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M D Lane

Publications and source records attributed to M D Lane.

At least 109 records · Page 6Linked to original sources
PubMed1987

Post-translational acquisition of ligand binding- and tyrosine kinase-domain function by the epidermal growth factor and insulin receptors.

The epidermal growth factor receptor (EGFR) and insulin receptor undergo slow post-translational modification by which they acquire hormone binding and tyrosine kinase (EGFR) function. The half-time for acquisition of EGF or insulin binding activity is 30-40 min and of tyrosine kinase activity (EGFR), is 10-15 min. Tunicamycin, an inhibitor of N-linked oligosaccharide addition, blocks acquisition of both EGF and insulin binding activity. With EGFR, activation precedes acquisition of resistance to endoglucosaminidase H (t1/2 approximately equal to 75 min), a medial Golgi event. Treatment of active high mannose receptor with endo H generates fully active aglyco-receptor; thus, core oligosaccharide addition is a prerequisite for activation, but not for EGF binding per se. EGFR is activated in and translocated from the endoplasmic reticulum (ER) slowly (t1/2 approximately equal to 75 min). Since translocation rate equals the rate for acquisition of endo H resistance, transit from the ER is rate limiting for EGFR maturation. Tunicamycin inhibits exit from the ER parallel to its effect on acquisition of binding activity. Insulin proreceptor, a 210 kDa high-mannose glycopolypeptide, acquires insulin binding function (t1/2 approximately equal to 45 min) then is proteolytically cleaved (t1/2 approximately equal to 3 hr) into subunits of the mature alpha 2 beta 2 receptor. Modification giving rise to insulin binding activity is due to a conformational change in the binding domain, since human autoimmune antibody recognizes only the active species, while rabbit polyclonal antibody recognizes all forms. Newly-translated EGF proreceptor lacks a functional tyrosine domain capable of autophosphorylation; 30-40 min after translation, while still in the ER, tyrosine kinase activity is acquired. Since the kinase domain is cytoplasmic, the receptor may become phosphorylated on tyrosine before reaching the plasma membrane.

Adipose Tissue↗
PubMed1986

Synthesis of epidermal growth factor receptor in human A431 cells. Glycosylation-dependent acquisition of ligand binding activity occurs post-translationally in the endoplasmic reticulum.

It was previously demonstrated that the epidermal growth factor (EGF) receptor in human A431 cells undergoes a slow post-translational modification by which it acquires EGF binding capacity (Slieker, L.J., and Lane, M.D. (1985) J. Biol. Chem. 260, 687-690). In this report, the role of glycosylation in the acquisition of ligand binding activity and in the intracellular translocation of the receptor precursor is characterized. Human A431 cells were incubated with [35S]methionine, and 35S-labeled EGF receptors were purified either by immunoprecipitation (total receptor) or by adsorption to an EGF affinity matrix (high affinity, or active receptor). The half-time for receptor activation is approximately 30 min and precedes its acquisition of resistance to endo-beta-N-acetylglucosaminidase H (t 1/2 = 75 min), a medial Golgi event. Activation is blocked by tunicamycin and is markedly slowed (t 1/2 = 120 min) by 1-deoxynojirimycin, an inhibitor of glucosidase I. In the latter case, the oligosaccharide chains are not further processed to complex forms. Treatment of the active high mannose receptor with endo-beta-N-acetylglucosaminidase H generates a fully active aglycoreceptor polypeptide, indicating that core oligosaccharide addition is a prerequisite for activation but that oligosaccharide chains are not intrinsically required for EGF binding. Subcellular fractionation studies showed that the EGF receptor is activated in the endoplasmic reticulum and that translocation from that organelle is extremely slow (t 1/2 = 75 min). Since the latter translocation rate approximates that for the acquisition of the resistance to endoglycosidase H, transit from the endoplasmic reticulum appears to be rate-limiting for the maturation of the receptor. Both tunicamycin and 1-deoxynojirimycin inhibit exit from the endoplasmic reticulum in parallel with their effects on the acquisition of binding activity. Immunoprecipitation of 35S-labeled EGF receptor with antiphosphotyrosine antibody in the presence of ATP suggested that the autophosphorylation activity of the receptor is also acquired post-translationally. The possible correlation of this to EGF binding activity is discussed.

