Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog.

The lipid bound to p60src, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [3H]myristic acid or [3H]palmitic acid. Incorporation of [3H]myristic acid was noticeably greater than incorporation of [3H]palmitic acid. All of the [3H]myristic acid-derived label in p60src was present as myristic acid. In contrast, none of the radioactivity derived from [3H]palmitic acid was recovered as palmitic acid. Instead, all 3H incorporated into p60src from [3H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-src also contains myristic acid. By comparison of the extent of myristylation of p60v-src with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, we estimate that greater than 80% of the molecules of p60v-src contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-src contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [3H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [3H]myristic acid-labeled p60src in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60src present in cells.     

Exogenous myristic acid acylates proteins in cultured rat hepatocytes

Fatty acid acylation is a functionally important modification of proteins. In the liver, however, acylated proteins remain largely unknown. This work was aimed at investigating fatty acid acylation of proteins in cultured rat hepatocytes. Incubation of these cells with [9,10-3H] myristic acid followed by two-dimensional electrophoresis separation of the delipidated cellular proteins and autoradiography evidenced the reproducible and selective incorporation of radioactivity from the precursor into 18 well-resolved proteins in the 10--120 kDa range and the 4--7 pH range. Radiolabeling of these proteins resulted from covalent linkage to the precursor [9,10-3H] myristic acid or to its elongation product, palmitic acid. The majority of the covalent linkages between the proteins and the fatty acids were broken by base hydrolysis, which indicated that the linkage was of thioester or ester-type. Only one of the studied proteins was attached to myristic acid via an amide linkage which resisted the basic treatment but was broken by acid hydrolysis. After incubation with [9,10-3H] palmitic acid, only two proteins previously detected with myristic acid were radiolabeled. Finally, the identified acylated proteins may be grouped into two classes: proteins involved in signal transduction (the alpha subunit of a heterotrimeric G protein and several small G proteins) and cytoskeletal proteins (cytokeratins, actin).     

Comparison of insulin-like growth factor I receptor and insulin receptor purified from human placental membranes

Insulin-like growth factor (IGF)-I receptor purified from human placental membranes as previously described (LeBon, T. R., Jacobs, S., Cuatrecasas, P., Kathuria, S., and Fujita-Yamaguchi, Y. (1986) J. Biol. Chem. 261, 7685-7689) was characterized. The IGF-I receptor was similar to the insulin receptor with respect to subunit structure (beta-alpha-alpha-beta), apparent sizes of deglycosylated alpha (Mr = approximately 88,000) and beta (Mr = approximately 67,000) subunits, and amino acid composition of the subunits. Monoclonal antibody specific to each receptor recognized its own receptor whereas polyclonal anti-human insulin receptor antibody cross-reacted with the IGF-I receptor, indicating that the receptors share one or more antigenic sites. Further characterization of the purified IGF-I receptor tyrosine-protein kinase activity indicated that by analogy with the insulin receptor the monomeric alpha beta form of the IGF-I receptor appears to have higher kinase activity than the intact receptor in the alpha 2 beta 2 form. The most significant difference between the two receptors was found in the N-terminal amino acid sequences of their alpha subunits, which apparently show 60% identity. The IGF-I receptor alpha subunit lacks residues corresponding to the N-terminal 4 amino acids of the insulin receptor alpha subunit. These results provide the first direct proof that the IGF-I receptor is a molecule distinct from the insulin receptor despite numerous similarities.     

