Biogenesis, transit, and functional properties of the insulin proreceptor and modified insulin receptors in 3T3-L1 adipocytes. Use of monensin to probe proreceptor cleavage and generate altered receptor subunits

The biogenesis, intracellular transport, and functional properties of the insulin proreceptor and modified insulin receptors were studied in hormone-responsive 3T3-L1 adipocytes. After control cells were labeled with [35S]Met for 7 min, the principal polypeptide that was precipitated by anti-insulin receptor antibodies had a molecular weight (Mr) of 180 000. This initial precursor was rapidly converted (t1/2 = 35 min) to a 200-kilodalton (kDa) polypeptide, designated the insulin proreceptor, by the apparent posttranslational addition of N-linked, high mannose core oligosaccharide units. Mature alpha (Mr 130 000) and beta (Mr 90 000) subunits were derived from sequences within the proreceptor by proteolytic cleavage and late processing steps, and these subunits appeared on the cell surface 2-3 h after synthesis of the 180-kDa precursor. The cation ionophore monensin was used in combination with metabolic labeling, affinity cross-linking, and external proteolysis to probe aspects of proreceptor function, transit, and the development of insulin sensitivity at the target cell surface. At 5 micrograms/mL, monensin potently inhibited the proteolytic cleavage step, and the 200-kDa polypeptide accumulated. Lower concentrations of the ionophore selectively blocked late processing steps in 3T3-L1 adipocytes so that apparently smaller alpha' (Mr 120 000) and beta' (Mr 85 000) subunits were produced. Proreceptor and alpha' and beta' subunits were translocated to the cell surface, indicating that the signal for intracellular transit occurs in the 200-kDa polypeptide and is independent of the posttranslational proteolysis and late processing steps. The alpha' subunit bound insulin both at the surface of intact cells and after solubilization with Triton X-100; the beta' subunit was phosphorylated in an insulin-stimulated manner. The detergent-solubilized 200-kDa proreceptor also exhibited both functional properties. However, the proreceptor that was transported to and exposed on the cell surface was incapable of binding insulin in intact adipocytes. Thus, late processing is not essential for the expression of functions associated with mature alpha and beta subunits. In contrast, it appears that the proteolytic generation of subunits is required for the correct orientation of the hormone binding site in the plasma membrane bilayer and the development of insulin responsiveness in 3T3-L1 adipocytes.     

Role of glycosylation in the processing of newly translated insulin proreceptor in 3T3-L1 adipocytes

A procedure was developed for the immunoprecipitation of glycosylated and nonglycosylated forms of the insulin receptor and its precursors without prior purification using lectins. 3T3-L1 adipocytes were labeled with [35S]methionine after which 35S-labeled receptor polypeptides were specifically immunoprecipitated and characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The first 35S-polypeptide detected was a 190-kDa glycosylated proreceptor which was rapidly (t1/2 approximately equal to 15 min) processed to a 210-kDa intermediate. The latter precursor was more slowly (t1/2 approximately equal to 2 h) proteolytically processed to 125-kDa (alpha') and 83-kDa (beta') precursors of the mature alpha- and beta-receptor subunits. Immediately prior to insertion into the plasma membrane, i.e. about 3 h after translation, the alpha'- and beta'-precursor polypeptides were converted to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits. The characteristics of the oligosaccharide moieties of the receptor precursors and products were investigated. The 210-kDa precursor and its two products, the 125-kDa alpha'- and 83-kDa beta'-species, and the mature alpha- and beta-receptor subunits bind tightly to wheat germ lectin, whereas the 190-kDa proreceptor species is not bound. Upon incubation with endoglycosidase H, both the 210- and 190-kDa species are converted to a 180-kDa species. The 125-kDa alpha'- and 83-kDa beta'-species are also cleaved by endoglycosidase H, being reduced in size to 97 and 79 kDa, respectively. Based on their sensitivity to endoglycosidase H and insensitivity to neuraminidase, the oligosaccharide chains of the receptor precursors (190, 210, 125, and 83 kDa) do not contain terminal sialic acid (or other capping sugars). However, near the time of insertion into the plasma membrane, capping of the alpha'- and beta'-species by sialic acid occurs, giving rise to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits, which are partially endoglycosidase H-resistant and neuraminidase-sensitive. When 3T3-L1 adipocytes are treated with tunicamycin, a 180-kDa proreceptor aglycopolypeptide is synthesized which is incapable of undergoing further processing and proteolytic cleavage to the alpha- and beta (or alpha'- and beta'-)-subunits. The 180-kDa species, which appears to be the aglyco-form of hte 190-kDa proreceptor generated by endoglycosidase H, is resistant to trypsin in the intact cell and apparently has not reached the cell surface. Thus, the oligosaccharide moieties of the insulin receptor precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin re     

