After insulin binds

Three recent advances pertinent to the mechanism of insulin action include (i) the discovery that the insulin receptor is an insulin-dependent protein tyrosine kinase, functionally related to certain growth factor receptors and oncogene-encoded proteins, (ii) the molecular cloning of the insulin proreceptor complementary DNA, and (iii) evidence that the protein tyrosine kinase activity of the receptor is essential for insulin action. Efforts are now focusing on the physiological substrates for the receptor kinase. Experience to date suggests that they will be rare proteins whose phosphorylation in intact cells may be transient. The advantages of attempting to dissect the initial biochemical pathway of insulin action include the wealth of information about the metabolic consequences of insulin action and the potential for genetic analysis in Drosophila and in man.     

Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance

Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.     

人胰岛素基因的克隆及其乳腺特异性表达载体的构建

从人血中提取人基因组DNA,根据GenBank登陆的人胰岛素基因序列设计引物,通过longPCR技术克隆了人胰岛素基因,并将该基因克隆入pMD18T载体构建pMI,将pMI和质粒pBEBT通过酶切连接构建成重组质粒pBEBI。结果成功扩出长为1.6kb的人胰岛素基因并构建了乳腺特异性表达载体。     

Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells

The sequence of the human epidermal growth factor (EGF) receptor shows great homology with the avian erythroblastosis virus v-erb B oncogene, raising the possibility that the receptor gene is identical to the c-erb B protooncogene. Human A431 epidermoid carcinoma cells, which have an unusually high number of EGF receptors, were examined to determine whether elevated EGF receptor levels correlate with gene amplification. Southern blots of genomic DNA's from A431 and other human cell lines were probed with either a v-erb B gene fragment or a human EGF receptor complementary DNA clone (pE7), previously isolated from an A431 complementary DNA library. When either probe was used to analyze Eco RI- or Hind III-generated DNA fragments, EGF receptor DNA sequences were amplified about 30-fold in A431. Differences in the banding pattern of A431 DNA fragments relative to normal fibroblast DNA indicate the occurrence of a rearrangement in the region of the receptor gene. Furthermore, A431 cells contain a characteristic, prominent 2.9-kilobase RNA. These results are consistent with the hypothesis that, in A431 cells, gene amplification, possibly associated with a translocation event, may result in the overproduction of EGF receptor protein or the appearance of the transformed phenotype (or both).     

Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing

A point mutation in the human insulin receptor gene in a patient with type A insulin resistance alters the amino acid sequence within the tetrabasic processing site of the proreceptor molecule from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser. Epstein-Barr virus-transformed lymphocytes from this patient synthesize an insulin receptor precursor that is normally glycosylated and inserted into the plasma membrane but is not cleaved to mature alpha and beta subunits. Insulin binding to these cells is severely reduced but can be increased about fivefold by gentle treatment with trypsin, accompanied by the appearance of normal alpha subunits. These results indicate that proteolysis of the proreceptor is necessary for its normal full insulin-binding sensitivity and signal-transducing activity and that a cellular protease that is more stringent in its specificity than trypsin is required to process the receptor precursor.     

A common PDGF receptor is activated by homodimeric A and B forms of PDGF

The human platelet-derived growth factor (PDGF) receptor complementary DNA was cloned and expressed by transfection of Chinese hamster ovary (CHO) fibroblasts. The ability of CHO cells expressing the human receptor complementary DNA (CHO-HR5) to interact with different recombinant forms of PDGF (AA and BB homodimers) was tested. Both forms of PDGF bind to the transfected receptor, stimulate the receptor tyrosine kinase activity, and elicit a mitogenic response in a manner that was indistinguishable from the responses of Balb/c 3T3 cells to AA and BB forms of PDGF can be attributed to a single type of receptor and show that the AA form, like the BB form, is a true mitogen.     

A family with severe insulin resistance and diabetes due to a mutation in AKT2

Inherited defects in signaling pathways downstream of the insulin receptor have long been suggested to contribute to human type 2 diabetes mellitus. Here we describe a mutation in the gene encoding the protein kinase AKT2/PKBbeta in a family that shows autosomal dominant inheritance of severe insulin resistance and diabetes mellitus. Expression of the mutant kinase in cultured cells disrupted insulin signaling to metabolic end points and inhibited the function of coexpressed, wild-type AKT. These findings demonstrate the central importance of AKT signaling to insulin sensitivity in humans.     

Sequence and expression of human estrogen receptor complementary DNA

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.     

Chromosomal location of human T-cell receptor gene Ti beta

A complementary DNA probe corresponding to the beta-chain gene of Ti, the human T lymphocyte receptor, has been molecularly cloned. The chromosomal origin of the Ti beta gene was determined with the complementary DNA by screening a series of 12 cell hybrid (mouse X human) DNA's containing overlapping subsets of human chromosomes. DNA hybridization (Southern) experiments showed that the human Ti beta gene resides on chromosome 7 and is thus not linked to the immunoglobulin loci or to the major histocompatibility locus in humans.     

