scholarly journals Insulin stimulates proteolysis of the α-subunit, but not the β-subunit, of its receptor at the cell surface in rat liver

1989 ◽  
Vol 261 (2) ◽  
pp. 333-340 ◽  
Author(s):  
K E Lipson ◽  
A A Kolhatkar ◽  
D B Donner
Keyword(s):  
Cell Surface ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Α Subunit ◽  
Receptor Complexes ◽  
Alpha Subunits ◽  
Alpha Beta

Insulin receptors in rat liver plasma membranes contain two alpha- and two beta-subunits held together by interchain disulphide bonds ([alpha beta]2 receptors). Affinity-labelled receptors were digested with chymotrypsin or elastase and then exposed to dithiothreitol before solubilization from membranes and SDS/polyacrylamide-gel electrophoresis. This resulted in partial reduction and isolation of Mr-225,000 alpha beta, Mr-200,000 alpha 1 beta, Mr-165,000 alpha beta 1 and Mr-145,000 alpha 1 beta 1 receptor halves containing intact (alpha, beta) or degraded (alpha 1, beta 1) subunits. The ability to identify half-receptor complexes containing intact or degraded subunits made it possible to assay each subunit simultaneously for insulin-induced proteolysis in isolated plasma membranes or during perfusion of rat liver in situ with insulin. In liver membranes, insulin binding increased the fraction of receptors containing degraded alpha-subunits to about one-third of the total population during 2 h of incubation at 23 degrees C. beta-Subunit proteolysis increased only minimally during this time. Plasma membranes isolated from livers perfused with insulin at 37 degrees C contained degraded alpha-subunits but only intact beta-subunits, showing that insulin induced cell-surface proteolysis of the binding, but not the kinase, domain of its receptor. Since previous observations [Lipson, Kolhatkar & Donner (1988) J. Biol. Chem 263, 10495-10501] have shown that receptors containing degraded alpha-subunits are internalized but do not recycle, it is possible that cell-surface degradation may play a role in the regulation of insulin-receptor number in hepatic tissue. Proteolysis of the beta-subunit is not a likely mechanism by which receptor-kinase activity may be attenuated under physiological conditions.

scholarly journals Protein kinase activity of the insulin receptor

1986 ◽  
Vol 235 (1) ◽  
pp. 1-11 ◽  
Author(s):  
S Gammeltoft ◽  
E Van Obberghen
Keyword(s):  
Insulin Receptor ◽  
Molecular Mechanisms ◽  
Kinase Activity ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Protein Kinase Activity ◽  
Intact Cells ◽  
Receptor Complexes ◽  
Alpha Subunits

The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural characterization of PACAP receptors on rat liver plasma membranes

1993 ◽  
Vol 265 (5) ◽  
pp. G811-G818
Author(s):  
T. D. Nguyen ◽  
G. G. Heintz ◽  
M. S. Wolfe
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Receptors ◽  
Receptor Complexes ◽  
High Affinity ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes ◽  
Pacap Receptors

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) and PACAP-27 are recently characterized hypothalamic peptides with marked homology with vasoactive intestinal peptide (VIP), which are concentrated in the innervation of the digestive tract. We now report that, on rat liver plasma membranes, PACAP interacts with at least two types of receptors: receptors demonstrating equally high affinity for PACAP and VIP and receptors with high affinity for PACAP but low affinity for VIP. In contrast, on rat intestinal epithelial cell laterobasal membranes, only receptors with high affinities for PACAP and VIP were observed. After 125I-labeled VIP or 125I-labeled PACAP-27 was cross-linked to the liver plasma membrane receptors with the use of either disuccinimidosuberate or disuccinimido dithiobis(propionate), analysis of the resulting ligand-receptor complexes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the structures of the VIP and PACAP receptors were similar: both ligand-receptor complexes displayed two radioactive bands with relative molecular weights of 80,000 and 56,000 under reducing conditions and of 75,000 and 53,000 under nonreducing conditions. These findings suggest that the receptors for the PACAP peptides and VIP are closely related, reflecting the marked homology between these peptides. The presence of receptors specific for PACAP on rat liver plasma membranes should stimulate further studies of the interaction between PACAP and the liver.

scholarly journals Inherited deficiency of the Mac-1, LFA-1, p150,95 glycoprotein family and its molecular basis.

