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In proliferating cells and tissues a number of checkpoints (G1/S and G2/M) preceding cell division (M-phase) require the signal provided by growth factors present in serum. IGFs (I and II) have been demonstrated to constitute key intrinsic components of the peptidic active fraction of mammalian serum. In vivo genetic ablation studies have shown that the cellular signal triggered by the IGFs through their cellular receptors represents a non-replaceable requirement for cell growth and cell cycle progression. Retroactive and current evaluation of published literature sheds light on the intracellular circuitry activated by these factors providing us with a better picture of the pleiotropic mechanistic actions by which IGFs regulate both cell size and mitogenesis under developmental growth as well as in malignant proliferation. The present work aims to summarize the cumulative knowledge learned from the IGF ligands/receptors and their intracellular signaling transducers towards control of cell size and cell-cycle with particular focus to their actionable circuits in human cancer. Furthermore, we bring novel perspectives on key functional discriminants of the IGF growth-mitogenic pathway allowing re-evaluation on some of its signal components based upon established evidences.

SHA is an l-rhamnose- and d-galactose-binding lectin that agglutinates human group B erythrocytes and was first purified almost 50 years ago. Although the original SHA-producing Streptomyces strain was lost, the primary structure of SHA was more recently solved by mass spectrometry of the archived protein, which matched it to a similar sequence in the Streptomyces lavendulae genome. Using genomic and protein biochemical analyses, this study aimed to identify SHA-secreting Streptomyces strains to further investigate the expression and binding activities of these putative proteins. Of 67 strains genetically related to S. lavendulae, 17 secreted pro-SHAs in culture. Seven SHA homologues were purified to homogeneity and then subjected to liquid chromatography–high-resolution multistage mass spectrometry (LC-MS/MS) and hemagglutination (HA) assays. Processing of pro-SHAs occurred during and after purification, indicating that associated proteases converted pro-SHAs into mature SHAs with molecular masses and HA activities similar to that of the archived SHA. Previously, the SHA monomer was shown to have two carbohydrate binding sites. The present study, however, found no HA activity in pro-SHAs, suggesting that pro-SHAs have only one binding site. Genetically, the SHA gene resides in conserved syntenic regions. The published genomes of 1,234 Streptomyces strains were analyzed, revealing 18 strains with SHA genes, 16 of which localized to a unique syntenic region. The SHA syntenic region consists of ∼17 open reading frames (ORFs) and is specific to S. lavendulae-related strains. Notably, a lipoprotein gene excludes SHA from the synteny in some strains, suggesting that horizontal gene transfer events during the course of evolution shaped the distribution of SHA genes. IMPORTANCE Lectins are extremely useful molecules for the study of glycans and carbohydrates. Here, we show that homologous genes encoding the l-rhamnose- and d-galactose-binding lectins, SHAs, are present in multiple bacterial strains, genetically related to Streptomyces lavendulae. SHA genes are expressed as precursor pro-SHA proteins that are truncated and mature into fully active lectins with two carbohydrate binding sites, which exhibit hemagglutination activity for type B red blood cells. The SHA gene is located within a conserved syntenic region, hinting at specific but yet-to-be-discovered biological roles of this carbohydrate-binding protein for its soil-dwelling microbial producer.

This study demonstrated that ARID5B mRNA is present in mouse cardiomyocyte HL-1 cells, and that ARID5B siRNA constantly knocked down ARID5B gene expression to the 40% level of control. AMPKα2 protein was elevated in such ARID5B knockdown HL-1 cells, and this was accompanied by an increase in the level of phosphorylated AMPKα. Since AMPKα2 mRNA levels did not change in ARID5B knockdown cells, the stability of AMPKα2 protein was investigated using inhibitors for protein synthesis and proteasomal degradation. Treatment of HL-1 cells with either cycloheximide or MG132 caused an appreciable increase in the amount of AMPKα2 protein in ARID5B knockdown cells, which suggests that knockdown of ARID5B mRNA extends the half-life of AMPKα2 protein in HL-1 cells via yet unidentified mechanisms. As for the expected downstream consequences of AMPKα2 activation, we found thus far that glucose uptake, fatty acid uptake, or fatty acid oxidation remained unchanged in HL-1 cells after knockdown of ARID5B. Further studies are required to understand the mechanisms for ARID5B knockdown and resulting AMPKα2 activation, and also to identify which metabolic pathways are affected by AMPKα2 activation in these cells. In summary, this study provided the foundation for an in vitro cell culture system to study possible roles of ARID5B in cardiomyocytes.