Post-translational acquisition of insulin binding activity by the insulin proreceptor. Correlation to recognition by autoimmune antibody

The post-translational acquisition of ligand binding activity by the insulin receptor was examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S] methionine, labeled receptor species were separated into "active" and "inactive" forms by affinity chromatography on insulin-agarose and then were characterized and quantitated. It was found that the newly translated high molecular weight proreceptor lacks the capacity to bind insulin. The acquisition of binding activity is relatively slow (t1/2 = 45 min) and occurs prior to conversion of the proreceptor to the mature alpha- and beta-subunits by proteolytic cleavage and maturation of its N-linked oligosaccharide chains (t1/2 = 3 h). Glycosylation appears to be required for this activation since the aglycoproreceptor, synthesized in the presence of tunicamycin, does not acquire insulin binding activity. However, once the proreceptor has acquired ligand binding activity, removal of its N-linked oligosaccharide chains with endoglycosidase H has no effect on the ability of the proreceptor to bind insulin. The modification of the proreceptor to bind insulin. The modification of the proreceptor that gives rise to insulin binding activity most likely involves a conformational change in the binding domain. A human autoimmune antibody that recognizes only the active insulin binding site does not interact with the inactive proreceptor, whereas a rabbit polyclonal antireceptor antibody recognizes all forms. Thus, the autoimmune antibody must recognize a new epitope created during conversion of the inactive proreceptor to the active form.     

Effect of alternative glycosylation on insulin receptor processing.

The mature insulin receptor is a cell surface heterotetrameric glycoprotein composed of two alpha- and two beta-subunits. In 3T3-L1 adipocytes as in other cell types, the receptor is synthesized as a single polypeptide consisting of uncleaved alpha- and beta-subunits, migrating as a 190-kDa glycoprotein. To examine the importance of N-linked glycosylation on insulin receptor processing, we have used glucose deprivation as a tool to alter protein glycosylation. Western blot analysis shows that glucose deprivation led to a time-dependent accumulation of an alternative proreceptor of 170 kDa in a subcellular fraction consistent with endoplasmic reticulum localization. Co-precipitation assays provide evidence that the alternative proreceptor bound GRP78, an endoplasmic reticulum molecular chaperone. N-Glycosidase F treatment shows that the alternative proreceptor contained N-linked oligosaccharides. Yet, endoglycosidase H insensitivity indicates an aberrant oligosaccharide structure. Using pulse-chase methodology, we show that the synthetic rate was similar between the normal and alternative proreceptor. However, the normal proreceptor was processed into alpha- and beta-subunits (t((1)/(2)) = 1.3 +/- 0.6 h), while the alternative proreceptor was degraded (t((1)/(2)) = 5.1 +/- 0.6 h). Upon refeeding cells that were initially deprived of glucose, the alternative proreceptor was processed to a higher molecular weight form and gained sensitivity to endoglycosidase H. This "intermediate" form of the proreceptor was also degraded, although a small fraction escaped degradation, resulting in cleavage to the alpha- and beta-subunits. These data provide evidence for the first time that glucose deprivation leads to the accumulation of an alternative proreceptor, which can be post-translationally glycosylated with the readdition of glucose inducing both accelerated degradation and maturation.     