1984 ◽  
Vol 160 (6) ◽  
pp. 1901-1918 ◽  
Author(s):  
T A Springer ◽  
W S Thompson ◽  
L J Miller ◽  
F C Schmalstieg ◽  
D C Anderson
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Molecular Basis ◽  
Autosomal Recessive Inheritance ◽  
Surface Expression ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Normal Surface ◽  
Alpha Subunits ◽  
Deceased Patient

Leukocyte surface glycoproteins that share a common beta subunit have been found to be congenitally deficient in three unrelated patients with recurring bacterial infection. The glycoproteins, Mac-1, LFA-1, and p150,95, have the subunit compositions alpha M beta, alpha L beta, and alpha X beta, respectively. Using subunit-specific monoclonal antibodies, both the alpha M and beta subunits of Mac-1, the alpha L and beta subunits of LFA-1, and at the least the beta subunit of p150,95, were found to be deficient at the cell surface by the techniques of immunofluorescence flow cytometry, radioimmunoassay, and immunoprecipitation. A latent pool of Mac-1 that can be expressed on granulocyte surfaces in response to secretory stimuli, such as f-Met-Leu-Phe, was also lacking in patients. Deficiency was found on all leukocytes tested, including granulocytes, monocytes, and T and B lymphocytes. Quantitation by immunofluorescence cytometry of subunits on granulocytes from parents of these patients and of a fourth deceased patient showed approximately half-normal surface expression, and, together with data on other siblings and a family with an affected father and children, demonstrate autosomal recessive inheritance. Deficiency appears to be quantitative rather than qualitative, with two patients expressing approximately 0.5% and one patient approximately 5% of normal amounts. The latter patient had alpha beta complexes on the cell surface detectable by immunoprecipitation. Biosynthesis experiments showed the presence of normal amounts of alpha'L intracellular precursor in lymphoid lines of all three patients. Together with surface deficiency of three molecules that share a common beta subunit but have differing alpha subunits, this suggests the primary deficiency is of the beta subunit. The lack of maturation of alpha'L to alpha L and the deficiency of the alpha subunits at the cell surface and in latent pools suggests that association with the beta subunit is required for alpha subunit processing and transport to the cell surface or to latent pools. The molecular basis of this disease is discussed in light of adhesion-related functional abnormalities in patients' leukocytes and the blockade of similar functions in healthy cells by monoclonal antibodies.

Solubilization of receptors for pancreatic polypeptide from rat liver membranes

1995 ◽  
Vol 268 (2) ◽  
pp. G215-G223
Author(s):  
T. D. Nguyen ◽  
M. S. Wolfe ◽  
G. G. Heintz
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Pancreatic Polypeptide ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Receptor Complexes ◽  
High Affinity

We have previously identified, on rat liver microsomes and plasma membranes, proteins that bind pancreatic polypeptide (PP) with high affinity and specificity and that may serve as receptors for a hepatic effect of PP (J. Biol. Chem. 267: 9416-9421, 1992). Further characterization of these proteins requires the solubilization of receptors with conserved ability to bind PP selectively and efficiently. In this report, using 6 mM of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), we solubilized, from liver microsomes, receptors that bound PP with high affinity (dissociation constant 6.15 +/- 1.6 nM) and specificity (no interaction with the homologous peptides neuropeptide Y and peptide YY). Gel filtration chromatography showed different degrees of receptor aggregation related to different concentrations of CHAPS in the eluent. To characterize the structure of these solubilized receptors, the chemical cross-linker N-(5-azido-2-nitrobenzoyloxy)succinimide was used to covalently bind these receptors to radiolabeled PP, and the resulting PP-receptor complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A radioactive band with an apparent molecular weight (M(r)) of 46,000 was detected that was inhibited by unlabeled PP with a half-maximal inhibitory concentration of approximately 10(-8) M. It most likely reflected a PP receptor with an estimated M(r) of 42,000, excluding the molecular weight of PP. The migration of this complex was not affected by the reducing agent dithiothreitol, suggesting the absence of disulfide bonding. The solubilization and identification of a bioactive hepatic PP receptor will allow further characterization and purification of this receptor and will lead to the clarification of the interaction between PP and the digestive system.