Cell Line↗
PubMed1986

Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes.

Insulin stimulates hexose transport and phosphorylation of the insulin receptor in monolayer cultures of intact 3T3-L1 adipocytes. To assess the phosphorylation state of the receptor in situ, cells were equilibrated with [32P]orthophosphate and then disrupted under denaturing conditions which preserved the phosphorylation state of the receptor established in the cell. The insulin receptor, isolated by lectin adsorption and two-dimensional nonreducing/reducing polyacrylamide gel electrophoresis, occurred as a single oligomeric species with an apparent alpha 2 beta 2 subunit composition. This oligomeric structure was not altered by treating cells with insulin. Only the beta-subunit of the receptor was phosphorylated; [32P]phosphoserine and [32P] phosphotyrosine were both identified in the beta-subunit from cells in the unstimulated state, but only [32P] phosphotyrosine increased in cells stimulated with insulin. Neither insulin-like growth factors I nor II stimulated insulin receptor beta-subunit phosphorylation, although both activated hexose transport. Upon the addition of insulin, [32P]orthophosphate incorporated into the beta-subunit increased 4.5-fold (7-fold with respect to [32P]tyrosine) and was complete within 1 min (t1/2 = 8 s). Following the removal of insulin from the monolayers, [32P]beta-subunit fell to the basal level (t1/2 = 2.5 min); there was no lag phase before either transition. The tyrosine protein kinase activity, measured in vitro with a model substrate, was higher with immunoaffinity-purified insulin receptor from insulin-stimulated cells than from cells in the basal state. Hexose transport rate, measured using 3-O-[methyl-14C]glucose, was half-maximally stimulated at 2 nM insulin. A 1-min latency period followed insulin addition, after which a 7-fold increase in the steady-state rate of hexose uptake was achieved within 5 min. Upon the removal of insulin, hexose transport continued at the stimulated steady-state rate for 2.5 min and then declined to the basal rate with a half-time of 8 min. These kinetic experiments in situ and protein kinase activity measurements in vitro support the hypothesis that beta-subunit phosphorylation is an intermediate step linking insulin binding to the increased glucose transport rate.

Adipose Tissue↗
PubMed1986

Insulin stimulates fluid-phase endocytosis and exocytosis in 3T3-L1 adipocytes.

Fluid phase endocytosis by monolayers of 3T3-L1 adipocytes has been followed by measuring [14C]sucrose uptake, a well characterized pinocytic marker. Insulin, at a maximal stimulatory concentration, increased the pinocytic rate by 2-fold within 5 min of its addition; this activation persisted for at least 2 h. The dose-response curve for the enhancement of fluid-phase endocytosis by insulin was identical with that for the stimulation of hexose transport, as measured by the uptake of 2-deoxyglucose. The concentration of insulin eliciting half-maximal effects was 6 nM. These results suggest that activation of endocytosis and hexose uptake by insulin are triggered by the same signalling event. Insulin-activated pinocytosis was not dependent upon the increased metabolism of D-glucose that occurs in response to the hormone, since the stimulation of fluid-phase endocytosis occurred in the absence of 5 nM glucose. Fluid-phase exocytosis was examined by loading cells with [14C]sucrose for various times and then measuring tracer efflux. The rate of sucrose release was biphasic; a portion of the internalized sucrose was rapidly released from the cell (t1/2 approximately 5 min), whereas the remainder was released slowly (t1/2 approximately to 5 h). These results are consistent with a sequential two-compartment model in which the [14C] sucrose first enters a compartment from which about 70% of the sucrose is rapidly released back into the medium and the remaining 30% is transferred to a second compartment. Therefore, the true rate of endocytosis is much greater than the observed accumulation rates, except after short uptake times. Insulin increases the rate of sucrose efflux from both compartments as well as the rate of transfer from the first compartment to the second compartment by about 2-fold. Furthermore, insulin increased the apparent size of the first and second compartments by 1.6- and 3-fold, respectively. The lysosomotropic agent chloroquine (200 muM) had only a small effect on fluid movements in these cells. The rapid and prolonged stimulation of fluid-phase endocytosis and exocytosis by insulin are hitherto unrecognized effects of this hormone.