Post-translational changes in tertiary and quaternary structure of the insulin proreceptor. Correlation with acquisition of function

Tertiary and quaternary structural changes that occur during post-translational processing of the insulin proreceptor were examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S]methionine, labeled insulin receptor species, isolated by immuno- and insulin-affinity adsorption, were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under conditions where intra- and intermolecular disulfide bonds remained intact or were cleaved by reduction. Reducing SDS-polyacrylamide gel electrophoresis confirmed that the insulin receptor is synthesized as a long-lived (t1/2 = 3 h) proreceptor precursor of 210 kDa which undergoes proteolytic cleavage and carbohydrate maturation to form the alpha- and beta-subunits of the mature receptor. The proreceptor acquires insulin binding activity through a subtle structural change (t1/2 = 45 min) detected only by an autoimmune antibody specific for an epitope of the active insulin binding site. Analysis of insulin receptor species by nonreducing SDS-polyacrylamide gel electrophoresis revealed that the proreceptor undergoes two additional structural changes not detected by reducing SDS-polyacrylamide gel electrophoresis. The proreceptor is synthesized as a monomer (M1) with an apparent molecular mass of 170 kDa that is converted by disulfide rearrangement to another monomeric form of 190-kDa apparent molecular mass (M2). N-Linked glycosylation is required for this transition, since aglycoproreceptor, synthesized in the presence of tunicamycin, does not undergo any detectable tertiary or quaternary structural changes. M2 self-associates to form a disulfide-linked proreceptor dimer (D) which is subsequently proteolytically processed, forming the mature, disulfide-linked alpha 2 beta 2 receptor tetramer. The mature receptor was distinguished from the three proreceptor species (M1, M2, and D) by its cell surface location and its ability to bind tightly to wheat germ agglutinin-agarose, indicating the presence of complex oligosaccharide chains. Subcellular fractionation indicated that both the M1 to M2 and M2 to D conversions occur in the endoplasmic reticulum. Separation of the nonreduced proreceptor species into "active" and "inactive" forms by affinity chromatography on insulin-agarose revealed that neither the transition of M1 to M2, nor of M2 to D, is correlated with the acquisition of insulin binding function. Rather, during its life-time, the M2 species acquires insulin binding activity and an epitope recognized by a binding site specific autoimmune antibody through a subtle structural change not detected by reducing or nonreducing SDS-polyacrylamide gel electrophoresis.     

Post-Translational Acquisition of Ligand Binding-and Tyrosine Kinase-Domain Function by the Epidermal Growth Factor and Insulin Receptors

Abstract

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 endoglucos-aminidase H (t_1/2 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 (t_1/2 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 (t_1/2 45 min) then is proteolytically cleaved (t_1/2 3 hr) into subunits of the mature α₂β₂ 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     

Substitution of arginine for histidine at position 209 in the alpha-subunit of the human insulin receptor. A mutation that impairs receptor dimerization and transport of receptors to the cell surface.

Receptors for insulin and epidermal growth factor contain cysteine-rich domains in the extracellular portion of the molecule. His209 (insulin receptor numbering system) is 1 of 2 amino acid residues that are identically conserved in the cysteine-rich domains of insulin receptors, epidermal growth factor receptors, and other homologous receptors. Previously, we have identified a patient with leprechaunism who is homozygous for a mutation substituting Arg for His209 in the insulin receptor gene (Kadowaki, T., Kadowaki, H., Rechler, M. M., Serrano-Rios, M., Roth, J., Gorden, P., and Taylor, S. I. (1990) J. Clin. Invest. 86, 254-264). In this investigation, the Arg209 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. The mutation impairs several steps in the post-translational processing of the insulin receptor:dimerization of 190-kDa proreceptors into a disulfide linked species, proteolytic cleavage of the proreceptor into alpha- and beta-subunits, and terminal processing of the high mannose form of N-linked oligosaccharide into complex carbohydrate. In addition, the defects in post-translational processing within the endoplasmic reticulum and Golgi apparatus are associated with a marked inhibition in transport of receptors to the plasma membrane. Nevertheless, a small number (approximately 10%) of the receptors are transported to the cell surface. These receptors on the cell surface bind insulin with normal affinity and have normal tyrosine kinase activity.     

