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1.
Somatomedin-C stimulates the phosphorylation of the beta-subunit of its own receptor 总被引:56,自引:0,他引:56
S Jacobs F C Kull H S Earp M E Svoboda J J Van Wyk P Cuatrecasas 《The Journal of biological chemistry》1983,258(16):9581-9584
Phosphorylation of the somatomedin-C receptor was investigated both in intact IM-9 cells and in IM-9 cells that had been solubilized with Triton X-100. Intact IM-9 cells were incubated with [32P]H3PO4 for 1 h and for an additional 5 min in the absence or presence of insulin or somatomedin-C. The cells were then solubilized and subjected to wheat germ agglutinin Sepharose chromatography. The extent of phosphorylation of insulin and somatomedin-C receptors was assessed by immunoprecipitating the wheat germ agglutinin Sepharose eluates with monoclonal antibodies specific for each receptor and analyzing the immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The beta-subunits of both receptors were phosphorylated in the absence of hormone, and the extent of phosphorylation of each receptor was enhanced by both hormones. However, each hormone was more potent than the other in enhancing phosphorylation of its own receptor. The beta-subunit of the somatomedin-C receptor was also phosphorylated when solubilized IM-9 cells that had been purified on wheat germ agglutinin Sepharose were incubated with [gamma-32P]ATP. In this soluble preparation, phosphorylation occurred on tyrosyl residues and was enhanced by concentrations of somatomedin-C in the range of 2.5 to 250 ng/ml, which is consistent with its receptor affinity. Tyrosyl phosphorylation of the somatomedin-C receptor also occurred when highly purified receptor, prepared by wheat germ agglutinin Sepharose affinity chromatography followed by immunoprecipitation, was incubated with [gamma-32P]ATP. This indicates that the responsible tyrosyl kinase activity is intrinsic to the receptor or tightly associated with it. 相似文献
2.
Insulin-like growth factor binding to the atypical insulin receptors of a human lymphoid-derived cell line (IM-9). 总被引:1,自引:0,他引:1
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The cells of the IM-9 human lymphocyte-derived line contain a sub-population of insulin-binding sites whose immunological and hormone-binding characteristics closely resemble those of the atypical insulin-binding sites of human placenta. These binding sites, which have moderately high affinity for multiplication-stimulating activity [MSA, the rat homologue of insulin-like growth factor (IGF) II] and IGF-I, are identified on IM-9 cells by 125I-MSA binding. They account for approximately 30% of the total insulin-receptor population, and do not react with a monoclonal antibody to the type I IGF receptor (alpha IR-3). The relative concentrations of unlabelled insulin, MSA and IGF-I required to displace 50% of 125I-MSA from these binding sites (1:4.7:29 respectively) are maintained for cells, particulate membranes, Triton-solubilized membranes precipitated either by poly(ethylene glycol) or a polyclonal antibody (B-10) to the insulin receptor, and receptors purified by insulin affinity chromatography. Because the atypical insulin/MSA-binding sites outnumber the type I IGF receptors in IM-9 cells by approximately 10-fold, they also compete with the latter receptors for 125I-IGF-I binding. Thus 125I-IGF-I binding to IM-9 cells is inhibited by moderately low concentrations of insulin (relative potency ratios for insulin compared with IGF-I are approx. 1/14 to 1/4) and is partially displaced (65-80%) by alpha IR-3. When type I IGF receptors are blocked by alpha IR-3 or removed by B-10 immunoprecipitation or insulin affinity chromatography, the hormone-displacement patterns for 125I-IGF-I binding resemble those of the atypical insulin/MSA-binding sites. 相似文献
3.