Blood type B-specific Streptomyces sp. 27S5 hemagglutinin (SHA) was discovered and characterized in the 1970s. Although strain 27S5 has been lost, the purified SHA protein survived intact under frozen conditions and retained its activity. Using modern techniques, here we further characterized SHA. Fourier-transform ion cyclotron resonance MS analysis determined the average molecular mass of SHA as 13,314.67 Da. MS of digested SHA peptides, Streptomyces genomic database matching, and N-terminal sequencing solved the 131-residue amino acid sequence of SHA. We found that SHA is homologous to N-terminally truncated hypothetical proteins encoded by the genomes of Streptomyces lavendulae, Streptomyces sp. Mg1, and others. The gene of the closest homologue in S. lavendulae, a putative polysaccharide deacetylase (PDSL), encodes 68 additional N-terminal amino acids, and its C terminus perfectly matched the SHA sequence, except for a single Ala-to-Glu amino acid difference. We expressed recombinant SHA(PDSL-A108E) (rSHA) as an enzymatically cleavable fusion protein in Escherichia coli, and glycan microarray analyses indicated that refolded rSHA exhibits the blood type B– and l-rhamnose–specific characteristics of authentic SHA, confirming that rSHA is essentially identical with SHA produced by Streptomyces sp. 27S5. We noted that SHA comprises three similar domains, representing 70% of the protein, and that these SHA domains partially overlap with annotated clostridial hydrophobic with conserved W domains. Furthermore, examination of GFP-tagged SHA revealed binding to microbial surfaces. rSHA may be useful both for studying the role of SHA/clostridial hydrophobic with conserved W domains in carbohydrate binding and for developing novel diagnostics and therapeutics for l-rhamnose–containing microorganisms.

Anti-mannotriose (Man3) antibodies were previously isolated from a Keio phage library displaying human single chain variable fragments (scFvs) using a neoglycolipid, Man3- dipalmitoylphosphatidylethanolamine. Of three genes constructed, the 5A3 clone was expressed in mouse myeloma NS0 cells as a conjugate with human IgG1 Fc (scFv-Fc) and characterized (Sakai, K., Shimizu, Y., Chiba, T., Matsumoto-Takasaki, A., Kusada, Y., Zhang, W., Nakata, M., Kojima, N., Toma, K., Takayanagi, A., Shimizu, N., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 253–262; Zhang, W., Matsumoto-Takasaki, A., Kusada, Y., Sakaue, H., Sakai, K., Nakata, M., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 263–270). Similarly, anti-Lex phages were screened from the same library with lacto-N-fucopentaose III (LNFPIII; Lex)-dipalmitoylphosphatidylethanolamine. Of five phage clones isolated, two scFv genes were constructed to express scFv-Fc proteins in NS0 cells. As was experienced with anti-Man3 scFv-Fc clones, only one anti-LNFPIII clone, 1F12, was successfully produced and purified as an scFv-Fc protein. Although anti-LNFPIII 1F12 and anti-Man3 5A3 scFv-Fc proteins were secreted into media, a decline in scFv-Fc production was observed with both stable clones during early passages. Transient expression of anti-LNFPIII and anti-Man3 scFv-Fc genes in COS-7 cells and subsequent analyses of scFv-Fc protein expression revealed accumulation of translated proteins in the endoplasmic reticulum for scFv-Fc proteins derived from clones that did not survive as stable clones. This report describes the following: (i) isolation of anti-LNFPIII scFv genes; (ii) purification of anti-LNFPIII scFv-Fc proteins from stably and transiently expressed cells; and (iii) extracellular or intracellular localization of two anti-LNFPIII and three anti-Man3 scFv-Fc proteins. The results suggest that expression of anti-Man3 and other anti-carbohydrate antibodies in mammalian cells is disadvantageous for cell growth.

Anti-influenza hemagglutinin (HA) Fabs were isolated from a phage display library using purified HA of influenza virus A/Puerto Rico/8/34 (PR8; H1N1) as an antigen. Four Fab clones displaying a 25-50-fold higher binding signal to PR8 HA than the control were selected for further analysis and comparison with anti-PR8 monoclonal antibody (mAb). All four Fabs and mAb recognized the PR8 HA under non-reducing conditions but rarely bound to reduced PR8 HA. Inhibition of influenza virus infection on MDCK cells was observed with Fab1 and mAb in a dose-dependent manner while Fab3 and 4 exhibited only a partial inhibitory effect. Moreover, Fab1 clone and mAb exhibited cross-reactivity with the A/Peking/262/95 (A/Peking; H1N1) strain. The inhibitory effects of mAb on both influenza strains were more potent than Fab1, which is likely attributed to its higher affinity for the antigen. SPR analyses, in fact, revealed that Fab1 and mAb have K(D) of 1.5 x 10(-8) and 3.2 x 10(-9)M, respectively. These results strongly suggest that phage library-derived Fabs can be readily prepared and that such HA-specific Fabs with inhibitory action on influenza infection may be used to treat influenza patients.

