Insulin receptors prepared with iodoacetamide show enhanced autophosphorylation and receptor kinase activity.

In this study, we found that adding iodoacetamide to the homogenization buffer used in the preparation of mouse or rat liver plasma membranes resulted in an increase of insulin receptor autophosphorylation by 4-5-fold and receptor kinase activity by about 2-fold. Similar effects were obtained with iodoacetate and p-chloromercuriphenyl sulfonate. The effect of iodoacetamide was minimal when it was added to membranes prepared without the thiol reagent. The enhancing effect of iodoacetamide on insulin receptor autophosphorylation was the result of a more than 2-fold decrease in the Km and a more than 3-fold increase in Vmax for ATP. The presence of iodoacetamide in the preparation of plasma membranes also greatly increased the solubilization of the insulin receptor from the plasma membrane by Triton X-100. We propose that iodoacetamide acts to alkylate some unknown thiols released during tissue homogenization and that in its absence these thiols formed mixed disulfides with the insulin receptor, thus adversely affecting the process of receptor activation by insulin.

Class I histocompatibility antigens and insulin receptors: evidence for interactions.

We provide evidence for an interaction between mouse class I major histocompatibility complex antigens and insulin receptors. Antibodies against class I but not class II major histocompatibility complex antigens immunoprecipitate photoaffinity-labeled hepatic insulin receptors. Haplotype specificity is demonstrated by reciprocal precipitation using anti-class I antibodies and three strains of mice. Antibodies against the 45-kDa products of either the H-2K or H-2D locus and rabbit anti-mouse beta 2-microglobulin antibodies were shown to precipitate insulin receptors. We also demonstrate the specific binding of 125I-labeled insulin and 125I-labeled epidermal growth factor, but not 125I-labeled glucagon or 125I-labeled atrial natriuretic factor, to solubilized plasma membranes immunoprecipitated with anti-H-2K antibody. These observations suggest a specific interaction between class I major histocompatibility complex antigens and certain hormone receptors.

High molecular weight forms of the insulin receptor.

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

Insulin receptor: its subunit structure as determined by photoaffinity labeling.

This communication reviews briefly the preparation of two photoreactive arylazido insulins and the use of these insulin derivatives to photolabel the insulin receptor of rat adipocytes. These experiments showed that the receptor contains disulfide linked subunits of 130,000, 90,000, and 40,000 daltons. When not reduced by dithiothreitol, three receptor species of 380,000, 300,000, and 230,000 daltons were detected. Based on the results of photolabeling we propose that the 300,000-dalton species is composed of one 130,000-, one 90,000-, and two 40,000-dalton subunits in disulfide linkages.

Structure of the insulin receptor of rat adipocytes. The three interconvertible redox forms.

Isolated intact rat adipocytes were photoaffinity labeled with radioactive and photoreactive N alpha B1-monoazidobenzoyl insulin (B1-MABI) or N epsilon B29-monoazidobenzoyl insulin (B29-MABI). Polyacrylamide gel electrophoresis of the labeled plasma membranes solubilized in sodium dodecyl sulfate revealed the specific labeling of three receptor species of 380 kDa, 300 kDa, and 230 kDa. Reduction of each species individually produced the subunits of 130 kDa, 90 kDa, and 40 kDa. Exposure of the adipocytes or plasma membranes after photolabeling to sulfhydryl alkylating agents such as N-ethylmaleimide or p-chloromercuriphenylsulfonate resulted in the appearance of the receptor quantitatively in the 380-kDa form. The effect of the sulfhydryl reagent was concentration dependent and in the case of p-chloromercuriphenylsulfonate the three receptor species reappeared when high concentrations of the reagent were used. Incubation of the adipocytes with low concentrations of dithiothreitol before photolabeling reduced these receptors to discrete lower-molecular-weight forms. In addition, an 85-kDa subunit was now photolabeled by B1-MABI. This subunit was demonstrated to be different from the 90-kDa subunit normally labeled by B29-MABI. We conclude that on the cell surface of the adipocyte, there is one molecular-weight form of insulin receptor of 380 kDa composed of one 130-kDa, one 90-kDa, one 85-kDa, and two 40-kDa subunits. The 300 kDa and 230 kDa are partially reduced forms of the 380-kDa species. We further postulate that a membrane factor or factors sensitive to sulfhydryl alkylating reagents may be involved in the partial reduction and oxidation of these three redox receptor species. The distribution of these redox receptor species may be related to the cellular or tissue sensitivity to insulin.