Commitment of mouse fibroblasts to adipocyte differentiation by DNA transfection

Cells of the mouse cell line 3T3-F442A can be induced by various hormones to differentiate into adipocytes, whereas cells of 3T3-C2, a subclone of 3T3, cannot. However, transfection of DNA from uninduced 3T3-F422A cells into 3T3-C2 cells permits recovery of 3T3-C2 transfectants that differentiate into adipocytes in the presence of insulin. DNA isolated from human fat tissue, when transfected into 3T3-C2 mouse cells, also gives rise to mouse transfectants that are induced to differentiate into adipocytes by the addition of insulin. Apparently, transfection of a trans-regulatory gene (or genes) from 3T3-F442A or human fat cells into 3T3-C2 cells is sufficient to commit 3T3-C2 cells to adipocyte differentiation.     

Defect in phosphorylation of insulin receptors in cells from an insulin-resistant patient with normal insulin binding

Mononuclear blood cells were obtained from a patient with type A insulin resistance. The cells showed a normal ability to bind iodine 125-labeled insulin. Analysis of solubilized insulin receptors from the patient's cells revealed a defect in insulin-stimulated tyrosine kinase activity, which is closely associated with the receptor itself. The enzyme failed to phosphorylate the insulin receptor and showed a markedly reduced ability to phosphorylate exogenously added substrates. It appears that receptors from this insulin-resistant patient have a defect distal to the insulin-binding site (the alpha subunit of the receptor). The defect could be located in the beta subunit, which has an adenosine triphosphate-binding site, or in another receptor component that transfers a signal of insulin binding into kinase activity. This dissociation between the normal binding and the defective protein kinase component of the insulin receptor represents the first biochemical defect of the receptor distal to ligand binding.     

Gene transfer and molecular cloning of the human NGF receptor

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.     

The insulin receptor contains a calmodulin-binding domain

Substantial evidence suggests that calcium has a pivotal role in regulating the initial events through which insulin alters plasma membrane metabolism. Because binding of insulin to its receptor represents the initial site of insulin action in the plasma membrane, studies were undertaken to determine whether the insulin receptor is a calmodulin-binding protein. Preparations enriched in the insulin receptor and calmodulin-binding proteins were isolated from detergent-solubilized rat adipocyte membranes by chromatography with wheat germ agglutinin agarose and calmodulin-conjugated Sepharose, respectively. Substantial purification of a manganese-dependent, insulin-sensitive phosphoprotein of 95K identified as the beta subunit of the insulin receptor was accomplished. Binding and photocovalent cross-linking of iodine-125-labeled calmodulin to these affinity-purified preparations and to isolated plasma membranes, followed by immunoadsorption with insulin receptor antibodies bound to protein A Sepharose, resulted in significant purification of a binding complex of 110K to 140K. These results indicate that the adipocyte insulin receptor or a polypeptide closely associated with the receptor is a calmodulin-binding protein.     

Axons guided by insulin receptor in Drosophila visual system

Insulin receptors are abundant in the central nervous system, but their roles remain elusive. Here we show that the insulin receptor functions in axon guidance. The Drosophila insulin receptor (DInR) is required for photoreceptor-cell (R-cell) axons to find their way from the retina to the brain during development of the visual system. DInR functions as a guidance receptor for the adapter protein Dock/Nck. This function is independent of Chico, the Drosophila insulin receptor substrate (IRS) homolog.     

Insulin receptor phosphorylation may not be a prerequisite for acute insulin action

An antiserum to the insulin receptor mimicked insulin's acute actions on glucose transport, phosphorylation of integral membrane proteins, and internalization of the insulin receptor in isolated rat adipose cells. These insulinomimetic actions of the antiserum occurred without the equivalent increase in phosphorylation of the beta subunit of the insulin receptor observed with insulin. Thus, a role of receptor phosphorylation in acute insulin action is now questioned.     

Genetic variation in the human insulin gene

Four recombinant lambda phages containing nucleotide sequences complementary to a cloned human preproinsulin DNA probe have been isolated from human DNA. Restriction analyses in conjunction with Southern hybridizations reveal two types of gene sequences. One isolate of each type was subjected to complete nucleotide sequence determination. The sequences contain the entire preproinsulin messenger RNA region, two intervening sequence. 260 nucleotides upstream from the messenger RNA capping site, and 35 nucleotides beyond the polyadenylate attachment site. Our results strongly suggest that these two gene types are allelic variants of a single insulin gene.     

Cloning of human androgen receptor complementary DNA and localization to the X chromosome

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.     

Regulation of the apolipoprotein AI gene by ARP-1, a novel member of the steroid receptor superfamily

Apolipoprotein AI (apoAI) is a lipid-binding protein that participates in the transport of cholesterol and other lipids in the plasma. A complementary DNA clone for a protein that bound to regulatory elements of the apoAI gene was isolated. This protein, designated apoAI regulatory protein-1 (ARP-1), is a novel member of the steroid hormone receptor superfamily. ARP-1 bound to DNA as a dimer, and its dimerization domain was localized to the COOH-terminal region. ARP-1 also bound to a thyroid hormone-responsive element and to regulatory regions of the apoB, apoCIII, insulin, and ovalbumin genes. In cotransfection experiments, ARP-1 downregulated the apoAI gene. The involvement of ARP-1 in the regulation of apoAI gene expression suggests that it may participate in lipid metabolism and cholesterol homeostasis.