Upregulation of insulin-like growth factor (IGF)-II expression has been reported for a variety of childhood and adulthood tumors. We determined IGF-II gene promoter usage in human cancerous and benign tissues by semiquantitative RT-PCR using P1–P4-specific primers. Although the human IGF-II gene structure is commonly thought to consist of nine exons and four promoters, we detected substantial utilization of a previously reported exon 4b, which is downstream of exon 4. Thus, exon 4b was intensively studied using 4b-specific primers. IGF-II gene promoter usage is highly variable in malignant and benign breast, prostate, and bladder tissues. While a majority of samples utilized P2–P4 promoters in a variety of combinations, when quantitated, P3 and P4 promoters were much more active than P2 promoter. This study not only demonstrated that IGF-II gene promoter usage is highly variable in malignant and benign tissues, but suggested that alternatively spliced exon 4b should be recognized as a 10th exon.

Phosphodiesterase type 3 isoforms, PDE3A and 3B, are expressed primarily in cardiovascular and adipose tissues, respectively. We previously reported a shorter transcript of 4.4-kb PDE3A which is predominantly transcribed in human placenta, whereas a full-length 7. 6-kb transcript corresponding to the cardiac PDE3A cDNA has not been characterized. Due to unfortunate circumstances created by changes in PDE3 nomenclature, PDE3B gene structure previously reported used PDE3A in its title. Here, we describe PDE3A gene structure, which comprises 16 exons spanning over 130 kb on chromosome 12p12. Two PDE3 isoforms share similar gene organization, but localize to different chromosomes. The most distal transcription initiation site of the PDE3A gene is approximately 1071 bases upstream of the ATG site, suggesting that exon 1 consists of 1071 and 960 bp of untranslated and translated sequences, respectively. The proximal 5'-flanking region, which does not contain TATA-like sequences, exhibited weak but significant promoter activity. Results suggest potential involvement of distal promoter/enhancer and translational regulation for expression of the 7.6-kb transcript.

Insulin-like growth factors (IGFs) are potent mitogens for a variety of cancer cells in vitro. A paracrine/autocrine role of IGF-II in the growth of breast and prostate cancer cells has been suggested. Information on cell-type-specific IGF-II expression in vivo in the breast and prostate is, however, limited. Thus, cell types expressing IGF-II mRNA and protein in tumors were identified by in situ hybridization and immunohistochemistry. Of 36 prostate, 17 breast, and 10 bladder cancers, and 9 paraganglioma tissues examined, IGF-II was expressed in more than 50% of prostate, breast, and bladder tumors, and in 100% of paraganglioma tumors. Expression levels of IGF-II were highest in the paraganglioma and bladder followed by prostate and breast tumors. In all the tumors expressing IGF-II, both mRNA and protein were localized to malignant cells, expression in the stroma being minimal. Since previous studies had indicated that an incompletely processed form of 15-kDa IGF-II exhibited higher mitogenic potency than the completely processed 7.5-kDa IGF-II form, the quantity and size of IGF-II proteins expressed in these tumors were analyzed by Western immunoblotting. Greater expression of 15-kDa IGF-II relative to the 7.5-kDa IGF-II form was clearly demonstrated in all six prostate cancers and in half of the two breast and four bladder cancers examined. The results are consistent with the hypothesis that the 15-kDa form of IGF-II expressed in cancerous cells contributes to autocrine cancer cell growth in vivo.

Receptors for insulin and insulin-like growth factor-I (IR and IGFIR) consisting of the alpha2beta2 structure are protein tyrosine kinases (PTKs). Carboxyl-terminal (CT) domains of their beta subunits are structurally diverse while the PTK domains share the highest homology. Interactions between CT and PTK domains of IR and IGFIR were studied by means of PTK activity, fluorescence energy transfer or surface plasmon resonance using BIAcore. We present evidence that IGFIR CT directly interacts with both IGFIR and IR. Although binding to both receptors, stimulation of PTK activity only occurs with IR but not IGFIR.

To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors, deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly8 for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The binding affinity and mitogenic activity of these rIGF-II mutants as well as commercially available des-(1-6)-rIGF-II were analyzed. While the relative affinity of des-(1-5)- and des-(1-6)-rIGF-II for purified human insulin and IGF-I receptors remained ator = 50% levels of that of rIGF-II, the affinity of des-(1-7)-rIGF-II decreased to approximately 10% and approximately 3%, respectively, of that of rIGF-II. When the octapeptide including Leu8 was removed prior to the Cys9-Cys47 intrachain bond, the relative affinity of this deletion mutant, des-(1-8)-rIGF-II, for these receptors dramatically decreased to1% of that of rIGF-II. Substituting Gly8 for Leu in rIGF-II decreased the affinity of this mutant for the IGF-I and insulin receptors to about the same extent. These results suggest that the side chains of Thr7 and Leu8 may play an important role in retaining all of the IGF-II functions. Decreases in the relative affinity for binding of the mutants to these receptors paralleled the decreases in their mitogenic potency for cultured Balb/c 3T3 cells. Although the relative affinity of des-(1-8)- or [Gly8]rIGF-II for rat IGF-II/CIM6-P (cation-independent mannose 6-phosphate) receptors was also1% of that of rIGF-II, the relative affinities of des-(1-5)-, des-(1-6)-, and des-(1-7)-rIGF-II for these receptors was significantly greater than that of rIGF-II. These results clearly demonstrate that Thr7 and Leu8 are important for binding to insulin and IGF-I receptors and Leu8 is critical for expression of all IGF-II functions.