J L Treadway B D Morrison I D Goldfine J E Pessin 《The Journal of biological chemistry》1989,264(36):21450-21453
Insulin and Mn/MgATP treatment of immunoaffinity-purified alpha beta heterodimeric insulin receptors induced the formation of an alpha 2 beta 2 heterotetrameric insulin receptor complex. In contrast, insulin-like growth factor-1 (IGF-1) treatment was completely ineffective in inducing the association of alpha beta heterodimeric insulin receptors. Similarly, IGF-1 or Mn/MgATP, but not insulin, treatment of immunoaffinity-purified alpha beta heterodimeric IGF-1 receptors induced the formation of an alpha 2 beta 2 heterotetrameric IGF-1 receptor complex. A monoclonal antibody specific for the insulin receptor (MA5) completely immunoprecipitated all the insulin binding activity from both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes but did not immunoprecipitate IGF-1 receptors. Conversely, the IGF-1 receptor-specific monoclonal antibody (alpha IR-3) immunoprecipitated all the IGF-1 binding activity, but not insulin receptors. The simultaneous treatment of pooled equal amounts of alpha beta heterodimeric insulin and IGF-1 receptors with a combination of insulin and IGF-1 resulted in the formation of alpha 2 beta 2 heterotetrameric insulin and IGF-1 receptor complexes. However, in the mixed alpha 2 beta 2 heterotetrameric receptor fraction MA5 immunoprecipitated 94% of the insulin binding in addition to 27% of the IGF-1 binding activity whereas alpha IR-3 immunoprecipitated 97% of the IGF-1 binding in addition to 38% of the insulin binding activity. Treatment of the mixed alpha beta heterodimeric insulin and IGF-1 receptors with Mn/MgATP also resulted in the formation of cross-immunoreactive (42-46%) alpha 2 beta 2 heterotetrameric receptors. These data directly demonstrate the formation of insulin/IGF-1 hybrid receptors by both a combination of insulin plus IGF-1 or Mn/MgATP treatment of purified human placenta alpha beta heterodimeric insulin and IGF-1 half-receptors in vitro. 相似文献
4.
Previous studies have suggested that protein kinase C (PKC) isoforms differentially influence the sensitivity of gamma-aminobutyric acid(A) (GABA(A) ) receptor responses in brain. Both PKCgamma and PKCepsilon knock-out mice exhibit altered ethanol potentiation of GABA(A) receptor mediated Cl(-) flux. Furthermore, chronic ethanol consumption alters GABA(A) receptor function and receptor subunit peptide expression by mechanisms that are not yet understood. The present study explored the possibility that PKC isoforms are directly associated with GABA(A) receptors, and this association is influenced by chronic ethanol exposure. GABA(A) receptors containing alpha1 or alpha4 subunits were immunoprecipitated from solubilized protein derived from the membrane fraction of rat cerebral cortex using selective antibodies. Immunoprecipitated receptors were screened by western blot analysis for the presence of PKCdelta, gamma and epsilon isoforms. We found pronounced labeling of PKCgamma but not PKCdelta or PKCepsilon in the alpha1 and alpha4 subunit immunoprecipitates. Immunoprecipitation with PKCgamma, but not with IgG antibody also yielded GABA(A) receptor alpha1 and alpha4 subunits in the immunoprecipitate. The association of PKCgamma with alpha1-containing receptors was decreased 44 +/- 11% after chronic ethanol consumption. In contrast, PKCgamma associated with alpha4-containing receptors was increased 32 +/- 7% after chronic ethanol consumption. These results suggest that PKCgamma may be involved in GABA(A) receptor adaptations following chronic ethanol consumption. 相似文献
5.
Somatostatin (SRIF) induces its biological effects by interacting with membrane-bound receptors that are linked to cellular effector systems via G proteins. We have studied SRIF receptor-G protein associations by solubilizing the SRIF receptor from rat brain and AtT-20 cells and immunoprecipitating the receptor-G protein complex with peptide-directed antisera against the different subunits of the G protein heterotrimer. Antiserum 8730, which selectively interacts with all Gi alpha subtypes, maximally and specifically immunoprecipitated SRIF receptor-Gi alpha complexes. To identify the subtypes of Gi alpha that are coupled to SRIF receptors, the subtype-selective antisera 3646, 1521, and 1518, which specifically interact with Gi alpha 1, Gi alpha 2, and Gi alpha 3, respectively, were used to immunoprecipitate SRIF receptor-Gi alpha complexes. Antiserum 3646 immunoprecipitated SRIF receptor-Gi alpha 1 complexes from both brain and AtT-20 cells. Antiserum 1521 immunoprecipitated Gi alpha 2 from both brain and AtT-20 cells but did not immunoprecipitate SRIF receptors from these tissues. Antiserum 1518 immunoprecipitated AtT-20 cell SRIF receptors but uncoupled brain SRIF receptor-G protein complexes. This result was confirmed with another peptide-selective antiserum, SQ, directed against Gi alpha 3. The findings from these studies indicate that Gi alpha 1 and Gi alpha 3 are coupled to SRIF receptors, whereas Gi alpha 2 is not. Even though brain and AtT-20 cell SRIF receptors were both coupled to Gi alpha, the receptors from these tissues differed in their coupling to Go alpha. Antiserum 2353, which is directed against Go alpha, immunoprecipitated SRIF receptors from AtT-20 cells, but did not immunoprecipitate or uncouple SRIF receptor-G protein complexes from rat brain. To determine the beta subunits associated with the SRIF receptor, antisera directed against G beta 36 and G beta 35 were used to immunoprecipitate SRIF receptor-G protein complexes from brain. Peptide-directed antiserum against G beta 36 selectively immunoprecipitated solubilized brain SRIF receptors. However, antiserum directed against the G beta 35 subunit did not immunoprecipitate brain SRIF receptors, suggesting that brain SRIF receptors may preferentially associate with G beta 36. In addition to coimmunoprecipitating with Gi alpha and G beta, brain SRIF receptors coimmunoprecipitated the G protein gamma subunits, G gamma 2 and G gamma 3. These results provide the first evidence that SRIF receptors are coupled to different subunits of G proteins and suggest that selectivity exists in the association of different G protein subunits with the SRIF receptor. 相似文献
6.