Adipose Tissue↗
PubMed1986

Identification and partial purification of the insulin-responsive glucose transporter from 3T3-L1 adipocytes.

The glucose transporter in 3T3-L1 adipocytes has been identified as a polypeptide of average Mr 51000 by means of its reaction with antibodies raised against the purified human erythrocyte glucose transporter and by photolabeling with [3H]cytochalasin B. The finding that the antibodies immunoprecipitated the photolabeled polypeptide demonstrated that both methods detected the same polypeptide. The 3T3-L1 adipocyte glucose transporter has been partially purified. The main steps in the purification procedure were the preparation of salt-washed cellular membranes, Triton X-100 solubilization, and immunoaffinity chromatography on affinity-purified antibodies against the human erythrocyte transporter. A simple method of affinity purification of these antibodies, which consists of adsorption from serum onto protein-depleted erythrocyte membranes and release with acid, and an assay for the 3T3-L1 adipocyte transporter polypeptide, which employs immunoblotting, have been developed.

Adipose Tissue↗
PubMed1986

Kinetic evidence for activating and non-activating components of autophosphorylation of the insulin receptor protein kinase.

Reduced and carboxamidomethylated-lysozyme (RCAM-lysozyme) is an excellent substrate (Km = 13 microM) and a potent inhibitor of receptor autophosphorylation (Ki = 0.6 microM). By using these properties of RCAM-lysozyme autophosphorylation was resolved into two kinetically and functionally distinct components involving formation of phosphotyrosine on the receptor's beta-subunits: 1. Insulin-stimulated autophosphorylation is independent of autophosphorylation at other sites; activation of insulin receptor-catalyzed substrate phosphorylation is dependent upon this component of autophosphorylation, which is inhibited by RCAM-lysozyme. 2. Autophosphorylation at saturating RCAM-lysozyme concentration is insensitive to insulin and has little effect on substrate phosphorylation. Thus, only insulin-dependent receptor autophosphorylation is responsible for activation of kinase-catalyzed substrate phosphorylation.

Catalysis↗
PubMed1986

Actinomyces adsorption mediated by type-1 fimbriae.

Monospecific antibody against the type-1 fimbriae of Actinomyces viscosus T14V reacted with 12 strains of A. viscosus (serotype 2), 12 strains of A. naeslundii (serotype 2 or serotype 3), but not with 11 serotype 1 strains of A. naeslundii. All strains positive for the type-1 fimbrial antigen adsorbed strongly to saliva-treated hydroxyapatite, and in many instances (18 of 24 strains) this interaction was inhibited by the monospecific antibody. These findings strongly suggest that only those strains of actinomyces which possess type-1 fimbriae adhere to the tooth surfaces, and that these fimbriae constitute the principal adhesin involved in this interaction.

Actinomyces↗
PubMed1985

The effects of cycloheximide and chloroquine on insulin receptor metabolism. Differential effects on receptor recycling and inactivation and insulin degradation.

