Characterization of a Novel Receptor in Toad Retina with Dual Specificity for Insulin and Insulin-Like Growth Factor I

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.     

Topography of human placental receptors for epidermal growth factor

These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.     

Insulin stimulates phosphorylation of serine residues in soluble insulin receptors

Using lectin affinity-purified receptor preparations from human hepatoma cells, insulin (10(-7)M) specifically stimulated phosphorylation of the 95,000 dalton (beta) subunit of its own receptor. Phospho-amino acid analysis of the receptor subunit revealed that insulin increased at least 2.5-fold the content of phosphoserine and of phosphotyrosine. In intact cells, the major effect of insulin is to increase the phosphoserine content of its receptor. These findings are the first demonstration of an insulin-stimulated serine kinase in a cell-free system.     

Immunological Identification of Multiple α-Like Subunits of the γ-Aminobutyric AcidA Receptor Complex Purified from Neonatal Rat Cortex

Antibodies were prepared against a synthetic peptide corresponding to amino acid sequences 174-203 of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1-subunit. The antibodies recognized this synthetic alpha 1-peptide, but failed to react with the homologous peptide sequence, 170-199, of the bovine beta 1-subunit. On Western blots, anti-alpha 1-subunit antibody recognized a 50-kilodalton (kDa) protein in affinity-purified receptor preparations from adult rat cortex and cerebellum. In receptor purified from neonatal cortex, the anti-alpha 1-antibody reacted with 50-kDa, 53-54-kDa, and 59-kDa proteins. After digestion with endoglycosidase F, these three protein bands retained differing electrophoretic mobilities. The 50-kDa and 59-kDa subunits of affinity-purified neonatal receptor, which were photoaffinity-labeled with [3H]flunitrazepam, were immunoprecipitated to different extents by alpha-subunit antibody. These data suggest the existence in GABAA receptor from neonatal cortex of three proteins (50 kDa, 53 kDa, and 59 kDa) which have immunological homology to alpha 1-subunit of bovine GABAA receptor. The presence of an alpha- and a beta-like subunit with similar mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis may account for the relatively high concentration of protein in the 53-54-kDa band which has been observed in receptor purified from neonatal cortex. The presence of multiple alpha-like subunits may be related to the presence of a relatively high concentration of type II GABA receptor in this tissue.     

Purification and characterization of the human brain insulin receptor

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.     

Monoclonal antibodies to receptors for insulin and somatomedin-C

Three monoclonal antibodies, designated alpha IR-1, alpha IR-2, and alpha IR-3, were prepared by fusing FO myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of insulin receptors from human placenta. These antibodies were characterized by their ability to immunoprecipitate solubilized receptors labeled with 125I-insulin or 125I-somatomedin-C in the presence or absence of various concentrations of unlabeled insulin or somatomedin-C. alpha IR-1 preferentially immunoprecipitates insulin receptors and also less effectively immunoprecipitates somatomedin-C receptors, while alpha IR-2 and alph IR-3 preferentially immunoprecipitate somatomedin-C receptors, but may also weakly immunoprecipitate insulin receptors. These three monoclonal antibodies, as well as A410, a rabbit polyclonal antibody, were used to immunoprecipitate insulin and somatomedin-C receptors from solubilized human lymphoid (IM-9) cells and human placenta membranes that had been 125I-labeled with lactoperoxidase. Analysis of the immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both receptors are composed of alpha and beta subunits. The beta subunit of the insulin receptor (immunoprecipitated by alpha IR-1 and A410) has a slightly more rapid mobility than the corresponding subunit of the somatomedin-C receptor (immunoprecipitated by alpha IR-2 and alpha IR-3). Interestingly, the alpha subunit of the placenta somatomedin-C receptor has a slightly faster mobility than its counterpart from IM-9 cells. Immunoprecipitation of receptor that had been reduced and denatured to generate isolated subunits indicates that alpha IR-2 and alpha IR-3 interact with the alpha subunit of the somatomedin-C receptor while A410 interacts with both subunits of the insulin receptor. alpha IR-1 failed to react with reduced and denatured receptors.     