Alternative glycosylation of the insulin receptor prevents oligomerization and acquisition of insulin-dependent tyrosine kinase activity

Glucose deprivation leads to the synthesis of an aberrantly glycosylated ('alternative') and inefficiently processed form of the insulin proreceptor in 3T3-L1 adipocytes. To further explore the effect of aberrant (rather than absent) N-linked glycosylation of the insulin receptor, we examined the relationship of processing to function. Our studies show that the alternative form of the proreceptor does not oligomerize nor does it acquire the ability to undergo insulin-sensitive autophosphorylation. This along with an interaction with the glucose-regulated stress protein GRP78/BiP implies inappropriate folding/dimerization and retention in the ER. Glucose refeeding causes the post-translational modification of the alternative form of the proreceptor to a novel 'intermediate' form which is independent of new protein synthesis. As little as 100 microM glucose (or mannose) can induce this modification. In vitro digestion of the alternative and intermediate proreceptors with SPC1/furin shows that both the alpha- and beta-subunit domains are glycosylated, albeit aberrantly. This implies that the aberrantly glycosylated proreceptor could serve as a substrate for SPC1 in a physiological setting if the receptor was able to interact with the enzyme in the appropriate compartment (i.e., the trans-Golgi network). Based on inhibitor studies, however, both the alternative and intermediate forms of the proreceptor appear to be primarily targeted to the proteasome for degradation.     

Similarities in glycosylation and transport between the secreted and plasma membrane forms of the epidermal growth factor receptor in A-431 cells

We have studied the synthesis and oligosaccharide processing of the 110,000 dalton form of the epidermal growth factor (EGF) receptor that is secreted into the medium of A-431 cells. Its 90,000 dalton precursor is soluble within the lumen of intracellular membrane vesicles shortly after synthesis, indicating that it lacks a membrane anchor. Analysis of labeled glycopeptides reveals that the glycosylation of the 110,000 dalton, secreted receptor is very similar to that of the 170,000 dalton, plasma membrane receptor. Based on Concanavalin A-Sepharose elution profiles of its glycopeptides, the secreted receptor has both complex and high-mannose N-linked oligosaccharides. Also, like the plasma membrane receptor, the secreted receptor contains N-acetylgalactosamine residues in its complex chains. Not only are major features of oligosaccharide processing of the soluble and membrane-bound forms of the receptor similar, but the kinetics of transport to the cell exterior is the same for each. These data indicate that the glycosylation pattern and kinetics of cellular transport of the EGF receptor are determined by factors other than the sequence of its cytoplasmic and transmembrane domains.     

Effects of castanospermine and 1-deoxynojirimycin on insulin receptor biogenesis. Evidence for a role of glucose removal from core oligosaccharides

The insulin proreceptor is a 190-kDa glycoprotein that is processed to mature alpha (135-kDa) and beta (95-kDa) subunits. In order to determine the role of carbohydrate chain processing in insulin receptor biogenesis, we investigated the effect of inhibiting glucose removal from core oligosaccharides of the insulin proreceptor with glucosidase inhibitors, castanospermine and 1-deoxynojirimycin. Cultured IM-9 lymphocytes treated with inhibitors had 50% reduction in surface insulin receptors as demonstrated by ligand binding, affinity cross-linking with 125I-insulin, and lactoperoxidase/Na 125I labeling studies. Degradation rates of surface labeled receptors were similar in both control and inhibitor-treated cells (t1/2 = 5 h); thus, accelerated receptor degradation could not account for this reduction. Biosynthetic labeling experiments with [3H]leucine and [3H]mannose identified an apparently higher molecular size proreceptor (approximately 205 kDa) that failed to show the characteristic decline with time as seen in the normal 190-kDa proreceptor. Along with this finding, the biosynthetic label appearing in the mature subunits was reduced in these inhibitor-treated cells. Endoglycosidase H treatment of both precursors produced identical 170-kDa bands. Carbohydrate chains released from the 205-kDa precursor by endoglycosidase H migrated in the same position as the Glc2-3Man9GlcNAc standards when separated by high performance liquid chromatography, whereas the 190-kDa proreceptor oligosaccharides migrated similar to the Man7-9GlcNAc chains. Although the mature subunits of control and inhibitor-treated cells demonstrated equal electrophoretic mobility, the endoglycosidase H-sensitive oligosaccharides of the mature subunits in treated cells also contained residues that migrated similar to the Glc2-3Man9GlcNAc standards. Thus, glucose removal from core oligosaccharides is apparently not necessary for the cleavage of the insulin proreceptor, but does delay processing of this precursor, which probably accounts for the reduction in cell-surface receptors.     

