Transmembrane domain of EphA1 receptor forms dimers in membrane-like environment

Eph receptor tyrosine kinases (RTKs) are activated by a ligand-mediated dimerization in the plasma membrane and subjected to clusterization at a high local density of receptors and their membrane-anchored ligands. Interactions between transmembrane domains (TMDs) were recognized to assist to the ligand-binding extracellular domains in the dimerization of some RTKs, whereas a functional role of Eph-receptor TMDs remains unknown. We have studied a propensity of EphA1-receptor TMDs (TMA1) to self-association in membrane-mimetic environment. Dimerization of TMA1 in SDS environment was revealed by SDS-PAGE and confirmed by FRET analysis of the fluorescently labeled peptide (K_d = 7.2 ± 0.4 μM at 1.5 mM SDS). TMA1 dimerization was also found in 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes (ΔG = −15.4 ± 0.5 kJ/mol). Stability of TMA1 dimers is comparable to the reported earlier stability of TMD dimers of fibroblast growth factor receptor 3 and tenfold weaker than the stability of TMD dimers of glycophorin A possessing high propensity to dimerization. Our results suggest that EphA1-receptor TMD contribute to the dimerization-mediated receptor activation. An assumed role of the TMD interactions is the efficient signal transduction due to TMD-driving mutual orientation of kinase domains in dimers, while a relatively low force of the TMD interactions does not prevent a ligand-controlled regulation of the receptor dimerization.     

The insulin-binding domain of insulin receptor is encoded by exon 2 and exon 3.

Insulin receptors are disulfide-linked oligotetramers composed of two heterodimers each containing a 130-kDa alpha subunit and a 90-kDa beta subunit. Insulin binds to the extracellular alpha subunit, and in the process stimulates the autophosphorylation of the beta subunit and the expression of tyrosine kinase activity. Studies combining the use of photoaffinity labeling and immunoprecipitation with anti-peptide antibody have directly demonstrated that the cysteine-rich domain, encoded by exon 3, in the alpha subunit is part of the insulin-binding site of the receptor. Experiments with chimeric insulin receptors and chimeric insulin-like growth factor I receptors have confirmed that the cysteine-rich domain constitutes a part of the insulin-binding site. In addition, results from these experiments suggest that the N-terminal sequence, encoded by exon 2, in the alpha subunit also participates in insulin binding. In this review it is proposed that, assuming two insulin-binding sites per each holoreceptor oligotetramer, each insulin-binding domain may contain respectively two sub-domains for hydrophobic and charge contact with insulin, and that high-affinity binding would require the interaction of both subunits with the possibility of each subunit reciprocally contributing one of the sub-domains.     

Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway

Estrogen and insulin-like-growth factor 1 (IGF-1) are potent mitogenic stimuli that share important properties in the control of cellular proliferation. However, the coupling between the signaling cascades of estrogen receptors alpha and beta and the IGF-1 receptor (IGF-1R) is poorly understood. Therefore, we selectively transfected estrogen receptor alpha or beta in COS7 and HEK293 cells, which contain IGF-1R. In presence of estrogen receptor alpha but not beta, 17beta-estradiol (E2) rapidly induces phosphorylation of the IGF-1R and the extracellular signal-regulated kinases 1/2. Furthermore, upon stimulation with E2, estrogen receptor alpha but not beta bound rapidly to the IGF-1R in COS7 as well as L6 cells, which express all investigated receptors endogenously. Control experiments in the IGF-1R-deficient fibroblast cell line R(-) showed that after stimulation with E2 only estrogen receptor alpha bound to the transfected IGF-1R. Overexpression of dominant negative mitogen-activated protein kinases kinase inhibited this effect. Finally, estrogen receptor alpha but not beta is required to induce the activation of the estrogen receptor-responsive reporter ERE-LUC in IGF-1-stimulated cells. Taken together, these data demonstrate that ligand bound estrogen receptor alpha is required for rapid activation of the IGF-1R signaling cascade.     

Novel imidazo[1,2-a]pyridine based inhibitors of the IGF-1 receptor tyrosine kinase: Optimization of the aniline

Following the discovery of imidazopyridine 1 as a potent IGF-1R tyrosine kinase inhibitor, the aniline part has been modified with the aim to optimize the properties of this series. The structure-activity relationships against IGF-1R kinase activity as well as inhibition of the hERG ion channel are discussed.     