The effects of protein synthesis inhibitors and the lysosomotropic agent chloroquine on the metabolism of the insulin receptor were examined. Through the use of the heavy-isotope density shift technique, cycloheximide was found to inhibit both the synthesis of new insulin receptor and the inactivation of old cellular insulin receptor. Upon investigation of the locus of this effect of protein synthesis inhibition, it was found that cycloheximide did not inhibit 1) the translocation of receptor from the cell surface to an intracellular site, 2) the recycling of receptor from the internal site back to the plasma membrane, nor 3) the degradation of insulin. Cycloheximide did, however, rapidly and completely inhibit the inactivation of the insulin receptor. In the presence of extracellular insulin, this effect of cycloheximide resulted in the long-term (6 h) accumulation of receptor in a trypsin-resistant intracellular compartment. Puromycin and pactamycin, protein synthesis inhibitors with mechanisms of action which differ from cycloheximide, produced the same effects on insulin receptor metabolism as cycloheximide, indicating that this effect on receptor metabolism is due to the inhibition of protein synthesis and not a secondary effect of cycloheximide. Actinomycin D also inhibited the inactivation of receptor. Chloroquine inhibited the receptor-mediated degradation of insulin, but had no effect on either the internalization or inactivation of the insulin receptor. The insulin-induced recycling of the internalized receptor was inhibited by chloroquine, possibly through the inhibition of the discharge of insulin from the insulin-receptor complex. From these observations, we suggest that 1) a protein factor is required to inactivate the insulin receptor, 2) this protein and the messenger RNA coding for the protein have short cellular half-lives, and 3) insulin degradation and insulin receptor inactivation are distinct, separable processes which not only occur at different rates, but possibly occur in distinct subcellular locations.

Animals↗
PubMed1985

Tissue specific expression of p422 protein, a putative lipid carrier, in mouse adipocytes.

The differentiation of 3T3-L1 preadipocytes leads to the expression of a new protein, p422, and its mRNA. This protein has 70% and 20-30% amino acid sequence homology to myelin P2 and the fatty acid binding proteins of liver and intestine, respectively. Investigation of the distribution in mouse tissues of p422 protein by immunoblotting and of p422 mRNA by cDNA hybridization indicates that they are expressed only in adipose tissue. Liver and intestinal fatty acid binding protein mRNA's were not detectable in mouse adipose tissue or in 3T3-L1 adipocytes. It is suggested that p422 functions as an adipocyte fatty acid binding protein.

Adipose Tissue↗
PubMed1985

Evidence for an increase in transcription of specific mRNAs during differentiation of 3T3-L1 preadipocytes.

Differentiation of 3T3-L1 preadipocytes in culture is accompanied by alterations in the abundance of several mRNAs and by the appearance of many new adipocyte-specific mRNAs. To investigate the processes responsible for these alterations, the kinetics of accumulation of several specific mRNAs were compared with their respective rates of nuclear runoff transcription. The mRNAs for fructose-1,6-bisphosphate aldolase and an unidentified 4800-base mRNA increase in abundance only moderately (2-4-fold) during differentiation. Runoff transcription by nuclei isolated from 3T3-L1 cells during the course of differentiation revealed very little or no change in the rates of transcription of these mRNAs. Similar results were obtained for the beta, alpha-actin and beta-tubulin mRNAs where no difference in nuclear runoff transcription rates were observed even though a 2-fold decrease in the steady-state levels of these mRNAs accompanies differentiation. In contrast, the steady-state levels of mRNAs for 3T3-L1 P2 protein, an adipocyte homologue of myelin P2 protein, and an unidentified 5000-base mRNA increased dramatically (greater than 20-fold) during adipose conversion. These large increases in abundance were correlated with marked rises (greater than 10-fold) in nuclear runoff transcription rates for these mRNAs during differentiation of 3T3-L1 preadipocytes. No change in runoff transcription activity for these mRNAs was detected by nuclei from control nondifferentiating 3T3-C2 cells. These results strongly suggest that an increased rate of specific transcription is primarily responsible for the accumulation of these mRNAs during preadipocyte differentiation.

Adipose Tissue↗
PubMed1985

Homogeneous functional insulin receptor from 3T3-L1 adipocytes. Purification using N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin and avidin-sepharose.