C F Burant M K Treutelaar N E Block M G Buse 《The Journal of biological chemistry》1986,261(31):14361-14364
The structure of insulin receptors, solubilized from rat skeletal muscle and liver, was studied. The alpha subunit was identified by specific cross-linking to A14 125I-insulin with disuccinimidyl suberate. Muscle- and liver-derived alpha subunits migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a Mr of 131,000 and 135,000, respectively. There was no significant difference in insulin binding affinity. Treatment of cross-linked, immunoprecipitated receptors with either neuraminidase or endoglycosidase H decreased the Mr of muscle- and liver-derived alpha subunits but did not affect the difference in Mr. Autophosphorylated beta subunits migrated with a Mr of 98,000 for muscle and 101,000 for liver. After partial V8 digestion of autophosphorylated, immunoprecipitated receptors the major phosphopeptide fragment migrated on SDS-PAGE at Mr 57,000 from muscle and 60,000 from liver. Glycosidase digestion of autophosphorylated receptors suggested that Mr heterogeneity was due in part to differences in the sialic acid content of beta subunits. Muscle and liver are the major target organs of insulin; the apparent heterogeneity of insulin receptor structure may be relevant to tissue-specific differences in insulin action. 相似文献
7.
Subtypes of insulin-growth factor I (IGF-I) receptors, including hybrid receptors containing insulin receptor alpha beta dimers associated with IGF-I receptor alpha beta dimers, have been described in a number of systems. The molecular basis of the multiple subtypes and their functional significance is not understood. Ligand-dependent phosphorylation of insulin and IGF-I receptors and immunoprecipitation with antipeptide and monoclonal antibodies have been used to characterize the subpopulations of these receptors in the human KB cell line. IGF-I receptors exhibit beta subunits of 95 and 102 kDa in these cells. IGF-I receptors containing 102-kDa beta subunits are immunoprecipitated by the IGF-I receptor-specific antibody alpha-IR3. Antibody alpha-IR3 does not appear to recognize a hybrid receptor in these cells. However, an antipeptide antibody against the carboxyl-terminal domain of the insulin receptor (AbP5) immunoprecipitates a population of receptors phosphorylated in response to IGF-I (1 nM) which contains both 95- and 102-kDa beta subunits. These receptors must be hybrid complexes because AbP5 does not recognize the 102-kDa beta subunit directly. The inability of antibody alpha-IR3 to recognize these complexes suggests that their IGF-I receptor alpha subunits must differ from typical IGF-I receptor alpha subunits either in primary sequence or conformation. Therefore, KB cells may contain more than one type of IGF-I receptor alpha subunit. Hybrid IGF-I receptors can also be distinguished from homotypic IGF-I receptors by their responsiveness to IGF-II. Stimulation of autophosphorylation in hybrid IGF-I receptors by IGF-I is 3-4-fold greater than that seen in response to IGF-II. In contrast, IGF-I and IGF-II are nearly equipotent in stimulating autophosphorylation in the alpha-IR3-reactive receptor population. This suggests the existence of functionally distinct receptor subtypes which may differ in their ability to mediate the biological effects of IGF-II. 相似文献
8.
Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor. 总被引:18,自引:7,他引:11
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M A Soos K Siddle M D Baron J M Heward J P Luzio J Bellatin E S Lennox 《The Biochemical journal》1986,235(1):199-208
Monoclonal antibodies for the human insulin receptor were produced following immunization of mice with IM-9 lymphocytes and/or purified placental receptor. Four separate fusions yielded 28 antibodies, all of which reacted with receptor from human placenta, liver and IM-9 cells. Some antibodies cross-reacted to varying degrees with receptor from rabbit, cow, pig and sheep, but none reacted with rat receptor. At least 10 distinct epitopes were recognized as indicated by species specificity and binding competition experiments. All of these epitopes appeared to be on extracellular domains of the receptor as shown by binding of antibodies to intact cells. In some cases the epitopes were further localized to alpha or beta subunits by immunoblotting. Several antibodies inhibited binding of 125I-insulin to the receptor, some had no effect on binding, and others enhanced the binding of 125I-insulin. It is concluded that these antibodies will be valuable probes of receptor structure and function. 相似文献
9.
Antibodies against the subunits of the dihydropyridine-sensitive L-type calcium channel of skeletal muscle were tested for their ability to immunoprecipitate the high affinity (Kd = 0.13 nM) 125I-omega-conotoxin GVIA receptor from rabbit brain membranes. Monoclonal antibody VD2(1) against the beta subunit of the dihydropyridine receptor from skeletal muscle specifically immunoprecipitated up to 86% of the 125I-omega-conotoxin receptor solubilized from brain membranes whereas specific antibodies against the alpha 1, alpha 2, and gamma subunits did not precipitate the brain receptor. Purified skeletal muscle dihydropyridine receptor inhibited the immunoprecipitation of the brain omega-conotoxin receptor by monoclonal antibody VD2(1). The dihydropyridine receptor from rabbit brain membranes was also precipitated by monoclonal antibody VD2(1). However, neither the neuronal ryanodine receptor nor the sodium channel was precipitated by monoclonal antibody VD2(1). The omega-conotoxin receptor immunoprecipitated by monoclonal antibody VD2(1) showed high affinity 125I-omega-conotoxin binding, which was inhibited by unlabeled omega-contoxin and by CaCl2 but not by nitrendipine or by diltiazem. An antibody against the beta subunit of the skeletal muscle dihydropyridine receptor stained 58- and 78-kDa proteins on immunoblot of the omega-conotoxin receptor, partially purified through heparin-agarose chromatography and VD2(1)-Sepharose chromatography. These results suggest that the brain omega-conotoxin-sensitive calcium channel contains a component homologous to the beta subunit of the dihydropyridine-sensitive calcium channel of skeletal muscle and brain. 相似文献
10.
A monoclonal antibody to human insulin receptor 总被引:1,自引:0,他引:1
Frederick C. Kull Steven Jacobs Ying-Fu Su Pedro Cuatrecasas 《Biochemical and biophysical research communications》1982,106(3):1019-1026
A murine hybridoma secreting antibody against human insulin receptor was produced by fusing FO myeloma cells with spleen and lymph node cells from a mouse that had been immunized with insulin receptor purified from human placenta. The secreted antibody was an IgG1 (κ), designated αIR-1. Like the previously described rabbit polyclonal antibody, αIR-1 did not inhibit insulin binding. It specifically immunoprecipitated 125I-insulin-receptor complexes as well as unoccupied receptor previously labeled directly with lactoperoxidase. Thus, αIR-1 interacts with the receptor at a site distinct from the insulin binding site. Unlike previously described anti-insulin receptor antibodies, αIR-1 exhibits strong tissue and species specificity. 相似文献
11.
Ludmerer SW Warren VA Williams BS Zheng Y Hunt DC Ayer MB Wallace MA Chaudhary AG Egan MA Meinke PT Dean DC Garcia ML Cully DF Smith MM 《Biochemistry》2002,41(20):6548-6560
35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits. 相似文献
12.
Cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors 总被引:2,自引:0,他引:2
Madin-Darby canine kidney (MDCK) cells were previously shown to have few or no plasma membrane insulin binding sites (Hofmann et al: J Biol Chem 258:11774, 1983]. Accordingly, neither insulin-stimulated incorporation of [14C]glucose into glycogen, nor insulin-induced uptake of radiolabeled alpha-aminoisobutyrate ([3H]AIB) could be demonstrated. To probe for receptors, MDCK cultures were surface-labeled with Na125I or were labeled with [35S]methionine. When solubilized cells were immunoprecipitated with sera containing antibodies to the insulin receptor, and immunoprecipitates were analyzed on SDS-gel electrophoresis, no evidence for insulin receptor components was found. Also, when intact MDCK cells wee incubated first with serum containing antibodies to the insulin receptor and then with 125I-protein A, no radiolabeling of insulin receptors occurred. Various agents reported to have insulin-like activity were tested on MDCK cells. The insulinomimetic lectins concanavalin A and wheat germ agglutinin as well as hydrogen peroxide enhanced incorporation of [14C]glucose into glycogen and induced stimulated [3H]AIB uptake, whereas trypsin, vanadate, and serum containing antibodies to the insulin receptor were without effects. Altogether, these results showed that MDCK cells had few or no insulin receptors and were correspondingly insulin-insensitive. However, since insulin-associated responses could be elicited by some insulin mimickers, the post-receptor limb of response in MDCK cells was apparently intact. 相似文献
13.
Identification of a monoclonal antibody which can distinguish between two distinct species of the type I receptor for insulin-like growth factor 总被引:1,自引:0,他引:1
A monoclonal antibody was identified which equally inhibits 125I-labeled insulin and insulin-like growth factor I (IGF-I) binding to their respective receptors in human IM-9 lymphoid cells and solubilized placenta receptor preparations. In contrast, this monoclonal antibody inhibits insulin but not IGF-I binding to human hepatoma (HepG2) cells, fibroblasts and muscle cells. These results indicate that there are two distinct species of the type I insulin-like growth factor receptor (which we have named type IA and type IB) and suggest that this monoclonal antibody may be useful in determining whether different biological effects are mediated through these two receptors. 相似文献
14.
《Journal of receptor and signal transduction research》2013,33(6-8):381-398
AbstractThe effect of three antibodies that interact with distinct regions of the insulin receptor (the a subunit (83-7), the juxtamembrane region near tyrosine 960 (960) or the carboxy terminal region of the I3 subunit (CT-1)) on insulin binding was examined. Detergent-solubilized insulin receptors from IM-9 cells immobilized on Sepharose beads by 960 antisera bound 2-3 times more IWinsulin tracer (25-60 pM) than receptors immobilized with either 83-7 or CT-1. &-incubation of solubilized receptors with either 83-7 or 960 resulted in equivalent depletion (90%) of insulin binding activity from solubilized IM-9 cell extracts, suggesting that both antibodies were in excess and capable of binding a similar population of receptors. Antibody 960, but not CT-1 or 83-7, also increased insulin binding 2 fold to solubilized receptors precipitated with polyethylene glycol. To determine whether the altered binding observed with antibody 960 was due to increased affinity of the receptor for insulin or appearance of more insulin binding sites, binding studies were performed over a wide range of insulin concentrations. Analysis of the resulting binding curves indicated that 960 increased the affinity of the receptor for insulin 3 fold over control (b= 0.3 nM for 960, and 0.9 nM for 83-7, respectively). The antibody 960 also specifically increased insulin binding to intact, saponin-permeabilized IM-9 cell membranes. These results indicate that binding of 960 antibody to the juxtamembrane region of the insulin receptor alters the affmity of the receptor for insulin. Since tyrosine 960 in the juxtamembrane region has been suggested to play a role in receptor signalling, changes in receptor conformation in this region that are likely to account for the change in affinity may play a role in signal transduction. 相似文献
15.