cDNAs for two distinct Type III cGMP-inhibited (cGI) cyclic nucleotide phosphodiesterases (PDE), designated cGIP1 and cGIP2, were previously cloned from rat adipose and human cardiac cDNA libraries, respectively. In this study, another cDNA (approximately 4.0 kilobase (kb)) encoding a cGI-PDE of 74 kDa (658 amino acids) was isolated from a human placental cDNA library. The nucleotide sequence of its open reading frame was virtually identical to a corresponding region in the 3' portion of the cardiac cGIP2 cDNA (approximately 7.6 kb) which encoded a approximately 125-kDa cGI-PDE (1141 amino acid). Northern blots and RNase protection assays revealed a prominent 4.4-kb transcript and a 7.6-kb transcript in human placenta. The transcription start site of the 4.4-kb transcript was assigned to cardiac cDNA nucleotide 1292, the putative beginning of exon 3 of the human cGIP2 gene, with a potential translation initiation site 183 bases downstream, as determined by RNase protection assay. The 5'-flanking region of the 4.4-kb transcript exhibited promoter activity in HeLa cells which expressed the 4.4-kb transcript, and contained a TATAA sequence 35 base pairs upstream from the tentative transcription start site. Recombinant cGI-PDEs, expressed in Sf9 cells from the 7.6- and 4.0-kb cDNA, exhibited differences in their subcellular localization and Km for cGMP. Thus, in human tissues, alternative transcription may contribute to generating at least two cGIP2 isoforms, cytosolic and membrane-associated cGI-PDEs with different Km values for cGMP.

Our previous study using a deletion mutant indicated that the COOH-terminal (CT) domain of the insulin receptor plays important roles in both catalytic efficiency and stability of the receptor kinase (Yan et al., J. Biol. Chem., 268 [1993] 22444). In this study, we purified the CT domain of 98 amino acids from bacterial cells over-expressing the CT domain and examined its effect on insulin and IGF-I receptor protein kinases. The purified CT domain stimulated the kinase activities of purified insulin receptor-transmembrane/cytoplasmic domain (IRTMTPK) and its CT domain-deletion mutant (IRTMTPK delta CT), 3.3-fold and 2.3-fold, respectively, while it was less effective in stimulating the kinase activity of purified IGF-I receptor transmembrane/cytoplasmic domain (IGFIRTMTPK) (1.4-fold). When the effect of the CT domain on autophosphorylation was examined, a marked increase in autophosphorylation was observed only with IRTMTPK delta CT. These results suggest that the CT domain specifically interacts with the insulin receptor cytoplasmic domain, thereby activating the kinase or autophosphorylation activity.

Stromal-epithelial interactions play a critical role in promoting tumorigenesis and invasion. To obtain detailed information on cancer cell behaviors on the stroma and kinetics of cell migration, which cannot be observed by conventionally-used Boyden chamber assays, this study was aimed at analyzing the cell invasion process in vitro using time-lapse microscopic observation. Serum-free conditions and reconstituted type I collagen gels which provided a basal membrane-stroma-like microenvironment were used to first establish a basal condition. Time-lapse microscopic observation for 30 h of cell invasion into the collagen gel revealed kinetic parameters and individualistic behavior of cancer cells. Of breast cancer MDA-MB-231 or MCF-7 cells and colon cancer LS180 or HT29 cells examined, MDA-MB-231 cells most rapidly disappeared from the collagen gel surface under basal conditions. Estrogen-dependent MCF-7 cells disappeared at a rate approximately two times slower than that of MDA-MB-231 cells under serum- and phenol red-free conditions. By the addition of 10 nM β-estradiol to the basal medium, MCF-7 cell invasion was facilitated to a rate similar to that of MDA-MB-231 cells. Microscopic analyses of collagen gel-sections demonstrated that most of the MDA-MB-231 and MCF-7 cells remained within 60 μm from the gel top under basal conditions, which is consistent with the observation obtained using Boyden chambers that no cells could cross the collagen I gel barrier unless 1% fetal calf serum was added to basal conditions. In summary, this study demonstrated future applicability of this method to understand the initial phase of cancer cell invasion processes.