Identification of a novel Mr = 76-kDa form of beta-adrenergic receptors

The structure of the human beta-adrenergic receptor in purified basal membranes of human placental syncytiotrophoblast was probed using photoaffinity labeling. Basal membranes display a high specific activity of receptors (4-5 pmol/mg protein) and possess both beta 1- and beta 2-adrenergic receptors subtypes. Autoradiography of membranes that were incubated with the beta-adrenergic antagonist [125I]iodoazidobenzylpindolol, photolyzed and then subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, identified four radiolabeled peptides, Mr = 65-kDa, 54-kDa, 43-kDa and a novel higher molecular weight 76-kDa form of the receptor. Photoaffinity labeling of each of these four peptides displayed the pharmacological properties expected for true beta-adrenergic receptors. The 76-kDa photoaffinity labeled receptor peptide observed in human placenta basal membranes has not been reported elsewhere. Competition studies with the beta1-selective ligand CGP-20712A demonstrate that the photoaffinity labeled receptor peptides are composed of both beta 1- and beta 2-adrenergic receptor subtypes.     

Isolation and partial characterization of high affinity laminin receptors in neural cells

Neural cells in culture (NG-108, PC12, chick dorsal root ganglion, chick spinal cord, and rat astrocytes) bind laminin with an apparent Kd of congruent to 10(-9) M. Laminin affinity chromatography of chick brain membranes washed with 150 mM NaCl and eluted with 0.2 M glycine buffer, pH 3.5, yields a single protein with an apparent molecular mass of 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Isoelectric focusing and peptide mapping indicate that the 67-kDa protein is distinct from bovine serum albumin (68 kDa) but indistinguishable from high affinity laminin receptors isolated from skeletal muscle. After electroblotting onto nitrocellulose paper and probing with 125I-laminin, this putative laminin receptor binds laminin specifically (100 ng/ml). A second protein (congruent to 120-140 kDa) is also detected with 125I-laminin (100 ng/ml) in the laminin affinity-purified membrane proteins. Both 67- and congruent to 120-140-kDa proteins can be laminin affinity-purified from cultures enriched for neurons (greater than 90%) following metabolic labeling with [35S]methionine. Our data suggest that neural cells (dorsal root ganglion, central nervous system neurons, astrocytes, and several neural cell lines) have high affinity binding sites for laminin and that two membrane proteins, 67- and congruent to 120-140-kDa, are responsible at least in part for this binding.     

Isolation of a proteolytically derived domain of the insulin receptor containing the major site of cross-linking/binding

Radiolabeled insulin was affinity cross-linked to purified insulin receptor with six separate bifunctional N-hydroxysuccinimide esters of different lengths. Results were qualitatively identical for each cross-linker in that insulin was predominantly cross-linked through its B chain to the receptor's alpha subunit. The maximum efficiencies of cross-linking were 10-15% for the most effective reagents, and this value was dependent upon the concentration and length of the cross-linker. In an effort to locate the cross-linking site, monoiodoinsulin was cross-linked to affinity-purified insulin receptor with disuccinimidyl suberate. Limited proteolysis of the hormone/receptor adduct with Staphylococcus aureus V8 protease, chymotrypsin, or thermolysin in an SDS-containing buffer rapidly generated a 55-kDa, insulin-labeled fragment as shown by SDS-polyacrylamide gel electrophoresis. We reported earlier that the 55-kDa chymotryptic fragment contained multiple internal disulfide bonds as evidenced by its shifting mobility on an SDS gel after dithiothreitol treatment [Boni-Schnetzler et al. (1987) J. Biol. Chem. 262, 8395-8401]. Here we show that the 55-kDa fragment is also formed by proteolysis of the receptor in the absence of prior insulin cross-linking. This fragment was prepared in amounts sufficient for sequence analysis and was purified by passage successively over gel permeation and reverse-phase HPLC columns. The sequence of the fragment's amino terminus corresponds to that of the amino terminus of the receptor's alpha subunit. This fragment also reacts with an antibody raised against a synthetic peptide corresponding to residues 242-253 of the receptor's alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase

In these studies we demonstrate that insulin stimulates both tyrosine and serine phosphorylation of the insulin receptor after its partial purification on wheat germ-agarose, and after affinity purification on insulin-agarose. Analysis of the serine phosphate incorporated into partially purified or highly purified insulin receptor suggests that an insulin-sensitive serine kinase (IRSK) copurifies with the insulin receptor. Following trypsin digestion, reversed-phase high pressure liquid chromatography (HPLC) analysis of the phosphorylated, affinity-purified insulin receptor preparation reveals phosphopeptide profiles similar to those of trypsin-digested receptors immunoprecipitated from 32P-labeled fibroblasts overexpressing the human insulin receptor. The major insulin-stimulated HPLC phosphopeptide peak from insulin receptors labeled in intact cells contains a hydrophilic phosphoserine-containing peptide which rapidly elutes from a C18 column. HPLC and two-dimensional separation indicate that the same phosphopeptide is obtained when affinity-purified insulin receptors are phosphorylated by IRSK. The serine containing tryptic peptide within the cytoplasmic domain of the human insulin receptor predicted to elute most rapidly upon HPLC had the sequence SSHCQR corresponding to residues 1293-1298. A synthetic peptide containing this sequence is phosphorylated by the insulin receptor/IRSK preparation. After alkylation and trypsin digestion, the synthetic phosphopeptide comigrates with the alkylated, tryptic phosphopeptide derived from insulin receptor phosphorylated in vitro by IRSK. We propose that serine 1293 or 1294 of the human insulin receptor is a major site(s) phosphorylated on the insulin receptor in intact cells and is phosphorylated by IRSK. Furthermore, insulin added directly to affinity-purified insulin receptor/IRSK preparations stimulates the phosphorylation of synthetic peptides corresponding to this receptor phosphorylation site and another containing threonine 1336. Kemptide phosphorylation is not stimulated by insulin under these conditions. No phosphorylation of peptide substrates for Ca2+/calmodulin-dependent protein kinase, protein kinase C, casein kinase II, or cGMP-dependent protein kinase by IRSK is detected. These data indicate that IRSK exhibits specificity for the insulin receptor and may be activated by the insulin receptor tyrosine kinase in an insulin-dependent manner.     

Altered placental insulin and insulin-like growth factor-I receptors in diabetes

We have compared the characteristics of IGF-I and insulin receptors in placentas of normals and insulin dependent diabetic patients. Specific binding of both IGF-I and insulin in placental membranes from patients with good glycemic control (as reflected by blood hemoglobin content) was unaltered while that in the placental membranes from the patients with poor glycemic control was increased to approximately 20% of the normals. This observed small but significant (p less than 0.05) increase in binding of IGF-I and insulin to placental membranes from diabetic patients with poor glycemic control was further magnified, approximately twice (p less than 0.001) the normal, when the membrane receptors were purified by lectin chromatography. The kinetic analysis of IGF-I and insulin binding in both membranes and lectin purified receptors revealed that the increased binding of insulin and IGF-I to the placentas from diabetic patients with poor glycemic control was due to an approximately 2 fold increase (p less than 0.001-0.05) in the receptor numbers without any significant changes of the affinities. The molecular characteristics of the receptors in these diabetic patients, as revealed by the cross-linking studies, did not reveal any changes when compared to the normals. The parallel changes of IGF-I and insulin receptors, shown here, are in accordance with the homologous nature of these two receptors. The increased receptor numbers of these two interrelated hormones in placentas of diabetics with poor glycemic control may be relevant to the altered placental functions in diabetic pregnancy.     

