalanyl-tRNA synthetase. Separately alpha- and beta-subunits of the enzyme exhibit no catalytic activity. Incubation of the mixture of alpha- and beta-subunits in conditions leading to reassociation of the oligomeric structure results in restoration of catalytic activity of the enzyme. In the presence of urea resin with immobilised analogs of ATP binds alpha- and beta-subunits of the enzyme. This testifies the presence of nucleotide-binding sites on both subunits. The possibility of using the affinity chromatography method to separate non-identical subunits of different enzymes is discussed.