A spectrin-like protein from mouse brain membranes: phosphorylation of the 235,000-dalton subunit

1984 ◽  
Vol 247 (1) ◽  
pp. C61-C73 ◽  
Author(s):  
S. R. Goodman ◽  
I. S. Zagon ◽  
C. F. Whitfield ◽  
L. A. Casoria ◽  
S. B. Shohet ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Mouse Erythrocyte ◽  
Alpha Beta ◽  
Beta 2 ◽  
Independent Protein ◽  
Brain Spectrin

A mouse brain spectrin-like protein, which was an immunoreactive analogue of erythrocyte spectrin, has been isolated from demyelinated membranes. This spectrin analogue was a 10.5 S, 972,000 molecular weight (Mr) (alpha beta)2 tetramer containing subunits of 240,000 (alpha) and 235,000 (beta) Mr. We demonstrated that in vivo only the 235,000 Mr beta subunit of the mouse brain spectrin-like protein was phosphorylated, which was an analogous situation to mouse erythrocyte spectrin in which only the 220,000 Mr beta subunit was phosphorylated. Incubation of isolated membrane fractions with [gamma-32P]ATP +/- adenosine 3',5'-cyclic monophosphate (cAMP) indicated that mouse brain spectrin-like protein, mouse erythrocyte spectrin, and human erythrocyte spectrin's beta subunits were all phosphorylated in vitro by membrane-associated cAMP-independent protein kinases.

scholarly journals Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins.

1994 ◽  
Vol 125 (4) ◽  
pp. 795-802 ◽  
Author(s):  
J L Thomas ◽  
D Holowka ◽  
B Baird ◽  
W W Webb
Keyword(s):  
Cell Surface ◽  
Large Scale ◽  
Plasma Membranes ◽  
Immunoglobulin E ◽  
Large Fraction ◽  
Surface Antigens ◽  
Specific Cell ◽  
Cell Surface Antigens ◽  
Receptor Complexes ◽  
Ige Receptors

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co-aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co-aggregated diI and IgE receptors. The results indicate that cross-linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre-existing membrane domains or causing their formation.

Analysis of subunit assembly of the Na-K-ATPase

1994 ◽  
Vol 266 (3) ◽  
pp. C579-C589 ◽  
Author(s):  
D. M. Fambrough ◽  
M. V. Lemas ◽  
M. Hamrick ◽  
M. Emerick ◽  
K. J. Renaud ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sodium Pump ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Limited Region ◽  
Subunit Assembly ◽  
Alpha Subunits ◽  
Subunit Isoforms ◽  
P Type

The Na-K-ATPase, or sodium pump, is comprised of two subunits, alpha and beta. Each subunit spans the lipid bilayer of the cell membrane. This review summarizes our efforts to determine how the two subunits interact to form the functional ion transporter. Our major approach has been to observe the potential for subunit assembly when one or both subunits are truncated or present as chimeras that retain only a limited region of the Na-K-ATPase. DNAs encoding these altered subunit forms of the avian Na-K-ATPase are expressed in mammalian cells. Monoclonal antibodies specific for the avian beta-subunit are then used to purify newly synthesized avian beta-subunits, and the presence of accompanying alpha-subunits indicates that subunit assembly has occurred. The ectodomain of the beta-subunit (approximately residues 62-304) is sufficient for assembly with the alpha-subunit, and a COOH-terminal truncation of the beta-subunit that lacks aminoacyl residues beyond 162 will assemble inefficiently. A maximum of 26 aminoacyl residues of the alpha-subunit are necessary for robust assembly with the beta-subunit, when this sequence replaces the COOH-terminal half of the loop between membrane spans 7 and 8 in the SERCA1 Ca-ATPase. This region of the Ca-ATPase faces the lumen of the endoplasmic reticulum. These findings encourage study of other related questions, including whether there is preferential assembly of certain subunit isoforms and how various P-type ATPases are targeted to their appropriate subcellular compartments.

scholarly journals Functional reconstitution of the human interleukin-3 receptor

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 84-90 ◽  
Author(s):  
T Kitamura ◽  
A Miyajima
Keyword(s):  
Ligand Binding ◽  
Growth Response ◽  
Human Factor ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Stable Transfectants ◽  
Interleukin 3 ◽  
Gm Csf ◽  
High Affinity ◽  
Alpha Subunits

The high-affinity receptors for human interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of alpha and beta subunits. The alpha subunits are primary ligand binding proteins specific for each ligand, whereas the three human receptors share a common beta subunit (beta c). In contrast to humans mice have two closely related genes, AIC2A and AIC2B, which are homologous to human beta c. The AIC2A gene encodes a low-affinity murine IL-3 binding protein, and the AIC2B protein is the beta subunit shared between murine GM-CSF receptors (mGMR) and IL-5 receptors (mIL- 5R). To examine the function of these receptor components, we established various stable transfectants of murine IL-2-dependent CTLL- 2 cells. CTLL-2 transfectants expressing both the alpha and beta subunits of the human IL-3 receptor (hIL-3R) proliferated in response to physiologic concentrations of hIL-3. Coexpression of hIL-3R alpha with AIC2B but not with AIC2A in CTLL-2 cells conferred a growth response to hIL-3. Although CTLL-2 transfectants expressing hIL-3R alpha alone did not proliferate in the presence of hIL-3, hIL-3- responsive sublines were repeatedly isolated. These sublines expressed endogenous AIC2B but not AIC2A. These results indicate that human beta c is essential for hIL-3 signaling and that AIC2B is a murine equivalent of human beta c. We also showed that hIL-3 and hGM-CSF induced tyrosine phosphorylation of several proteins in CTLL transfectants, similar to those observed in human factor-dependent TF-1 cells stimulated with hIL-3 and hGM-CSF.