The receptors for insulin and insulin-like growth factor-I (IGF-I) are closely related in primary sequence and overall structure. We have examined the immunological relationships between these receptors by testing the reactivity of anti-(insulin receptor) monoclonal antibodies with IGF-I receptors in various tissues and cell lines. Antibodies for six distinct epitopes reacted with a subfraction of IGF-I receptors, as shown by inhibition of 125I-IGF-I binding, precipitation of 125I-IGF-I-receptor complexes or immunodepletion of receptor from tissue extracts before binding assays. Both immunoreactive and non-immunoreactive subfractions displayed the expected properties of 'classical' IGF-I receptors, in terms of relative affinities for IGF-I and insulin. The proportion of total IGF-I receptors which was immunoreactive varied in different cell types, being approx. 40% in Hep G2 cells, 35-40% in placental membranes and 75-85% in IM-9 cells. The immunoreactive fraction was somewhat higher in solubilized receptors than in the corresponding intact cells or membranes. A previously described monoclonal antibody, alpha-IR-3, specific for IGF-I receptors, inhibited IGF-I binding by more than 80% in all preparations. When solubilized placental receptors were pretreated with dithiothreitol (DTT) under conditions reported to reduce intramolecular (class I) disulphide bonds, the immunoreactivity of IGF-I receptors was abolished although total IGF-I binding was little affected. Under the same conditions insulin receptors remained fully immunoreactive. When solubilized receptor preparations were fractionated by gel filtration, both IGF-I and insulin receptors ran as symmetrical peaks of identical mobility. After DTT treatment, the IGF-I receptor was partially converted to a lower molecular mass form which was not immunoreactive. The insulin receptor peak showed a much less pronounced skewing and remained fully immunoreactive in all fractions. It is concluded that the anti- (insulin receptor) antibodies do not react directly with IGF-I receptor polypeptide, and that the apparent immunoreactivity of a subfraction of IGF-I receptors reflects their physical association with insulin receptors, both in cell extracts and in intact cells. The most likely basis for this association appears to be a 'hybrid' receptor containing one half (alpha beta) of insulin receptor polypeptide and the other (alpha' beta') of IGF-I receptor polypeptide within the native (alpha beta beta' alpha') heterotetrameric structure. 相似文献
16.
Cross-linking of insulin receptors to MHC antigens in human B lymphocytes: evidence for selective molecular interactions 总被引:3,自引:0,他引:3
Molecular interactions between insulin receptors and MHC antigens were investigated in human B cells. Two B lymphoblastoid cell lines, IM-9 and 526, chosen for their high insulin binding capacity, were found to express 15,000 and 25,000 insulin receptors per cell, respectively. Insulin receptors were labeled with a 125I-photoreactive insulin analogue, and all other surface proteins by lactoperoxidase-catalyzed radioiodination. Neighbor proteins were cross-linked with a cleavable homobifunctional reagent dithio-bis-(succinimidyl propionate) (DSP) and solubilized before immunoprecipitation by anti-HLA monoclonal antibodies. Gel analysis of the precipitated proteins showed that 90% of insulin receptors precipitable by anti-insulin receptor antibodies were precipitated by anti-class I antibodies (anti-heavy chain and anti-beta 2-microglobulin) after cross-linking with 2 mM DSP. In neither IM-9- nor 526 cells could HLA antigens be precipitated by anti-insulin receptor antibodies, suggesting that the concentration of class I antigens largely exceeds the concentration of insulin receptors at the cell surface. In 526 lymphocytes, class I MHC antigens were also found to adjoin class II antigens, since both molecules could be coprecipitated with anti-HLA A, B, C and with anti-HLA-DR antibodies after chemical cross-linking. Down-regulation of insulin receptors by chronic exposure of IM-9 cells to insulin did not affect the amount of MHC molecules present on the cell surface, and conversely, class I MHC molecules were internalized in 526 cells irrespective of the presence of insulin. These results thus show that insulin receptors and MHC antigens form multimolecular complexes in the plasma membrane of cultured human B cells. These interactions, which do not appear to influence the regulation of these proteins on the cell surface, may be involved in the mechanism of hormone signaling. 相似文献
17.
Characterization of insulin receptor carbohydrate by comparison of chemical and enzymatic deglycosylation 总被引:4,自引:0,他引:4
V L Herzberg F Grigorescu A S Edge R G Spiro C R Kahn 《Biochemical and biophysical research communications》1985,129(3):789-796
To characterize the carbohydrate moieties of the insulin receptor on IM-9 lymphocytes, the cells were surface iodinated and solubilized, and the insulin receptors were precipitated with anti-receptor antibody. The precipitates were resuspended, subjected to either enzymatic digestion or chemical treatment with trifluoromethanesulfonic acid and the relative mobilities of the alpha and beta subunits before and after treatment were analyzed by polyacrylamide gel electrophoresis and autoradiography. The results indicate that the alpha subunit possesses primarily N-linked carbohydrate which is both complex (Endoglycosidase F sensitive) and polymannose (Endoglycosidase H sensitive). The beta subunit also contains polymannose oligosaccharide units and has, in addition, a substantial amount of carbohydrate which is removed by chemical treatment but is not susceptible to Endoglycosidase F, suggesting the presence of O-linked saccharides. The apparent molecular weights of the core protein of the mature alpha and beta subunits as determined by gel electrophoresis following complete deglycosylation are 98 kDa and 80 kDa, respectively. 相似文献
18.