Evidence for exogenous substrate phosphorylation and dimer formation by the activated 190 kDa insulin proreceptor in vitro

The insulin receptor is synthesized as a single chain, 190 kDa glycoprotein precursor, which undergoes proteolytic cleavage, carbohydrate processing, and fatty acylation to generate the mature receptor on the plasma membrane. The relationship of these post-translational modifications to the acquisition of receptor function, i.e. ligand binding and phosphokinase activity, is not fully understood. Therefore, the 190 kDa proreceptor and mature receptor kinase activities were separately examined in vitro, and their phosphorylation properties compared. The solubilized receptor precursor from IM-9 lymphocytes was purified by sequential lectin chromatography and, following site specific anti-receptor antibody immunoprecipitation, phosphokinase studies performed. The isolated proreceptor was activated by insulin and phosphorylated exogenous substrate alpha-casein, as similarly observed for the mature receptor. Structurally, the phosphorylated proreceptor was identified as a 360 kDa homodimer under non-reducing condition.     

Biosynthetic and glycosylation studies of cell surface platelet-derived growth factor receptors

The cell surface pool of metabolically labeled platelet-derived growth factor (PDGF) receptors in BALB/c3T3 fibroblasts was studied using an antiphosphotyrosine antibody. Exposure of intact cells to PDGF stimulates autophosphorylation of surface PDGF receptors and allowed immunoaffinity purification of only PDGF-activated receptors. Pulse-chase experiments demonstrated appearance of newly synthesized receptors in a surface activatable pool within 30-45 min of synthesis. In the absence of exogenous PDGF, the apparent half-life of this pool was 2 h. The presence of both N- and O-linked oligosaccharide chains on cell surface PDGF receptors was demonstrated. Enzymatic removal of the N-linked oligosaccharide chains reduced the receptor's apparent Mr by approximately 40 kDa and removal of O-linked oligosaccharide caused approximately a 7-kDa reduction. Activation of receptor tyrosine autophosphorylation by PDGF did not require either processing of high-mannose N-linked oligosaccharides to complex forms or the presence of sialic acid on receptor oligosaccharide chains. Tryptic cleavage of PDGF-activated surface receptors in intact cells yielded two discrete phosphotyrosine-containing fragments of 107 and 85 kDa. Cleveland digest patterns from each fragment indicate that both are derived from the intact PDGF receptor. These data indicate that PDGF receptors are synthesized and turn over rapidly in the absence of ligand. Partial characterization of the extracellular domain oligosaccharide contribution to receptor function and trypsin susceptibility is provided.     

Post-translational protein modification and expression of ankyrin-binding site(s) in GP85 (Pgp-1/CD44) and its biosynthetic precursors during T-lymphoma membrane biosynthesis.

In this study, we have investigated the biosynthesis and processing of GP85 (Pgp-1/CD44), a lymphoma transmembrane glycoprotein known to contain ankyrin-binding site(s). Using a standard pulse-chase protocol, we have detected a 52-kDa polypeptide precursor (p52) within the first 5 min of pulse labeling which contains a high mannose-type N-linked oligosaccharide chains. The conversion of p52 to GP85 requires further glycosylation (both complex type N-linked and O-linked) which takes place in the Golgi complex within 10-20 min after p52 is synthesized. GP85 is then incorporated into the plasma membrane where its turnover rate is relatively slow, a t1/2 of approximately 8 h. Following tunicamycin treatment, we have detected two other precursor proteins: p42 which is unglycosylated and p58 which is O-glycosylated. p42 appears to be an immediate precursor of p52 because p52 is converted to p42 upon deglycosylation. Therefore, the biosynthesis of GP85 appears to occur in the following sequence: p42 in equilibrium to p52 in equilibrium to GP85. Further analysis reveals that all of the GP85 precursors (i.e. p42, p52, and p58) contain ankyrin-binding site(s). Chemical composition analysis of GP85 indicates that this molecule contains approximately 3 N-linked and 4-5 O-linked oligosaccharide chains. Although neither N-glycosylation nor O-glycosylation appears to play an important role in the formation of ankyrin-binding site(s), O-glycosylation (and to a lesser extent N-glycosylation) of GP85 is required for T-lymphoma cell surface interaction with both collagen and hyaluronic acid. These findings suggest that GP85 (Pgp-1/CD44) and its biosynthetic precursors play a pivotal role in regulating adhesion functions such as lymphocyte homing and binding to the extracellular matrix.     

Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function

A-431 cells were treated with inhibitors of either N-linked glycosylation (tunicamycin or glucosamine) or of N-linked oligosaccharide processing (swainsonine or monensin) to examine the glycosylation of epidermal growth factor (EGF) receptors and to determine the effect of glycosylation modification on receptor function. The receptor was found to be an Mr = 130,000 polypeptide to which a relatively large amount of carbohydrate is added co-translationally in the form of N-linked oligosaccharides. Processing of these oligosaccharides accounts for the 10,000-dalton difference in electrophoretic migration between the Mr = 160,000 precursor and Mr = 170,000 mature forms of the receptor. No evidence was found for O-linked oligosaccharides on the receptor. Mr = 160,000 receptors resulting from swainsonine or monensin treatment were present on the cell surface and retained full function, as judged by 125I-EGF binding to intact cells and detergent-solubilized extracts and by in vitro phosphorylation in the absence or presence of EGF. On the other hand, when cells were treated with tunicamycin or glucosamine, ligand binding was reduced by more than 50% in either intact cells or solubilized cell extracts. The Mr = 130,000 receptors synthesized in the presence of these inhibitors were not found on the cell surface. In addition, no Mr = 130,000 phosphoprotein was detected in the in vitro phosphorylation of tunicamycin or glucosamine-treated cells. It appears, therefore, that although terminal processing of N-linked oligosaccharides is not necessary for proper translocation or function of the EGF receptor, the addition of N-linked oligosaccharides is required.     

N-linked oligosaccharide chains of the insulin receptor beta subunit are essential for transmembrane signaling.

Insulin receptor (IR) is a glycoprotein possessing N-linked oligosaccharide side chains on both alpha and beta subunits. The present study focuses for the first time on the potential contribution of N-linked oligosaccharides of the beta subunit in the processing, structure, and function of the insulin receptor. To investigate this point, a receptor mutant (IR beta N1234) was obtained by stable transfection into Chinese hamster ovary cells of an IR cDNA modified by site-directed mutagenesis on the four potential N-glycosylation sites (Asn-X-Ser/Thr) of the beta subunit. The mutated receptor presents an alpha subunit of 135 kDa, indistinguishable from the wild type alpha subunit, but the beta subunit has a reduced molecular mass (80 kDa instead of 95 kDa) most likely due to the absence of N-glycosylation. Metabolic labeling experiments indicate a normal processing and maturation of this mutated receptor which is normally expressed at the surface of the cells as demonstrated by indirect immunofluorescence. The affinity of the mutant for insulin (Kd = 0.12 nM) is similar to that of the wild type receptor (Kd = 0.12 nM). However, a major defect of the mutated IR tyrosine kinase was assessed both in vitro and in vivo by (i) the absence of insulin-stimulated phosphorylation of the poly(Glu-Tyr) substrate in vitro; (ii) the reduction of the insulin maximal stimulation of the mutated IR autophosphorylation in vitro (2-fold stimulation for the mutant receptor as compared to a 7-fold stimulation for the wild type); and (iii) a more complex alteration of the mutated receptor tyrosine autophosphorylation in vivo (3-fold increase of the basal phosphorylation and a 4-fold simulation of this phosphorylation as compared to the wild type receptor, the phosphorylation of which is stimulated 14-fold by insulin). The physiological consequences of this defect were tested on three classical insulin cellular actions; in Chinese hamster ovary IR beta N1234, glucose transport, glycogen synthesis, and DNA synthesis were all unable to be stimulated by insulin indicating the absence of insulin transduction through this mutated receptor. These data provide the first direct evidence for a critical role of oligosaccharide side chains of the beta subunit in the molecular events responsible for the IR enzymatic activation and signal transduction.     