Acylation of Rat Brain Myelin Proteolipid Protein with Different Fatty Acids

The acylation of rat brain proteolipid protein (PLP) with tritiated palmitic, oleic, and myristic acids was studied in vivo and in vitro and compared with the acylation of lipids. Twenty-four hours after intracranial injection of [3H]myristic acid, only 16% of the PLP-bound label appeared as myristic acid, with 66% as palmitic, 9% as stearic, and 6% as oleic acid, whereas greater than 63% of the label in total or myelin phospholipid was in the form of myristic acid. In contrast, after labelling with [3H]palmitic or oleic acids, 75% and 86%, respectively, of the radioactivity in PLP remained in the original form. When brain tissue slices were incubated for short periods of time, the incorporation of palmitic and oleic acids into PLP exceeded that of myristic acid by a factor of 8. In both systems and with all precursors studied, the label associated with PLP was shown to be in ester linkage. The results suggest a preferential acylation of PLP with palmitic and oleic acids as compared with myristic acid. This is consistent with the fatty acid composition of the isolated PLP.     

src kinase catalyzes the phosphorylation and activation of the insulin receptor kinase

When a partially purified insulin receptor preparation immobilized on insulin-agarose is incubated with [gamma-32P]ATP, Mn2+, and Mg2+ ions, the receptor beta subunit becomes 32P-labeled. The 32P-labeling of the insulin receptor beta subunit is increased by 2-3-fold when src kinase is included in the phosphorylation reaction. In addition, the presence of src kinase results in the phosphorylation of a Mr = 125,000 species. The Mr = 93,000 receptor beta subunit and the Mr = 125,000 32P-labeled bands are absent when an insulin receptor-deficient sample, prepared by the inclusion of excess free insulin to inhibit the adsorption of the receptor to the insulin-agarose, is phosphorylated in the presence of the src kinase. These results indicate that the insulin receptor alpha and beta subunits are phosphorylated by the src kinase. The src kinase-catalyzed phosphorylation of the insulin receptor is not due to the activation of receptor autophosphorylation because a N-ethylmaleimide-treated receptor preparation devoid of receptor kinase activity is also phosphorylated by the src kinase. Conversely, the insulin receptor kinase does not catalyze phosphorylation of the active or N-ethylmaleimide-inactivated src kinase. Subsequent to src kinase-mediated tyrosine phosphorylation, the insulin receptor, either immobilized on insulin-agarose or in detergent extracts, exhibits a 2-fold increase in associated kinase activity using histone as substrate. src kinase mediates phosphorylation of predominantly tyrosine residues on both alpha and beta subunits of the insulin receptor. Tryptic peptide mapping of the 32P-labeled receptor alpha and beta subunits by high pressure liquid chromatography reveals that the src kinase-mediated phosphorylation sites on both receptor subunits exhibit elution profiles identical with those phosphorylated by the receptor kinase. Furthermore, the HPLC elution profile of the receptor auto- or src kinase-catalyzed phosphorylation sites on the receptor alpha subunit are also identical with that on the receptor beta subunit. These results indicate that: the src kinase catalyzes tyrosine phosphorylation of the insulin receptor alpha and beta subunits; and src kinase-catalyzed phosphorylation of insulin receptor can mimic the action of autophosphorylation to activate the insulin receptor kinase in vitro, although whether this occurs in intact cells remains to be determined.     

Mammalian red cell membrane Rh polypeptides are selectively palmitoylated subunits of a macromolecular complex.

Incubation of [3H]palmitic acid, ATP, and CoA with inside-out membrane vesicles prepared from human or other mammalian red cells resulted in nearly exclusive 3H-palmitoylation of the Mr = 32,000 Rh polypeptides. [3H]Palmitic, [3H]myristic, and [3H]oleic acids were comparably esterified onto Rh polypeptides in inside-out membrane vesicles in the presence of ATP and CoA, although [3H]palmitic acid was preferentially incorporated by intact human red cells. Experiments using sulfhydryl reagents or tryptic digestions suggested that multiple sulfhydryl groups on the Rh polypeptides located near the cytoplasmic leaflet of the lipid bilayer were 3H-palmitoylated; the exofacial sulfhydryl group essential for Rh antigenic reactivity was not 3H-palmitoylated. Transfer of fatty acid from [14C]palmitoyl-CoA to sites on the Rh polypeptides occurred even after previous incubation of inside-out membrane vesicles at 95 degrees C or after solubilization of inside-out membrane vesicles in Triton X-100. Hydrodynamic analyses of Triton X-100-solubilized membranes surprisingly demonstrated that 3H-palmitoylated Rh polypeptides behaved as a protein of apparent Mr = 170,000. These in vitro studies suggest that palmitoylation of Rh polypeptides occurs within a macromolecular complex by a highly selective but possibly nonenzymatic mechanism.     