IGF-2r的结构和功能及其在胚胎发育中的作用

胰岛素样生长因子-2受体（insulin-like growth factor-2 receptor,IGF-2r）和非阳离子依赖型甘露糖-6-磷酸受体（cation-independent mannose-6-phosphate receptor,CI-MPR）为同一分子,属多结构域跨膜糖蛋白。IGF．2r与阳离子依赖型M6P受体（cation-dependent mannose-6-phosphotase receptor,CD,MPR）共同介导溶酶体酶的分选和转运过程,对溶酶体的形成起重要作用。IGF-2／M6P受体除能与溶酶体酶、TGF-β前体等M6P配体结合外,还能与非糖基化的IGF-2、视黄酸等作用,调节蛋白质的转运和跨膜信号传导等活动。IGF-2r为父源性印迹（paternal imprinted）基因,基因的印迹、表达受基因印迹控制区（imprinting control region,ICR）的甲基化差异修饰所调控。IGF-2r基因表达缺失出现胎儿肥大、心脏和胎盘发育不全、围产期死亡等异常现象,证明IGF-2r在胚胎发育过程中起重要作用。     

1.42 Å crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1.     

The insulin-like growth factor 1 (IGF-1) receptor is a substrate for gamma-secretase-mediated intramembrane proteolysis

Several type-1 membrane proteins undergo regulated intramembrane proteolysis resulting in the generation of biologically active protein fragments. Presenilin-dependant gamma-secretase activity is central to this event and includes amyloid precursor protein (APP), Notch and ErbB4 as substrates. Here we show that the insulin-like growth factor 1 receptor (IGF-IR) undergoes regulated intramembrane proteolysis. A metalloprotease-dependant ectodomain-shedding event generates a approximately 52 kDa IGF-IR-carboxyl terminal domain (CTD). The IGF-IR-CTD is consequentially a substrate for gamma-secretase cleavage, liberating a approximately 50 kDa intracellular domain (ICD) that can be inhibited by a specific gamma-secretase inhibitor. This study suggests that the IGF-IR is a substrate for gamma-secretase and may mediate a function independent of its role as a receptor tyrosine kinase.     

Estrogen signals via an extra-nuclear pathway involving IGF-1R and EGFR in tamoxifen-sensitive and -resistant breast cancer cells

Activation of IGF-1R can activate metalloproteinases which release heparin-binding EGF (Hb-EGF) and lead to EGFR-dependent MAPK activation in certain tissues. We postulated that this pathway is operative in E₂-induced MAPK activation in breast cancer tissues. As evidence, we showed that E₂ rapidly induced the phosphorylation of both IGF-1R and EGFR and that siRNA knockdown or selective inhibitors against either growth factor receptor inhibited E₂-induced MAPK activation. The selective inhibitors or knockdown of either IGF-1R or EGFR significantly inhibited cell growth and reversed cell death protection induced by E₂ in MCF-7 cells. Our data support the conclusion that the IGF-1R acts upstream of EGFR in a linear pathway which mediates E₂ action on MAPK activation, cell growth stimulation and anti-apoptosis in breast cancer cells. During the process of development of tamoxifen resistance this pathway is up-regulated with increased sensitivity to activate EGFR for cell growth and protection against apoptosis. Surprisingly, translocation of ERα out of the nucleus into the cytoplasm, mediated by c-Src, occurs during development of resistance. This effect can be abrogated by administration of the c-Src inhibitor, PP2 which also restores sensitivity to tamoxifen.     

Focal adhesion kinase (FAK) activates and stabilizes IGF-1 receptor

Recent studies have shown a direct association between IGF-1R and FAK, two important mediators of cell growth, survival and migration. However, the mechanism by which FAK affects IGF-1R function remains unknown. This study investigates the potential role of FAK in mediating activation and stability of IGF-1R. Autophosphorylation and phosphorylation capacities of wild type and mutant IGF-1R were studied. Surprisingly, we found that the mutant IGF-1R lacking the three core tyrosine residues in the activation-loop can be phosphorylated although it is unable to undergo autophosphorylation, suggesting that another kinase possesses the ability to phosphorylate IGF-1R. By using wild type MEFs and FAK−/− MEFs we could demonstrate that FAK mediates activation-loop independent phosphorylation, as well as Akt and ERK activation. Furthermore, the stability of IGF-1R was decreased upon FAK siRNA or inactivation. Taken together, our data suggest a role for FAK in phosphorylation, signaling and stability of the IGF-1R.     

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor,a human GPCR

The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His)₆ tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.     