Direct demonstration of insulin receptor internalization. A quantitative electron microscopic study of covalently bound 125I-photoreactive insulin incubated with isolated hepatocytes.

When 125I-insulin is incubated with isolated rodent hepatocytes at 37 degrees C, the ligand initially binds to the plasma membrane of the cell and is subsequently internalized by adsorptive endocytosis. To confirm directly that the insulin receptor is internalized with the ligand, we covalently linked photoreactive 125I-N sigma B29 (azidobenzoyl) insulin to its specific hepatocyte receptor and followed its fate by quantitative electron microscopic autoradiography. We found that the covalently linked photoreactive insulin is internalized by the cell in fashion analogous to the internalization of ordinary 125I-insulin, indicating that, at least under these conditions, the insulin receptor is internalized with the ligand.

Subunit structure of insulin receptor of rat adipocytes as demonstrated by photoaffinity labeling.

Isolated rat adipocytes were incubated in the dark with either one or two radioiodinated photoreactive insulin derivatives, N epsilon B29-(azidobenzoyl) insulin (B29-MABI) and N alpha B1-(azidobenzoyl) insulin (B1-MABI), and were then exposed to light. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and radioautography of the crude plasma membrane fraction after reduction showed that B29-MABI labeled specifically three proteins of Mr 130 000, 90 000, and 40 000 whereas B1-MABI labeled specifically two proteins of Mr 130 000 and 40 000. B1-MABI also variably labeled some bands of intermediate Mr between 130 000 and 90 000. In contrast, the labeling of the 40-kilodalton protein was not observed in our previous studies in which photolabeling was carried our on isolated plasma membrane preparations [Yip, C. C., Yeung, C. W. T. & Moule, M. L. (1980) Biochemistry 19, 70-76; Yeung, C. W. T., Moule, M. L., & Yip, C. C. (1980) Biochemistry 19, 2196-2203]. Without reduction, an Mr 300 000 band and a larger band which barely entered a 5-15% gradient gel were specifically labeled by both photoreactive insulins. Reduction of these two high molecular weight bands gave rise to the 130-, 90-, and 40-kilodalton bands. The labeling of these proteins was affected neither by the time or temperature of incubation nor by the addition of methylamine, chloroquine, bacitracin, phenylmethanesulfonyl fluoride, p-(chloromercuri) benzenesulfonic acid, Trasylol, N-ethylmaleimide, or benzamidine. The labeling of these proteins by the photoreactive insulin derivatives was inhibited by first incubating the adipocytes with a human autoimmune serum to insulin receptor. We therefore conclude that these proteins are subunits of the insulin receptor in intact adipocytes.

Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations.

The photoreactive insulin derivatives N epsilon B29-(azidobenzoyl)insulin (MAB-insulin) and N alpha A1, N epsilon B29-di(azidobenzoyl)insulin (DAB-insulin) were synthesized by reacting bovine insulin with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. These derivatives were purified by ion exchange chromatography on SP-Sephadex, and their identities were established by polyacrylamide gel electrophoresis, amino acid analysis, and end-group determination. Their biological activities were measured by receptor binding assay and fat cell assay. The photoreactivity of these two derivatives was demonstrated by spectral changes and by the formation of covalent polymers of high molecular weight when exposed to light. Radioactive MAB-insulin and DAB-insulin were prepared by iodination with [125I]iodine. These radioactive derivatives were characterized for their photoreactivity, immunoreactivity, and receptor binding to liver plasma membrane. Liver plasma membrane preparations of rat, mouse, and guinea pig were incubated with these radioactive insulin derivatives and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization and reduction showed that two proteins were specifically labeled. The molecular weights of the two radioactive bands were estimated to be about 130 000 and 90 000 in all three species of animals.