The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus.

[3H]Myristic and [3H]palmitic acid were compared as tracers for the fatty acylation of cellular lipids and viral glycoproteins in chicken embryo cells infected with fowl plague and Semliki Forest virus (SFV). Both of these substrates are incorporated into glycerolipids to a similar extent, whereas sphingolipids show much higher levels of palmitate than myristate after a 20 h labeling period. Both fatty acid species were found to be subject to metabolic conversions into longer chain fatty acids yielding 11.7% C16:0 from [3H]myristic and 11.8% C18:0 from [3H]palmitic acid. The reverse, a metabolic shortening of the exogenous acyl-chains yielding, for instance, significant levels of myristic acid from palmitic acid was not observed. Out of the various [3H]fatty acids present after in vivo labeling with [3H]myristic acid (C14:0) the elongated acyl-species arising from metabolic conversion (e.g., C16:0; C18:0) are preferred over myristic acid in the acylation of SFV E1 and E2 and of the influenza viral hemagglutinin (HA2). During acylation of exogenous E1 from SFV in vitro incorporation of palmitic acid from palmitoyl CoA exceeds that of myristic acid from myristoyl CoA by a factor of 37. This indicates that specificity for the incorporation of fatty acids into viral membrane proteins occurs at the level of the polypeptide acyltransferase(s).     

Alpha and beta subunits of the nicotinic acetylcholine receptor contain covalently bound lipid

Labeling of the BC3H1 muscle-like cell line with [3H] palmitate, followed by immunoprecipitation of the acetylcholine receptor, indicated that the alpha and beta subunits of the receptor contain covalently bound fatty acid. After acid hydrolysis, fatty acid methyl esters could be recovered from the isolated [3H]palmitate-labeled alpha subunit. Treatment of differentiated BC3H1 cells with cerulenin, an inhibitor of fatty acid and sterol synthesis and fatty acid acylation of proteins, resulted in a 50% inhibition in expression of the acetylcholine receptor on the cell surface under conditions where there was minimal inhibition of protein synthesis. We conclude that this previously undetected post-translational modification may play a role in assembly and/or surface expression of the acetylcholine receptor.     

Fatty acid acylation of proteins in Physarum polycephalum

We have investigated the occurrence of protein-fatty acid acylation by metabolic incorporation of [3H]myristic and [3H]palmitic acids in Physarum polycephalum. We show that this organism contains fatty acylated proteins with mainly myristic acid covalently attached in alkali-stable linkages, probably amides. We find no evidence for ester-linked fatty acids, in contrast to the situation in vertebrate cells.     

Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an Mr 95,000 protein (identified as the insulin receptor beta subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) antisera) and an Mr 180,000 protein (which was unreactive with all anti-insulin receptor antibodies). After purification and tryptic digestion of the Mr 95,000 protein, tryptic peptides containing Tyr(P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. The partial amino acid sequence obtained by gas- and solid-phase Edman degradation was compared to the amino acid sequence of the intracellular extension of the rat insulin receptor deduced from the genomic sequence. Approximately 80% of all beta subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg from the tyrosine kinase domain, which is recovered mainly as the double phosphorylated species (predominantly in the form with Tyr(P) at residues 3 and 7 from the amino terminus; the remainder with Tyr(P) at residues 3 and 8), with 10-15% as the triple phosphorylated species. A second tryptic peptide is located near the carboxyl terminus, contains 2 tyrosines, and has the sequence, Thr-Tyr-Asp-Glu-His-Ile-Pro-Tyr-Thr-; this contains 20-30% of beta subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. Approximately 10% of beta subunit [32P]Tyr(P) resides on an unidentified tryptic peptide of Mr 4,000-5,000. The insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells thus involves at least 6 of the 13 tyrosine residues located on the beta subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin. This multisite regulatory tyrosine phosphorylation is the initial intracellular event in insulin action.     