Insulin receptor was purified 10,000-fold from cultured mouse 3T3-L1 adipocytes in 35% overall yield. The specific activities of 125I-insulin binding and autophosphorylation increased in parallel, following the initial Triton X-100 extraction of membranes. The isolation protocol, performed entirely at pH 8.45, entailed adsorption by avidin-Sepharose CL-4B of a complex formed between Triton X-100-solubilized insulin receptor and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin, and the specific elution of the complex with biotin. The avidin-Sepharose CL-4B was a partially denatured preparation, showing estimated dissociation constants of 0.2 microM for biotin and approximately 1 microM for the bifunctional ligand at, pH 7, 4 degrees C. The bifunctional ligand was characterized by 70% competency in binding to avidin, 100% competency in binding to solubilized insulin receptor, full stimulation of autophosphorylation of the isolated receptor, and maximal stimulation of hexose uptake by intact 3T3-L1 adipocytes. The insulin binding properties of the insulin receptor were uniform throughout this purification procedure. At pH 8.45, 4 degrees C, an average Kd = 0.72 nM was determined for a single class of noninteracting insulin binding sites. The apparent autophosphorylation of the beta-subunit was also unchanged following affinity chromatography. A single oligomeric structure was established for the purified receptor, composed only of 135,000- and 95,000-Da subunits, whose association was lost by denaturation in the presence of reducing agent. This single structure occurred in the initial Triton X-100 extract. The purified insulin receptor was capable of autophosphorylating the beta-subunit and catalyzed phosphorylation of protein substrates.

Adipose Tissue↗
PubMed1985

Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes.

Following the differentiation of 3T3-L1 preadipocytes insulin acutely activates the rate of 2-deoxy-[1-14C]glucose uptake in the mature 3T3-L1 adipocyte by 15- to 20-fold. Phenylarsine oxide, a trivalent arsenical that forms stable ring complexes with vicinal dithiols, prevents insulin-activated hexose uptake in a concentration-dependent manner (Ki = 7 microM) but has no inhibitory effect on basal hexose uptake. 2,3-Dimercaptopropanol at a level nearly stoichiometric to that of phenylarsine oxide prevents or rapidly reverses the inhibition of hexose uptake; 2-mercaptoethanol, even in high stoichiometric excess over the arsenical, does not reverse inhibition of hexose uptake. When phenylarsine oxide is added after adipocytes have been fully activated by insulin, 2-deoxy-[1-14C]glucose uptake rate decays slowly at a rate corresponding to that caused by the withdrawal of insulin (t1/2 = 10 min). Using the same conditions under which phenylarsine oxide blocked activation, the Km for deoxyglucose uptake, the rate at which 125I-insulin became cell-associated, and the 125I-insulin binding isotherm for solubilized insulin receptor were not affected by phenylarsine oxide. These results support the transporter translocation model for insulin-activated hexose transport and implicate vicinal sulfhydryl groups in a post-insulin binding event essential for the translocation of glucose transporters to the plasma membrane.

3-O-Methylglucose↗
PubMed1985

Post-translational processing of the epidermal growth factor receptor. Glycosylation-dependent acquisition of ligand-binding capacity.

The post-translational processing of the epidermal growth factor receptor in human A431 epidermoid carcinoma cells has been investigated. By employing the affinity matrix epidermal growth factor Affi-Gel in conjunction with immunoprecipitation, it has been demonstrated that core oligosaccharide addition is essential for the acquisition of epidermal growth factor-binding activity. Furthermore, the initial 160-kDa translation product was observed to undergo a processing step by which ligand-binding activity was acquired with a half-time of approximately 30 min while exhibiting no apparent change in mobility on sodium dodecyl sulfate-polyacrylamide gels. This was shown not to involve the conversion of high-mannose chains to complex chains which have been capped with fucose and sialic acid. Possible explanations for this activation in terms of translocation of intermediates and/or formation of disulfide bonds are discussed.

Carbohydrate Metabolism↗
PubMed1985

Posttranslational processing of the insulin proreceptor.