scholarly journals Functional reconstitution of the human interleukin-3 receptor

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 84-90 ◽  
Author(s):  
T Kitamura ◽  
A Miyajima
Keyword(s):  
Ligand Binding ◽  
Growth Response ◽  
Human Factor ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Stable Transfectants ◽  
Interleukin 3 ◽  
Gm Csf ◽  
High Affinity ◽  
Alpha Subunits

Abstract The high-affinity receptors for human interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of alpha and beta subunits. The alpha subunits are primary ligand binding proteins specific for each ligand, whereas the three human receptors share a common beta subunit (beta c). In contrast to humans mice have two closely related genes, AIC2A and AIC2B, which are homologous to human beta c. The AIC2A gene encodes a low-affinity murine IL-3 binding protein, and the AIC2B protein is the beta subunit shared between murine GM-CSF receptors (mGMR) and IL-5 receptors (mIL- 5R). To examine the function of these receptor components, we established various stable transfectants of murine IL-2-dependent CTLL- 2 cells. CTLL-2 transfectants expressing both the alpha and beta subunits of the human IL-3 receptor (hIL-3R) proliferated in response to physiologic concentrations of hIL-3. Coexpression of hIL-3R alpha with AIC2B but not with AIC2A in CTLL-2 cells conferred a growth response to hIL-3. Although CTLL-2 transfectants expressing hIL-3R alpha alone did not proliferate in the presence of hIL-3, hIL-3- responsive sublines were repeatedly isolated. These sublines expressed endogenous AIC2B but not AIC2A. These results indicate that human beta c is essential for hIL-3 signaling and that AIC2B is a murine equivalent of human beta c. We also showed that hIL-3 and hGM-CSF induced tyrosine phosphorylation of several proteins in CTLL transfectants, similar to those observed in human factor-dependent TF-1 cells stimulated with hIL-3 and hGM-CSF.

scholarly journals Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity

1994 ◽  
Vol 300 (1) ◽  
pp. 37-43 ◽  
Author(s):  
P E Milhiet ◽  
D Corbeil ◽  
V Simon ◽  
A J Kenny ◽  
P Crine ◽  
...  
Keyword(s):  
Cell Surface ◽  
Culture Media ◽  
Enzymic Activity ◽  
Beta Subunit ◽  
Membrane Topology ◽  
Site Directed Mutagenesis ◽  
Alpha Subunit ◽  
Cell Extracts ◽  
Membrane Bound ◽  
Alpha Beta

Endopeptidase-24.18 (E-24.18; EC 3.4.24.18) is a metallopeptidase of the astacin family and is highly expressed in kidney brush-border membranes of rodents. Rat E-24.18 consists of two disulphide-linked alpha/beta dimers [(alpha/beta)2]. In order to investigate the mechanisms of assembly and the importance of each subunit in the enzymic process, the cloned cDNAs for the rat alpha and beta subunits were transiently expressed either alone or together in COS-1 cells. Immunoblotting of cell extracts and spent culture media showed that, when expressed alone, the alpha subunit is secreted, whereas the beta subunit is membrane-bound. In alpha/beta-transfected cells, the alpha subunit remained membrane-bound, but could be released from the cell surface after papain treatment or after incubation with 10 mM dithiothreitol. Furthermore, mutants of the alpha subunit in which the putative C-terminal anchor domain was deleted could still form cell-associated alpha/beta dimers. These results are consistent with a topological model of E-24.18 in which the beta subunit is anchored in the plasma membrane and the alpha subunit is retained at the cell surface through disulphide bridge(s) with the beta subunit. Both the alpha and beta recombinant subunits expressed in COS-1 cells showed little azocasein-degrading activity. However, activity of either individual subunits of alpha/beta dimers was increased after mild trypsin digestion, suggesting that in COS-1 cells the enzymes are synthesized as zymogens. Finally, inactivation of the alpha subunit by site-directed mutagenesis of Glu-157, which is believed to play a role in catalysis, showed that both subunits participate in the enzymic activity of the heterodimer.

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