High molecular weight forms of the insulin receptor

The insulin receptor of liver, adipose, and placental plasma membranes was photoaffinity labeled with radioiodinated N epsilon B29-(monoazidobenzoyl)insulin. Three specifically labeled bands of 450, 360, and 260 kilodaltons (kDa) were identified in each tissue by polyacrylamide gel electrophoresis of the membranes solubilized in sodium dodecyl sulfate (SDS). The 360- and 260-kDa bands corresponded to partially reduced forms of the 450-kDa band. The distribution of radioactivity between the three insulin receptor bands was dependent on the tissue, the purity of the receptor preparation, and the conditions of solubilization in SDS. The 360- and 260-kDa bands became more prominent in each tissue with an increasing time of solubilization in SDS. However, with a short solubilization time in SDS, the 450-, 360-, and 260-kDa bands of the receptor were distributed approximately in a ratio of 85:15:0 in all three tissues. Inclusion of sulfhydryl alkylating reagents during solubilization in SDS altered this ratio to about 95:5:0. We conclude that the 450-kDa band represents the predominant form of the photolabeled insulin receptor and that the 260-kDa and probably the 360-kDa form as well were generated during the experimental manipulations preceding identification of the receptor. However, the appearance of the 360- and 260-kDa bands was not due to reductant present in SDS or buffer solutions and could not be accounted for by proteolytic degradation of the receptor. Furthermore, purification of the receptor over 2000-fold did not prevent the appearance of the 360- and 260-kDa bands.(ABSTRACT TRUNCATED AT 250 WORDS)     

An insulin-like growth factor I/insulin hybrid exhibiting high potency for interaction with the type I insulin-like growth factor and insulin receptors of placental plasma membranes

We have prepared by semisynthetic methods a two-chain insulin/insulin-like growth factor I hybrid that contains a synthetic peptide related to residues 22-41 of insulin-like growth factor I linked via peptide bond to ArgB22 of des-octapeptide-(B23-B30)-insulin and have applied the analog to the analysis of ligand interactions with the type I insulin-like growth factor and insulin receptors of placental plasma membranes. Relative potencies for the inhibition of 125I-labeled insulin-like growth factor I binding to type I insulin-like growth factor receptors were 1.0:0.20:0.003 for insulin-like growth factor I, the hybrid analog, and insulin, respectively. Corresponding relative potencies for the inhibition of 125I-labeled insulin binding to insulin receptors were 0.007:0.28:1 for the three respective peptides. Additional studies identified that the hybrid analog interacts with only one of two populations of insulin-like growth factor I binding sites on placental plasma membranes and permitted the analysis of insulin-like growth factor I interactions with the separate populations of binding sites. We conclude that (a) des-octapeptide-(B23-B30)-insulin can serve well as a scaffold to support structural elements of insulin-like growth factor I and insulin necessary for high affinity binding to their receptors, (b) major aspects of structure relevant to the conferral of receptor binding affinity lie in the COOH-terminal region of the insulin B chain and in the COOH-terminal region of the insulin-like growth factor I B domain and in its C domain, and (c) the evolution of ligand-receptor specificity in these systems has relied as much on restricting interactions (through the selective introduction of negative structural elements) as it has on enhancing interactions (through the introduction of affinity conferring elements of structure).     

Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein.

Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes.     

Ontogeny of insulin receptors in the rat hemochorial placenta

Binding of 125I-insulin to rat placental membranes was time and protein concentration dependent, reversible, and specific. Unlabeled porcine insulin competed for 125I-insulin binding with an IC50 of 65 nM, while IGF-I was much less potent with an IC50 of 2.12 mM. Specific binding of 125I-insulin decreased during the second half of gestation from Days 11 to 19. Scatchard analysis of the binding data for membranes prepared from Gestation Days 11 and 19 yielded typical curvilinear plots which showed a marked decrease in the number of binding sites in late gestation placenta. Beginning on Day 14, insulin binding was characterized with isolated labyrinth and basal zone portions of the hemochorial placenta. There was no evidence for differences in Kd values or the number of binding sites in these two functionally distinct portions of the rat placenta. Crosslinking of 125I-insulin followed by SDS-PAGE showed a single protein with a molecular weight of 130,000 from placental tissues on Gestation Days 11 and 19 and confirmed a gestational decrease in the number of insulin receptors. In solubilized, lectin-purified preparations from placenta and liver membranes, insulin stimulated the phosphorylation of a Mr 95,000 protein. 32P-incorporation into this 95,000 protein was stimulated fivefold by insulin in Day 11 placenta receptor, whereas no detectable 32P-incorporation was found in Day 19 placenta. Thus, while the alpha- and beta-subunits of insulin receptors in mid and late gestation placenta have molecular weights which are similar to receptors in maternal liver, data indicate the presence of a functional difference in insulin-stimulated kinase activities.     