Following translation of the insulin proreceptor by 3T3-L1 adipocytes, about 1.5 hours is required for its conversion into active receptor; an additional 1.5 hours is needed for the active receptor to reach the plasma membrane. During this 3-hour period the proreceptor undergoes a complex series of processing events, glycosylation being an essential processing step. Thus, treatment of 3T3-L1 adipocytes with tunicamycin causes the depletion of cellular insulin binding activity and the accumulation of an inactive aglyco proreceptor. To investigate posttranslational processing of normal proreceptor and the role of glycosylation in active receptor formation, metabolic labeling experiments were conducted. The first 35S-labeled intermediate detected is a 190-kDa polypeptide (proreceptor) which is rapidly (t1/2 = 15 minutes) processed into a 210-kDa species. Both polypeptides contain N-linked core oligosaccharide chains, but in the latter case these chains appear to contain terminal N-acetylglucosamine. The 210-kDa precursor is converted slowly (t1/2 = 2 hours) by proteolytic processing into a 125-kDa (alpha') and 83-kDa (beta') species. Immediately prior to insertion into the plasma membrane, 3 hours after its synthesis, the alpha' and beta' precursors are converted to mature receptor composed of alpha (135 kDa) and beta (95 kDa) subunits. The 125-kDa alpha' and 83-kDa beta' precursors are endoglycosidase H-sensitive and their oligosaccharide chains do not contain terminal sialic acid. Just prior to insertion into the plasma membrane the alpha' and beta' precursors are sialylated, giving rise to the 135-kDa alpha and 95-kDa beta receptor subunits and becoming Endo H resistant and neuraminidase sensitive. In the presence of tunicamycin, a 180-kDa aglyco receptor polypeptide accumulates which is not further processed and does not reach the cell surface. It is concluded that N-linked oligosaccharide chains on the proreceptor are required either for its intracellular translocation to the proteolytic cleavage site or for its identification as a target of the cleavage enzyme. Thus, glycosylation of the insulin proreceptor is crucial for proper processing and formation of functional receptor.

Cell Membrane↗
PubMed1985

Kinetics of citrate-induced activation and polymerization of chick liver acetyl-CoA carboxylase.

The kinetic time course of citrate-induced activation and polymerization (into filaments) of the protomeric form of acetyl-CoA carboxylase were compared to assess the concertedness of the two processes. Rapid-quench techniques were employed to measure the kinetics of activation of the carboxylase-catalyzed reaction by citrate. When enzyme was preincubated with citrate prior to initiating the steady state turnover reaction with acetyl-CoA in the rapid-quench device, the observed rate of carboxylation of acetyl-CoA was apparently linear from the moment of mixing. However, when enzyme was mixed with citrate to initiate the reaction, a lag (t1/2 = 0.7 s) occurred in the approach to steady state carboxylation rate. This lag was independent of enzyme concentration over a 230-fold range and was marginally dependent upon citrate concentration. Over the same range of enzyme concentration, polymerization of carboxylase protomers, as determined by right angle light scattering, was enzyme concentration-dependent in a manner predicted by a single protomer activation step, followed by a rate-limiting dimerization of active protomer and subsequent polymerization. Polymerization is a second order process, with a second order rate constant of 597,000 M-1 s-1. There appear to be two steps that limit polymerization of the inactive carboxylase protomer: a rapid citrate-induced conformational change, which is independent of enzyme concentration and leads to an active protomeric form of the enzyme and the dimerization of the active protomer, which constitutes the first step of polymerization and is enzyme concentration-dependent. Dimerization is the rate-limiting step of acetyl-CoA carboxylase polymerization. On the basis these results, it is concluded that activation of catalysis and the polymerization of carboxylase protomers are not concerted. Furthermore, activation of carboxylation leading to the formation of an active protomer was faster than polymerization under all conditions, and therefore precedes polymerization. It was also shown that the activation constant (Kact) for citrate is altered in a predictable manner by the accumulation of the reaction product, malonyl-CoA, the Kact increasing with malonyl-CoA concentration. Depolymerization of fully polymerized acetyl-CoA carboxylase is caused by malonyl-CoA or ATP.Mg (and HCO3-). Both malonyl-CoA and ATP.Mg (and HCO3-) compete with citrate in the maintenance of a given state of the protomer-polymer equilibrium apparently by carboxylating the enzyme to form enzyme-biotin CO2- which destabilizes the polymeric form.

Acetyl-CoA Carboxylase↗