Recently, we have reported three monoclonal antibodies (mAbs) against purified human placental insulin-like growth factor (IGF)-I receptors. These antibodies, in contrast to the well-studied mAb alpha IR-3, stimulate binding of IGF-I and IGF-II to the receptor and DNA synthesis as well [Xiong, et al., Proc. Natl. Acad. Sci. U.S.A. 1992(89), 5356]. Here we describe two additional mAbs, 1H7 and 2C8, against the IGF-I receptor that have characteristics different from either alpha IR-3 or our previously reported mAbs. Both 1H7 and 2C8 bind to the alpha subunit of the IGF-I receptor as determined by immunoblotting. MAb 1H7 inhibited the binding of IGF-I and IGF-II to the IGF-I receptor while 2C8 had no effect on the binding of either ligand to the receptor. When their effects on DNA synthesis were examined using NIH 3T3 cells expressing human IGF-I receptors, 1H7 inhibited basal and IGF-I- or IGF-II-stimulated DNA synthesis whereas 2C8 stimulated basal DNA synthesis but provided no synergism in the presence of IGF-I or IGF-II.

We have previously expressed the human insulin receptor beta subunit domain containing transmembrane and cytoplasmic domains (IRTMTPK) in insect cells, and showed that the purified IRTMTPK was highly active (Li, S. L., Yan, P.-F., Pax, I. B., and Fujita-Yamaguchi, Y. (1992) Biochemistry 31, 12455-12462). To investigate the role of COOH-terminal and acidic domains of the insulin receptor kinase, we have expressed deletion mutants IRTMTPK delta CT (delta 76 amino acids) and IRTMTPK delta Acid (delta 19 amino acids). Both enzymes were purified by a one-step method using the same immunoaffinity column as used for IRTMTPK. While Km and Vmax for prephosphorylated IRTMTPK and delta Acid mutant enzyme determined using poly(Glu, Tyr)(4:1) were similar, catalytic efficiency of the delta CT mutant enzyme was significantly lower than those of IRTMTPK and delta Acid mutant enzyme as judged by Km and Vmax. Experiments for thermostability and susceptibility to proteases revealed that Tm of delta CT mutant enzyme was 3.5 degrees C lower than that of IRTMTPK enzyme (= 33.3 degrees C) and that delta CT mutant enzyme was digested by either trypsin or Lys-C into a 28,000 core domain much faster than IRTMTPK. Activation of delta CT mutant enzyme by polylysine was less significant than that of IRTMTPK and delta Acid mutant enzyme, approximately 4-versus approximately 17-fold. These studies suggested that the COOH-terminal domain plays important roles in both catalytic efficiency and stability of the insulin receptor kinase, and that the acidic domain by itself is not responsible for kinase activation by polylysine.

Five mutants of recombinant insulin-like growth factor-II (rIGF-II) that bound with high affinity to either the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CIM6-P) or the IGF-I receptor were prepared by site-directed mutagenic procedures, expressed as fusion proteins in the larva of Bombyx mori or Escherichia coli, purified to homogeneity, renatured, and characterized in terms of their receptor binding affinities and specificities as well as their biological activities. Class I mutants in which Phe26, Tyr27, and Val43 were substituted with Ser, Leu, and Leu, respectively, bound to enriched preparations of rat placental IGF-II/CIM6-P receptors with apparent equilibrium dissociation constants (Kd(app)) that were only slightly greater, i.e. 0.10, 0.05, and 0.06 nM, than that of rIGF-II (0.04 nM) or hIGF-II (0.03 nM). In contrast, replacing Phe26 with Ser resulted in 5- and 20-fold decreases in the affinities of this mutant for highly purified human placental IGF-I and insulin receptors, respectively. The affinities of the two other Class I mutants, [Leu27]- and [Leu43]rIGF-IIs, for these two receptors were reduced 80- to 220-fold. The affinities of Class II mutants, i.e. [Thr48,Ser49,Ile50]- and [Arg54,Arg55] rIGF-IIs, for IGF-I receptors were as potent as rIGF-II; however, they bound very poorly or not at all to the IGF-II/CIM6-P receptor. In the binding study of those mutant rIGF-IIs, IGF-II was observed to have an unexpectedly high affinity for pure human placental insulin receptor preparations. For example, the affinities of hIGF-II, rIGF-II, and two Class II rIGF-II mutants for the insulin receptor were only 3-, 9-, and 5-fold less, respectively, than that of porcine insulin. In two biological assay systems, i.e. the stimulation of DNA synthesis in Balb/c 3T3 cells and glycogen synthesis in HepG2 cells, the Kd(app) of the rIGF-II mutants for the IGF-I receptor but not the IGF-II/CIM6-P receptor correlated with their abilities to produce biological responses.