Cation-independent mannose 6-phosphate receptor contains covalently bound fatty acid

The cation-independent mannose 6-phosphate receptor (215,000 daltons) was isolated from embryonic bovine tracheal cells and embryonic human skin fibroblasts labelled with [3H]palmitic acid. The tritium label was detected in the protein upon fluorographic analysis of SDS-polyacrylamide gels of the purified receptor. The label was not sensitive to hydroxylamine, methanolic KOH, or beta-mercaptoethanol, but labelled fatty acid was recovered from the protein by acidic methanolysis. Labelled receptor protein could not be isolated from cells grown in the presence of [3H]myristic acid. The results suggest the presence of amide-linked palmitic acid in the structure of the cation-independent mannose 6-phosphate receptor.     

The beta subunit of the insulin receptor is an insulin-activated protein kinase

In the presence of adenosine 5'-[gamma-32P]triphosphate ([gamma-32P]ATP) and a partially purified human placental insulin receptor preparation, insulin stimulates the phosphorylation of an Mr 94000 protein in a time- and dose-dependent manner. Half-maximal stimulation of 32P incorporation occurs at (2-3) X 10(-9) M insulin, a concentration identical with the Kd for insulin binding in this preparation. Immunoprecipitations with monoclonal anti-insulin receptor antibody demonstrate that the Mr 94000 protein kinase substrate is a component of the insulin receptor, the beta subunit. If the partially purified, soluble placental receptor preparation is immunoprecipitated and then exposed to [gamma-32P]ATP and insulin, phosphorylation of the Mr 94000 protein is maintained. The photoincorporation of 8-azido[alpha-32P]ATP into placental insulin receptor preparations was carried out to identify the ATP binding site responsible for the protein kinase activity. Photoincorporation into numerous proteins was observed, including both subunits of the insulin receptor. However, when photolabeling was performed in the presence of excess adenosine 5'-(beta, gamma-imidotriphosphate), a nonhydrolyzable ATP derivative, the beta subunit of the insulin receptor was the only species protected from label incorporation. These data indicate that the beta subunit of the insulin receptor has insulin-dependent protein kinase activity. Phosphotyrosine formation is the primary result of this activity in placental insulin receptor preparations.     

GABAA receptor subtypes immunopurified from rat brain with alpha subunit-specific antibodies have unique pharmacological properties.

The unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor were expressed in bacterial and used to produce subunit-specific polyclonal antisera. Antibodies immobilized on protein A-Sepharose were used to isolate naturally occurring alpha-specific populations of GABAA receptors from rat brain that retained the ability to bind [3H]muscimol, [3H]flunitrazepam, [3H]Ro15-1788, and [125I]iodo-clonazepam with high affinity. Pharmacological characterization of these subtypes revealed marked differences between the isolated receptor populations and was generally in agreement with the reported pharmacological profiles of GABAA receptors in cells transiently transfected with alpha 1 beta 1 gamma 2, alpha 2 beta 1 gamma 2, alpha 3 beta 1 gamma 2, and alpha 5 beta 1 gamma 2 combinations of subunits. Additional subtypes were also identified that bind [3H]muscimol but do not bind benzodiazepines with high affinity. The majority of GABAA receptor oligomers contains only a single type of alpha subunit, and we conclude that alpha 1, alpha 2, alpha 3, and alpha 5 subunits exist in vivo in combination with the beta subunit and gamma 2 subunit.     