Photoaffinity labeling of insulin receptor of rat adiopocyte plasma membrane.

A photosensitive insulin derivative was synthesized by reacting radioactive iodinated bovine insulin with N-hydroxysuccinimide ester of 4-azidobenzolic acid. The photo-sensitivity and specificity of this insulin derivative were established by its covalent nonspecific cross-link to albumin and its covalent specific cross-link to the heavy and light chains of anti-insulin immunoglobulin. Plasma membrane preparations of rate adipocytes were incubated with the photosensitive insulin derivative and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization with sodium dodecyl sulfate and reduction with beta-mercaptoethanol showed that a protein having a molecular weight of 130,000 was specifically labeled by the radioactive photosensitive insulin, suggesting that this protein may be the insulin receptor.

Structure and function of insulin: preparation and biological activity of guinea pig des B-Asp30,des-A-Asn21-insulin.

Bovine des-B-Ala30,des-A-Asn21-insulin and guinea pig des-B-Asp30,des-A-Asn21-insulin were prepared from bovine and guinea pig insulin by digestion with carboxypeptidase A (EC 3.4.12.2). As reported by other investigators, the biological activity of bovine des-Ala30,des-Asn21-insulin was less than 10% that of bovine insulin. Contrary to theoretical consideration, removal of A-Asn21 and B-Asp30 from the carbosyl termini of guinea pig insulin resulted in a loss of more than 90% of the biological activity. Receptor binding studies of these insulin derivatives demonstrated a good correlation between the loss of biological activity and the decrease in binding affinity. It is suggested that the carboxyl terminal A-Asn21 of insulin may interact directly with the insulin receptor.

Insulin biosynthesis in the bullhead, Ictalurus nebulosus, and the effect of temperature.

Insulin biosynthesis in the brown bullhead, Ictalurus nebulosus (Le Sueur), was studied by measuring the incorporation in vitro of [(3)H]leucine into proteins of the principal islet. The tissue was incubated for 6-15h in Krebs-Ringer bicarbonate buffer with [(3)H]leucine, supplemented with amino acids and glucose. Proteins, precipitated with trichloroacetic acid and extracted with acid ethanol, were separated by gel-filtration on Biogel P-30 in 3m-acetic acid. Three major components were found after incubation of the islets at 22 degrees C. On the basis of the results of sulphitolysis, biological activity and the demonstrated precursor-product relationship, components I and II were identified as proinsulin and insulin respectively. The third component was not identified. At 12 degrees C, [(3)H]leucine was incorporated only into proinsulin. No radioactivity was found in insulin or the unidentified component III at 12 degrees C as was found after incubation at 22 degrees C. When the temperature was lowered from 22 degrees to 12 degrees C after 3h of a 15h incubation, decreased conversion of proinsulin into insulin resulted at the lower temperature compared with the control tissue maintained at 22 degrees C. When the temperature was raised from 12 degrees to 22 degrees C at 3h of a 15h incubation, conversion of proinsulin into insulin occurred. No conversion occurred in the control tissue with the temperature maintained at 12 degrees C. No qualitative difference in the incorporation of [(3)H]leucine into proinsulin and its conversion into insulin at 12 degrees and 22 degrees C could be demonstrated between islet tissue from fish acclimated to less than 12 degrees C or to 22 degrees C. The results suggest that the enzyme(s) responsible for converting proinsulin into insulin in the bullhead may be temperature sensitive with low activity at 12 degrees C.