Human placental insulin receptor contains 47 Cys per an alpha beta dimer. Most of the 94 Cys in an intact alpha 2 beta 2 receptor are expected to form interchain or intrachain disulfide bonds, since there appears to be only one free cysteine residue in each beta subunit. In order to gain more insight into the three-dimensional organization of the insulin receptor, we have used limited trypsin digestion, SDS-PAGE, and protein microsequencing. The present study revealed the following; major tryptic cleavages occurred at alpha 164, alpha 270, alpha 582, and beta 1115, generating Mr 175,000, 130,000, 100,000, 70,000, and 55,000 disulfide-linked complexes. Under reducing conditions, tryptic fragments of Mr values = 30,000, 70,000, 20,000, 55,000, and 20,000 were identified to be alpha(1-164), alpha(165-582), alpha(165-270), alpha(271-582), and alpha(583-C-terminal), respectively. The major beta subunit tryptic fragment of Mr = 55,000 was assumed to have beta(724-1115) or beta(N-terminal-392). The Mr 175,000 complex appeared to contain two alpha(1-164) and two alpha(165-582), whereas the Mr 70,000 complex contained alpha(583-C-terminal) and beta(724-1115). Tryptic cleavage at alpha 582 apparently produced one Mr 175,000 and two Mr 70,000 complexes, suggesting that the alpha(583-C-terminal) domain interacts with the extracellular domain of the beta subunit by disulfide bonds. Tryptic cleavage at alpha 270 resulting in a formation of one Mr 100,000 complex consisting of two alpha(1-270) and two Mr 130,000 complexes consisting of alpha(271-C-terminal) and beta(724-1115) suggests that Cys residues involved with disulfide bonds between the two alpha subunits are located in the alpha(1-270) domain. The identification of the Mr 55,000 complex consisting of small tryptic fragments between alpha(122-270) indicates that 40 Cys residues in the two alpha(122-270) domains are inter- and intramolecularly associated by disulfide bonds. The alpha(1-121) domain does not appear to be linked to any other domains by disulfide bonds. These results are consistent with the structural model that the N-terminal domains of alpha subunits (122-270) are disulfide-linked together while the C-terminal domain (583-C-terminal) of the alpha subunit is linked to the N-terminal domain of the beta subunit by disulfide bonds.

Local epithelial cell growth is involved in physiological and pathological processes such as development, morphogenesis and carcinogenesis. Cell growth is triggered by the binding of growth factors, such as epidermal growth factor (EGF) to their own receptors (i.e. EGFR). In confluent slates, EGFRs are distributed on the basal-lateral side of polarized cells. Therefore, the delivery of EGF to EGFR is thought to be regulated by both tight junctions between the cells, as well as by the basement membrane to which the cells are attached. This research constructed a new culture device with which EGF can be delivered to either the basal-lateral or apical side of confluent epithelial cells that are cultured on an artificial Matrigel basement membrane. Cells in a confluent state started to regrow as a result of incubation with basal medium containing EGF, but not 'as a result of incubation with apical medium containing EGF. The rate of confluent epithelial cell regrowth depended on EGF concentrations supplied via the basement membrane. In contrast, cells in which tight junctions were disrupted by pre-treatment with EGTA displayed regrowth as a result of incubation with apical medium containing EGF. These results suggest that EGF signaling from the basal-lateral side is regulated by the basement membrane, while that from the apical side is blocked by the tight junctions. Thus, this culture model can be readily used to study the control of local growth of epithelial cells in physiological and pathological processes.

Type 3 cyclic nucleotide phosphodiesterase (PDE-3) isoforms exhibit a high affinity ("low K(m)") for cAMP and are specifically inhibited by cGMP and a number of pharmacological agents, which increase myocardial contractility, inhibit platelet aggregation, and increase smooth muscle relaxation. The PDE-3 family consists of at least two isozymes, PDE-3A (cardiac type) and PDE-3B (adipocyte type), with distinct tissue-specific distributions. PDE-3A mRNA is highly expressed in the cardiovascular system, whereas PDE-3B mRNA is primarily expressed in adipocytes and hepatocytes. Toward understanding potential roles of PDE-3 in diabetes mellitus, we have established a specific and sensitive RNase protection assay (RPA) for quantitating PDE-3A and PDE-3B mRNA in rat diabetic models. In fatty Zucker diabetic (ZDF) rats, PDE-3A mRNA, but not PDE-3B mRNA, was expressed in heart, whereas liver and white and brown fat tissues predominantly expressed PDE-3B mRNA. Unexpectedly, PDE-3B mRNA expression was approximately 2.5 times higher than PDE-3A mRNA in aorta from both ZDF and Sprague-Dawley (SD) rats. In contrast, expression levels of PDE-3A mRNA in heart were similar in both species. With this RPA, we were thus able to compare PDE-3A and -3B mRNA levels in different tissues as well as in different rat species.