Subunit structure of the purified human placental insulin receptor. Intramolecular subunit dissociation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Insulin receptors purified from human placental membranes by gel-filtration and insulin-agarose affinity chromatography were found to be composed of eight different high molecular weight complexes as identified by nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The subunit stoichiometry of these different high molecular weight forms of the insulin receptor were determined by comparisons of silver-stained gel profiles with the autoradiograms of 125I-insulin specifically cross-linked to the alpha subunit and [gamma-32P]ATP specifically autophosphorylated beta subunit gel profiles. Two-dimensional SDS-polyacrylamide gel electrophoresis in the absence and presence of reductant confirmed the subunit stoichiometries as alpha 2 beta 2, alpha 2 beta beta 1, alpha 2 (beta 1)2, alpha 2 beta, alpha 2 beta 1, alpha 2, alpha beta, and beta, where alpha is the Mr = 130,000 subunit, beta is the Mr = 95,000 subunit, and beta 1 is the Mr = 45,000 subunit. Treatment of the insulin receptor preparations with oxidized glutathione or N-ethylmaleimide prior to SDS-polyacrylamide gel electrophoresis increased the relative amount of the alpha 2 beta 2 complex concomitant with a total disappearance of the alpha 2 beta, alpha 2 beta 1, alpha 2, and free beta forms. The effects of oxidized glutathione were found to be completely reversible upon extensive washing of the treated insulin receptors. In contrast, the effects of N-ethylmaleimide were totally irreversible by washing, consistent with known sulfhydryl alkylating properties of this reagent. The formation of these lower molecular weight insulin receptor subunit complexes was further demonstrated to be due to SDS/heat-dependent intramolecular sulfhydryl-disulfide exchange occurring within the alpha 2 beta 2 complex. These studies demonstrate that the largest disulfide-linked complex (alpha 2 beta 2) is the predominant insulin receptor form purified from the human placenta with the other complexes being generated by proteolysis and by internal subunit dissociation.     

Biosynthesis and glycosylation of p150,95 and related leukocyte adhesion proteins

The p150,95 cell surface protein is a member of a family of heterodimeric leukocyte adhesion proteins that have homologous alpha subunits, each noncovalently associated with a common beta subunit. In this report we have metabolically labeled the U937 cell line at various timepoints during its phorbol myristic acetate-induced maturation to examine the kinetics of synthesis of these proteins during monocytic differentiation, and their maturation and glycosylation. The p150,95 alpha subunit was immunoprecipitated with p150,95-specific monoclonal antibody (MAb), or an antiserum to the denatured, purified alpha X subunit. The glycosylation and polypeptide chain length of the p150,95, Mac-1, and lymphocyte function associated antigen (LFA-1) alpha and beta subunits were compared by immunoprecipitation with subunit specific MAb and antisera, and by digestion with Endo H and N-glycanase. The p150,95 alpha subunit is synthesized as a precursor of 146,000 Mr, has five to six N-linked oligosaccharides, and has a polypeptide chain backbone of 132,000 Mr. Over 50% of the carbohydrate on the mature alpha subunit of 150,000 Mr was sensitive to Endo H digestion. The p150,95 alpha and beta precursors can associate before maturation into the mature form. Conversion to the mature form was accompanied by loss of reactivity with the antiserum to the denatured alpha X subunit, suggesting a change in conformation. Mac-1 and LFA-1 alpha subunits have precursors of 160,000 Mr and 165,000 Mr, respectively, and contain N-linked carbohydrates. The polypeptide chain length for the Mac-1 alpha subunit is 137,000 Mr, and for LFA-1 is 149,000 Mr. Only 14% of the oligosaccharide on the mature LFA-1 alpha subunit was sensitive to Endo H, suggesting that unlike p150,95, most is converted to the complex type. The differences noted in the Mr of the three homologous alpha subunits are therefore due to differences in both polypeptide chain length and carbohydrate processing during biosynthesis.     

N-myristoylation of the rod outer segment G protein, transducin, in cultured retinas.