Insulin receptors in rat brain: insulin stimulates phosphorylation of its receptor beta-subunit

In rat brain cortex synaptosomes insulin stimulated the phosphorylation of its own receptor beta-subunit (94 kDa) as identified by immunoprecipitation with anti-insulin or anti-receptor antiserum. The receptor alpha-subunit (115 kDa) was characterized by specific labeling with 125I-labeled photoreactive insulin. These observations indicate that: (i) insulin receptors in brain are composed of alpha-subunits which bind insulin, and beta-subunits, the phosphorylation of which can be stimulated by insulin; (ii) the size of alpha-subunits in brain is significantly smaller than in other tissues (115 vs 130 kDa), whereas beta-subunits (94 kDa) are identical. We suggest that brain insulin receptors represent a subtype regarding their binding function, whereas their enzyme function is more conserved.     

Adipocyte insulin receptor. Generation of a cryptic domain of the alpha-subunit during internalization of hormone-receptor complexes.

The dynamics of the internalization of photoaffinity-labelled insulin-receptor complexes was investigated in isolated rat adipocytes by using tryptic proteolysis to probe both the orientation and cellular location of the labelled complexes. In cells that were labelled at 16 degrees C and not prewarmed, 150 micrograms of trypsin/ml rapidly degraded the labelled 125 kDa insulin-receptor subunit into a major proteolytic fragment of 70 kDa and minor amounts of 90- and 50-kDa fragments. With milder trypsin treatment conditions (100 micrograms of trypsin/ml, 15 s at 37 degrees C), the 90 kDa peptide (different from the 90 kDa beta-subunit of the insulin receptor) appeared as a major intermediate proteolytic product, but this species was rapidly and completely converted into the 70- and 50-kDa fragments with continued exposure to trypsin, such that it did not accumulate to appreciable amounts in cells that were not prewarmed before trypsin exposure. By contrast, trypsin treatment of cells prewarmed to 37 degrees C for various times showed that: first, a proportion of the labelled 125 kDa receptors was internalized (became trypsin-insensitive); secondly, the 90 kDa tryptic peptide was formed in large amounts, with proportionate decreases occurring in the amounts of the 70- and 50-kDa tryptic peptides. The increased accumulation of the 90 kDa tryptic peptide from cells preincubated at 37 degrees C, but not at 16 degrees C, indicated that trypsin cleavage sites within the 90 kDa segment of the insulin-receptor alpha-subunit that were exposed at 16 degrees C were made inaccessible by incubation at 37 degrees C, a finding that is consistent with generation of a cryptic domain of the receptor subunit. The tryptic generation of the 90 kDa peptide at 37 degrees C was rapid, becoming half-maximal in 4.4 +/- 0.6 min and maximal in 15-20 min, preceded the intracellular accumulation of labelled receptors (half-maximal in 12.6 +/- 0.7 min and maximal in 30-40 min), was highly correlated with receptor internalization, and was not observed in cultured IM-9 lymphocytes, a cell line in which photolabelled insulin receptors are primarily lost by shedding into the incubation media. These results show that, in adipocytes incubated at 37 degrees C, rapid masking of a previously (at 16 degrees C) accessible domain of the insulin-receptor alpha-subunit occurs and that this dynamic process happens at an early stage in the internalization of insulin-receptor complexes.     