Asparagine-linked oligosaccharides on formyl peptide chemotactic receptors of human phagocytic cells

Formyl peptide chemotactic receptors affinity-labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (where Nle represents norleucine) and ethylene glycol bis(succinimidyl succinate) consist of two isoelectric forms with cell type differences in both apparent size and charge (neutrophils: 55-70 kDa, pI 5.8, and 6.2.; monocytes: 60-75 kDa, pI 5.6 and 6.0; differentiated HL-60 cells: 62-85 kDa, pI 5.6 and 6.0). Endo-beta-N-acetylglucosaminidase F (endo F) cleavage of N-linked oligosaccharides from formyl peptide receptor generates 40-50- and 33-kDa products that can be affinity-labeled. Whereas both pI forms of this receptor from neutrophils are cleaved by endo F to 33-kDa final products, this cleavage does not eliminate pI differences. Tunicamycin decreases expression of formyl peptide receptor on differentiating HL-60 and causes a dose-dependent decrease in size of the major product seen after affinity labeling (0.5 micrograms/ml: 38-48 kDa; 2 micrograms/ml: 32 kDa). Thus, the formyl peptide receptor polypeptide backbone from all three cell types contains at least two N-linked oligosaccharide side chains which contribute to the cell type differences in Mr and are not required for ligand binding. Papain treatment of intact cells generates a membrane-bound formyl peptide receptor fragment that can be affinity-labeled and is of similar size (29-31 kDa) in all three cell types. Endo F treatment of the affinity-labeled papain fragment of formyl peptide receptor does not alter its size, suggesting that this fragment does not contain the N-linked oligosaccharide cleaved by endo F from intact receptor. The results indicate that at least two N-linked oligosaccharide chains are located on the distal 1-3-kDa portion of the receptor polypeptide backbone.     

Biosynthesis of the epidermal growth factor receptor: post-translational glycosylation-independent acquisition of tyrosine kinase autophosphorylation activity

We previously showed that the epidermal growth factor (EGF) receptor in human A431 epidermoid carcinoma cells undergoes a slow post-translational modification whereby it acquires (t1/2 = 30-40 min) EGF binding capacity (Slieker, L.J., et. al. (1986) J. Biol. Chem., 261, 15233-15241). This activation occurs in the endoplasmic reticulum and requires core N-linked glycosylation. By employing both anti-EGF receptor and anti-phosphotyrosine antibodies to immunoprecipitate receptor pulse-labeled with [35S]methionine, we demonstrate here that the EGF receptor also acquires tyrosine kinase autophosphorylation activity post-translationally (t1/2 = 10-15 min). The acquisition of tyrosine kinase activity is independent of the acquisition of EGF binding capacity, since it precedes the latter process and does not require N-linked glycosylation.     

Secretion of rat hepatic lipase is blocked by inhibition of oligosaccharide processing at the stage of glucosidase I

Rat hepatic lipase is a glycoprotein bearing two N-linked oligosaccharide chains. The importance of glycosylation in the secretion of hepatic lipase was studied using freshly isolated rat hepatocytes. Various inhibitors of oligosaccharide synthesis and processing were used at concentrations that selectively interfere with protein glycosylation. Secretion of hepatic lipase activity was abolished by tunicamycin, castanospermine, and N-methyldeoxynojirimycin. No evidence was found by ELISA or Western blotting for secretion of inactive protein. Inhibition of secretion became apparent after a 30-min lag, corresponding to the time of intracellular transport of pre-existing protein. Simultaneously, intracellular hepatic lipase activity ws depleted. Secretion of hepatic lipase protein and activity was not affected by deoxymannojirimycin and swainsonine. Upon SDS-polyacrylamide gel electrophoresis, hepatic lipase secretion by deoxymannojirimycin- or swainsonine-treated cells showed an apparent Mr of 53 kDa and 55 kDa, respectively, which was distinct from hepatic lipase secreted by untreated cells (Mr = 58 kDa). We conclude that glycosylation and subsequent oligosaccharide processing play a permissive role in the secretion of hepatic lipase. As secretion is prevented by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin, but not by inhibitors of subsequent oligosaccharide trimming, the removal of glucose residues from the high-mannose oligosaccharide intermediate in the rough endoplasmic reticulum appears the determining step.