Bovine retinas incubated with [3H]myristic acid incorporated detectable radiolabel into only a few proteins. The most heavily labeled was the alpha subunit of the rod outer segment G protein transducin (Gt alpha). The radiolabeled protein was specifically eluted from illuminated membranes in the presence of GTP, displaying the unique solubility properties of Gt alpha. It comigrated with Gt alpha in electrophoresis and chromatography and was immunoprecipitated by Gt alpha-specific antibodies. The radiolabel was confirmed by hydrolysis, chemical derivatization, and chromatography to be amide-linked myristic acid. The solubility of the myristoylated Gt alpha indicates that myristoylation is not sufficient to cause tight membrane association of this normally membrane-bound subunit. Incorporation of [3H]myristate was blocked by the protein synthesis inhibitor cycloheximide, suggesting that that fatty acid group is introduced during or soon after translation in the rod inner segment.     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits

The biosynthesis and carbohydrate processing of the insulin receptor were studied in cultured human lymphocytes by means of metabolic and cell surface labeling, immunoprecipitation with anti-receptor autoantibodies, and analysis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. In addition to the two major subunits of Mr = 135,000 and Mr = 95,000, two higher molecular weight bands were detected of Mr = 210,000 and Mr = 190,000. The Mr = 210,000 band and the two major subunits were labeled by [3H]mannose, [3H]glucosamine, [3H]galactose, and [3H]fucose, and were bound by immobilized lentil, wheat germ, and ricin I lectins. On the other hand, the Mr = 190,000 band was labeled only by [3H]mannose and [3H]glucosamine and was bound only by lentil lectin. All four components could be labeled with [35S] methionine; however, in contrast with the other three polypeptides, the Mr = 190,000 band was not labeled by cell surface iodination with lactoperoxidase, suggesting that it is not exposed at the outer surface of the plasma membrane. Pulse-chase studies with [3H]mannose showed that the Mr = 190,000 was the earliest labeled component of the receptor; radioactivity in this band reached a maximum 1 h after the pulse, clearly preceded the appearance of the other components, and had a very brief half-life (t1/2 = 2.5 h). The Mr = 210,000, Mr = 135,000, and Mr = 95,000 bands were next in appearance and reached a maximum 6 h in the chase period. Monensin, an ionophore which interferes with maturation of some proteins, blocked both the disappearance of the Mr = 190,000 protein and the appearance of the Mr = 135,000 and Mr = 95,000 subunits. The mannose incorporated in the Mr = 190,000 component was fully sensitive to treatment with endoglycosidase H while that in the Mr = 210,000 band and the two major subunits was only partially sensitive. Tryptic fingerprints of the 125I-labeled Mr = 210,000 band suggested that this component contains peptides of both the Mr = 135,000 and Mr = 95,000 subunits. In conclusion, the Mr = 190,000 component appears to represent the high mannose precursor form of the insulin receptor that undergoes carbohydrate processing and proteolytic cleavage to generate the two major subunits. In addition, the Mr = 210,000 band is probably the fully glycosylated form of the precursor that escapes cleavage and is expressed in the plasma membrane.     

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.     

Direct identification of palmitic acid as the lipid attached to p21ras.

p21v-H-ras, the transforming protein of Harvey murine sarcoma virus, contains a covalently attached lipid. Using thin-layer chromatography, we identified the acyl group as the 16-carbon saturated fatty acid palmitic acid. No myristic acid was detected in fatty acids released from in vivo-labeled p21v-H-ras. The p21v-K-ras protein encoded by Kirsten sarcoma virus was also palmitylated. The processing and acylation of p21v-K-ras however differed from that of p21v-H-ras. Three forms of [3H]palmitic acid-labeled p21ras proteins were detected in Kirsten sarcoma virus-transformed cells. This contrasted with Harvey sarcoma virus, in which two forms of p21v-H-ras contained palmitic acid. Analysis by partial proteolysis of p21v-H-ras labeled with [3H]palmitic acid suggested that all of the lipid found in intact p21v-H-ras was located in the C-terminal region. On sodium dodecyl sulfate-polyacrylamide gels, p21v-H-ras labeled with [3H]palmitic acid migrated slightly ahead of the majority of p21v-H-ras. Of the mature forms of p21v-H-ras, apparently only a subpopulation contains palmitic acid.