Muscarinic Acetylcholine Receptors from Avian Retina and Heart Undergo Different Patterns of Molecular Maturation

Muscarinic acetylcholine receptors (mAChRs) from the avian CNS exist in two molecular weight forms whose concentrations change during development. Here, we have compared the development of mAChRs from embryonic hearts with those of the CNS. Analysis of [3H]-propylbenzilylcholine mustard (PrBCM)-labeled retina and heart mAChRs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two atropine-sensitive peaks for each tissue. Apparent molecular masses of retina mAChRs, 86 +/- 0.7 kilodaltons (kDa) and 72 +/- 0.7 kDa, were different from those of heart mAChRs, 77 +/- 1.0 kDa and 52 +/- 0.9 kDa. During retina development, the major receptor type changed from 86 kDa to 72 kDa. No such change occurred during heart development. Furthermore, the 52-kDa species appeared to be generated by endogenous proteolysis, as prolonged incubation of heart membranes at 37 degrees C increased the amount of 52-kDa peptide with a decrease of 77-kDa peptide. Protease inhibitors blocked this conversion. Incubation of retina membranes at 37 degrees C did not result in a conversion of the 86-kDa peptide into the 72-kDa peptide, but it did cause the appearance of a minor amount of 52-kDa peptide. The proteolysis of retina mAChRs was not enhanced by cohomogenizing them with heart tissue, arguing against the presence of releasable proteases in heart. Membrane-bound retina and heart mAChRs displayed similar sensitivity to exogenous (Staphylococcus aureus V8) protease, indicating that heart receptors were not unusually susceptible to proteolytic attack; analysis of the labeled polypeptides with the V8 protease showed different patterns of digestion for the retina and heart receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of rat liver cell multiplication-stimulating activity (MSA) to human placenta and serum proteins.

Multiplication-stimulation activity (MSA) from the medium of BRL-3A rat liver cells in culture binds to cell membrane and cytosol receptors from human placenta and to serum proteins. The binding of MSA to placental cell membranes is dependent on time, temperature, pH and divalent ion concentration. MSA bound to placental cytosol receptor and serum is not displaced by insulin, whereas that bound to placental cell membranes is displaced by insulin and insulin-like peptides. The affinity of the three receptors for MSA is similar [approximately 10(8) M(-1)]. An assay using 125I-MSA and placental membrane receptor detects somatomedin-like receoptor activity (SmLRA) in unextracted sera from man and animals. A binding protein in serum that competes for 125I-MSA with receptor could not be completely separated from SmLRA by heating, acidification, charcoal treatment and gel chromatography of the serum. The relative activities of SmLRA and serum binding protein remained constant in three disorders of human growth (acromegaly, growth hormone deficiency and Laron's dwarfism) in which values of SmLRA varied widely. However, the binding protein is only partly responsible for the apparent SmLRA of unextracted serum. It is concluded that MSA is a suitable radioligand for the investigation of somatomedin disorders in man either by receptor assays or by studies of tissue receptors.     

Purification and properties of a membrane-bound insulin binding protein, a putative receptor, from Neurospora crassa.

The protein that is responsible for specific, high-affinity binding of insulin to the surface of Neurospora crassa cells has been purified to homogeneity. The insulin binding activity of solubilized plasma membranes resembled that of intact cells with regard to affinity of binding, specificity for mammalian insulins, and amount of insulin bound per cell. Insulin binding activity was purified from Triton X-100 solubilized membranes in two steps: FPLC on a MonoQ HR5/5 column; and affinity chromatography on insulin-agarose. The pure material migrated as a single band of ca. 66 kDa on SDS gels, pI = 7.4 by isoelectric focusing. The protein bound 5.34 pmol of insulin/micrograms, or 35% of that expected for univalent binding. Cross-linking of 125I-insulin to pure protein or to solubilized membranes revealed a single labeled band of 67-70 kDa on SDS gels. In nonreducing native gels, two labeled bands of ca. 55 and 110 kDa were produced after cross-linking, and two bands of similar molecular weight bound iodinated insulin after transfer to nitrocellulose filters. These may correspond to active monomer and dimer forms. The pure protein possessed no protein kinase activity against itself, or against exogenous substrates (histone H2, casein, or the synthetic peptide Glu80-Tyr20), and possessed no detectable phosphorylated amino acids. It is suggested, however, that this 66-kDa protein is the "receptor" that mediates insulin-induced downstream metabolic effects.