Since the studies on tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro suggested that protamine and poly(L-lysine) may activate phosphorylation of the receptor beta subunit [Sacks & McDonald (1988) J. Biol. Chem. 263, 2377-2383], we examined the effects of a variety of basic polycations/proteins and polyamines on insulin receptor kinase activity. The insulin receptor purified from human placental membranes was incubated with each basic polycation/protein or polyamine and assayed for tyrosine-specific protein kinase activity by measuring 32P incorporation into the src-related peptide. At a concentration of 1 microM, poly(L-lysine) and poly(L-ornithine) markedly stimulated kinase activity, whereas poly(L-arginine) and histones H1 and H2B inhibited insulin receptor kinase. In contrast, at a concentration of 1 mM, three polyamines (spermine, spermidine and putrescine) did not alter kinase activity. Poly(L-lysine) and poly(L-ornithine) stimulated the insulin receptor kinase by 5-10-fold at concentrations of 0.1-1 microM. Protamine sulphate also showed a significant stimulatory effect at a concentration of 100 microM. Preincubation of the receptor with poly(L-lysine) or poly(L-ornithine) for 20-60 min resulted in maximal kinase activation. Poly(L-lysine), the most effective activator of the receptor kinase, was used to characterize further the mechanisms of the kinase activation. Poly(L-lysine) activates the insulin receptor kinase by increasing the Vmax. without changing the Km. Poly(L-lysine) markedly stimulates the kinase activity of insulin receptor preparations that have lost both basal kinase activity and the ability to be stimulated by insulin. Insulin and poly(L-lysine) also differed in their ability to stimulate the kinase activity of prephosphorylated receptors. Prephosphorylation of the receptors did not affect the stimulation of the kinase by insulin. In contrast, prephosphorylation of receptors resulted in a markedly enhanced ability of poly(L-lysine) to stimulate kinase activity. These studies suggest that the mechanisms by which poly(L-lysine) and insulin activate the kinase are different. In conjunction with other additional evidence, it is suggested that poly(L-lysine) interacts directly with the beta-subunit of the receptor, thereby activating the receptor kinase.

The receptors for insulin and insulin-like growth factor (IGF)-I carry intrinsic tyrosine-specific protein kinases (TPK) in their cytoplasmic domains that show 84% homology. Our previous studies using tyrosine-containing synthetic polymers (Arch. Biochem. Biophys. 260, 416, 1988) revealed subtle differences between the two receptor TPKs. In the present study, low molecular weight kinase inhibitors were used to compare the two receptor TPKs purified from human placenta. Staurosporine was the most potent inhibitor of both receptor TPKs among the three inhibitors tested. It was 100 times more inhibitory to insulin receptor TPK (ED50 = 61nM) than IGF-I receptor TPK (ED50 = 6.2 microM). Apigenin and kaempferol showed approximately the same inhibitory potency toward both TPKs with a range of 10 approximately 1000 microM. Staurosporine is thus an excellent tool to biochemically characterize the two receptor TPKs as well as to selectively inhibit insulin-activated TPK in intact cells.

Phosphocellulose paper has been found to be the paper of choice in assaying protein kinase activities using [gamma-32P]ATP by the trichloroacetic acid method of precipitation and washing. A study of binding of ATP of increasing concentrations at constant specific activity with Whatman 3MM or ATP-coated Whatman 3 MM papers (in vogue) versus phosphocellulose paper (proposed here) has shown that the latter has the least affinity for ATP when washing is done with either trichloracetic acid or trichloroacetic acid containing pyrophosphate. In an experiment where the placental cytosolic protein kinase was serially diluted, the phosphocellulose paper was found to give higher signal/noise ratios at all dilutions studied compared to the other two papers. With regard to the technological side of washing the papers, we have found that the traditional method of cutting papers into small squares before loading the samples is perhaps not the best. Instead, we propose the use of a flat sheet matrix for loading the samples because this method ensures uniformity of washing among the samples while shaking is performed on a simple shaker. In addition, the whole paper matrix can provide an almost instantaneous autoradiogram of hundreds of samples facilitating biochemical experimentation with protein kinases.

Summary Insulin was found to stimulate the phosphorylation of the 21,000-dalton protein encoded by the ras oncogene of Harvey murine sarcoma virus in membrane fraction both in vivo and in vitro . When the human ras proteins expressed in E. coli were reconstituted with purified human insulin receptor, GTPase activity of normal or its mutated oncogenic ras protein was not stimulated by the addition of insulin. Likewise, tyrosine kinase activity or insulin binding capaeity of the receptor was not influenced when assayed in the presence of the ras proteins. These results suggest that ras proteins may be coupled with the insulin receptor system through some unidentified membrane factors.

Insulin-like growth factor (IGF) I receptor was purified from Triton X-100-solubilized human placental membranes by wheat germ agglutinin-Sepharose chromatography followed by immunoaffinity chromatography using alpha IR-3, a monoclonal antibody directed against the IGF-I receptor. Purification of 3200-fold and 2800-fold was achieved from wheat germ agglutinin-Sepharose eluates with regard to IGF-I binding and kinase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed two major protein bands corresponding to the alpha and beta subunits of the receptor, which accounted for at least 90% of the protein content. The purified receptor bound 10-20 micrograms of IGF-I/mg of protein and was more than 95% free of contamination by insulin receptor. It sedimented in glycerol gradients as a single species with a sedimentation coefficient of 13.7 S and gave three protein bands with Mr = approximately 300,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that alpha 2 beta 2 is an intact form of the IGF-I receptor. The purified receptor, when incubated with [gamma-32P] ATP, became phosphorylated at tyrosine residues of its beta subunit. This was stimulated 3-fold by IGF-I. It also had IGF-I-stimulated tyrosine kinase activity (5264 pmol of 32P incorporated/min/mg of protein) toward a synthetic peptide corresponding to the autophosphorylation site of pp60src. These data strongly suggest that it is a tyrosine-specific protein kinase.