R H Whitson G K Grimditch E Sternlicht S A Kaplan R J Barnard K Itakura 《The Journal of biological chemistry》1988,263(10):4789-4794
When insulin receptors of rat skeletal muscle sarcolemmal vesicles were solubilized with Triton X-100, the specific binding of 125I-labeled insulin increased by more than 10-fold over that seen in the intact vesicles. Partial purification of the skeletal muscle insulin receptors on wheat germ agglutinin affinity columns increased the total insulin binding activity by 7-fold and reduced the Kd for insulin binding from 1.92 to 0.20 nM, suggesting that an inhibitor of insulin binding was removed by this purification step. This was confirmed when the unbound fractions of the affinity column were dialyzed and reconstituted with the insulin receptors. The inhibitory activity in the sarcolemmal extract could not be accounted for by the presence of Triton X-100. The skeletal muscle inhibitor was more potent in inhibiting insulin binding to skeletal muscle insulin receptors than to liver or adipose receptors. The inhibitor was very effective in inhibiting insulin binding to wheat germ agglutinin-purified IM-9 receptors, but had negligible effects on insulin binding to intact IM-9 cells. The properties of the alpha and beta subunits of the skeletal muscle insulin receptors appear to be the same as those of insulin receptors of other tissues: cross-linking of 125I-labeled insulin to the receptor revealed a band of 130,000 daltons, and insulin stimulated the phosphorylation of bands of 90,000 and 95,000 daltons in the receptor preparation. The skeletal muscle insulin binding inhibitor elutes from molecular sieves in a major 160,000-dalton peak and minor 75,000-dalton peak. The binding inhibitor is not inactivated by heat, by mercaptoethanol, or by trypsin, pepsin, or proteinase K. Collectively, these data suggest that the inhibitor may be a small molecule that aggregates with itself, with larger proteins, or with detergent micelles. 相似文献
19.
Ming Guo Lawrence T. Kim Steven K. Akiyama Harvey R. Gralnick Kenneth M. Yamada Frederick Grinnell 《Experimental cell research》1991,195(2):315-322
We used monoclonal antibodies against specific integrin subunits to examine the role of integrin receptors in keratinocyte activation. We found that before activation, beta 1 subunits in keratinocytes showed a diffuse distribution, whereas after activation, keratinocytes organized beta 1 receptors into marginal adhesion plaques. In immunoprecipitation experiments with antibodies against beta 1 integrin subunits, we found mostly immature subunits synthesized in keratinocytes freshly harvested from skin. Moreover, integrin receptor complexes immunoprecipitated from these cells by monoclonal antibodies against alpha 2, alpha 3, or alpha 5 subunits contained only immature beta 1 subunits. With keratinocytes cultured 4-7 days, anti-beta 1 antibodies immunoprecipitated mostly mature beta 1 subunits, and integrin complexes immunoprecipitated from cultured cells by anti-alpha subunit antibodies contained mostly mature beta 1 subunits. Antibodies directed against beta 1 subunits also inhibited keratinocyte migration. Based on these results, we suggest that up-regulation of migration by activated keratinocytes depends on changes in processing of pre-beta 1 subunits to mature beta 1 subunits. We also studied the distribution of integrin subunits in skin and on keratinocytes migrating out of skin explants. Whereas beta 1, alpha 2, and alpha 3 subunits were detected in keratinocytes in skin and migrating out of explants, alpha 5 subunits were observed only in migrating cells. 相似文献
20.
M11D杂交瘤细胞株是由人胎盘细胞膜纯化所得胰岛素受体免疫BALB/C小鼠后,取其脾细胞与同系小鼠骨髓瘤细胞株NS-1细胞融合所得。该杂交瘤细胞分泌的抗体经ELISA及放射免疫沉淀法证实为胰岛素受体特异的单克隆抗体。该抗体经Protein A-Sepharose亲和层析分离、纯化,SDS-聚丙烯酰胺梯度凝胶电泳鉴定得分子量分别为53000及23000的两条区带,免疫双扩证明为IgGl。该抗体特异地沉淀125Ⅰ-人胎盘细胞膜胰岛素受体,沉淀经SDS-聚丙烯酰胺凝胶电泳后放射自显影得分子量为135000的特异显影带,与胰岛素受体α亚基分子量相同,说明M11D为抗胰岛素受体α亚基的单克隆抗体。 相似文献