Added N alpha-p-tosyl-l-arginine methyl ester or N alpha-benzoyl-l-arginine ethyl ester inhibited the stimulation by insulin of phosphorylation of the 95,000 dalton subunit of the insulin receptor both in a partially purified insulin receptor fraction from rat adipocytes and in a highly purified insulin receptor preparation from human placenta. N-alpha-p-tosyl-l-lysine chloromethyl ketone, N alpha-p-tosyl-l-lysine methyl ester, or N-acetyl-l-phenylalanine ethyl ester were much less potent, while N-benzoyl-1-alanine methyl ester was without effect. Inhibition of the phosphorylation by the arginine analogues did not require preincubation of the insulin receptor with inhibitors in the presence of insulin prior to phosphorylation. Inhibition by N alpha-p-tosyl-l-arginine methyl ester was decreased by preincubation of the receptor fraction with cold ATP and MnCl2. These results suggest that N alpha-p-tosyl-l-arginine methyl ester inhibits an initial ATP and Mn2+ dependent reaction in insulin-stimulated phosphorylation process.

Since tyrosine-specific protein kinase (TPK) is much less abundant than Ser Thr -specific kinases in cells, determination of TPK activity in crude cell extracts or column chromatography eluates has been difficult. This is compouded by the absence of a rapid, economical method for the separation of high endogenous protein phosphorylation background from exogenously added tyrosine-containing substrates. We have developed a new solid-phase assay, which provides high sensitivity and efficiency at a low cost for assaying the TPK activity of crude enzyme preparations. This assay utilizes immobilized tyrosine-containing synthetic polymers such as (Glu:Tyr, 4:1)n in polyacrylamide gels. The kinase reaction is started by adding crude enzyme solutions and [τ-32P]ATP-metal ion mixtures into microtiter-size wells made in the gels. After the phosphorylation reaction, the reaction mixtures are removed and the gels are prewashed in water followed by electrophoresis to completely remove free radioactive ATP. 32P incorporation into the immobilized TPK-specific substrate can be detected by autoradiography and quantitated by cutting the gel pieces and counting them with a liquid scintillation counter. The simple, rapid method should facilitate screening of TPK inhibitors and activators as well as examining the substrate specificity of TPKs. Other enzymes, including Ser Thr -specific protein kinases, can also be analyzed by this technique.

Neuraminidase treatment of the purified insulin receptor resulted in an increase in both insulin-binding and kinase activities. Neuraminidase-treated alpha and beta subunits moved further than native subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The enhancement of insulin-binding and kinase activities and increased mobility of the subunits on SDS-PAGE were not observed when the receptor was treated with neuraminidase in the presence of neuraminidase inhibitor. These results suggest that terminal sialic acid residues have a significant role in insulin-binding and kinase activities. The involvement of sialic acid residues in the activities of the receptor has not been detected by previous studies.

The notion of an insulin-dependent translocation of the glucose transporter in rat adipocytes was confirmed by immunoblotting and reconstitution of glucose transport activity of subcellular fractions. Quantitatively, however, significantly different results were obtained with these two techniques; when compared with reconstitution, immunoblotting detected translocation of a larger amount of the transporter from a low density microsome fraction to a plasma membrane fraction. The acidtropic agents chloroquine and dibucaine, which have been reported to inhibit the recycling of various receptors, were utilized to study the detailed translocation mechanism of the glucose transporter and the insulin receptor. These acidtropic agents caused accumulation of 125I-insulin in a subcellular fraction probably corresponding to lysosomes. They did not, however, significantly affect either the insulin-induced activation of glucose transport or the recycling of the transporter and the insulin receptor as detected by immunoblotting. About 50% of radioactivity released from adipocytes which were allowed to internalize insulin was due to intact insulin, and chloroquine did not change the release rate of intact insulin, raising the possibility of receptor-mediated exocytosis of insulin. The release of degraded insulin decreased with chloroquine treatment. The results are consistent with the idea that these acidtropic agents mainly act to inhibit degradation of insulin in lysosomes, and their effect on the recycling of the glucose transporter and the insulin receptor is minimal, indicating that the recycling of these membrane proteins proceeds irrespective of organelle acidification. Electron micrographs showed vesicles underneath the plasma membranes, with sizes similar to those of the low density microsome fraction where the internalized glucose transporter and the insulin receptor were located.

Summary Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues by the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant Km. The β (235 K) subunit of fodrin, but not the α (240 K) subunit, was phosphorylated by the kinase. Neither the α (240 K) subunit nor the β (220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction.

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.