IGF-1 receptor inhibition by picropodophyllin in medulloblastoma

The insulin-like growth factor-1 receptor (Igf1r) is a multifunctional membrane-associated tyrosine kinase associated with regulation of transformation, proliferation, differentiation and apoptosis. Increased IGF pathway activity has been reported in human and murine medulloblastoma. Tumors from our genetically-engineered medulloblastoma mouse model over-express Igf1r, and thus this mouse model is a good platform with which to study the role of Igf1r in tumor progression. We hypothesize that inhibition of IGF pathway in medulloblastoma can slow or inhibit tumor growth and metastasis. To test our hypothesis, we tested the role of IGF in tumor growth in vitro by treatment with the tyrosine kinase small molecule inhibitor, picropodophyllin (PPP), which strongly inhibits the IGF pathway. Our results demonstrate that PPP-mediated downregulation of the IGF pathway inhibits mouse tumor cell growth and induces apoptotic cell death in vitro in primary medulloblastoma cultures that are most reflective of tumor cell behavior in vivo.     

Inhibition of estradiol binding to its receptor by the cupric ion

 Treatment of human recombinant estrogen receptor (hER) expressed in yeast with very low concentrations of the cupric ion decreased its ability to bind [³H]estradiol ([³H]E₂) and [¹²⁵I]tamoxifen aziridine (minimal Cu²⁺ concentration: 1 nM). This decrease was reflected in a loss of immunoreactivity for monoclonal antibodies raised against the hormone binding domain (HBD). An ER recombinant expressing solely the HBD confirmed that the ion operated at this level. Cysteines located within this domain contributed to the inhibitory action of the Cu²⁺ in view of a partial restoration of the [³H]E₂ binding activity with β-mercaptoethanol. Histidines were also implicated since the influence of Cu²⁺ on the [³H]E₂ binding parameters (Scatchard plot analysis) was maintained after oxidation of thiol groups by methyl methanethiosulfonate, and partly reversed by imidazole. Received: 10 December 1997 / Accepted: 26 June 1998     

Ligand-induced ErbB receptor dimerization

Structural studies have provided important new insights into how ligand binding promotes homodimerization and activation of the EGF receptor and the other members of the ErbB family of receptor tyrosine kinases. These structures have also suggested possible explanations for the unique properties of ErbB2, which has no known ligand and can cause cell transformation (and tumorigenesis) by simple overexpression. In parallel with these advances, studies of the EGF receptor at the cell surface increasingly argue that the structural studies are missing key mechanistic components. This is particularly evident in the structural prediction that EGF binding linked to receptor dimerization should be positively cooperative, whereas cell-surface EGF-binding studies suggest negative cooperativity. In this review, I summarize studies of ErbB receptor extracellular regions in solution and of intact receptors at the cell surface, and attempt to reconcile the differences suggested by the two approaches. By combining results obtained with receptor ‘parts’, it is qualitatively possible to explain some models for the properties of the whole receptor. These considerations underline the need to consider the intact ErbB receptors as intact allosterically regulated enzymes, and to combine cellular and structural studies into a complete picture.     

Mammary Specific Transgenic Over-expression of Insulin-like Growth Factor-I (IGF-I) Increases Pig Milk IGF-I and IGF Binding Proteins,with no Effect on Milk Composition or Yield

IGF-I regulates lactation by stimulating mammary mitogenesis, inhibiting apoptosis, and partially mediating the effects of growth hormone on lactogenesis. Herein, lactation performance during first and second parity was assessed in transgenic swine (TG) that over-expressed human IGF-I in milk under the control of the bovine α-lactalbumin promoter, regulatory regions and signal peptide coding sequence. Milk samples were collected throughout lactation (farrowing to d24) from TG sows and non-transgenic littermates (CON) and IGF-I, IGF-II, and IGFBP determined. Colostral (<24 h postpartum) IGF-I content was 26-fold greater (p < 0.001) in TG sows (949 ± 107 μg/L; range 228–1600 μg/L) than CON (36 ± 17.8 μg/L) and was 50- to 90-fold greater (p < 0.001) in mature milk (d2-24 postpartum). There was no effect of parity on milk IGF-I content. Milk IGF-II concentration was unaffected by IGF-I over-expression. Low molecular weight IGFBP (IGFBP-2 and -5) in the milk of TG sows were higher (p = 0.02) than CON in the early postpartum period, but did not differ in mature milk. Milk yield, determined by weigh-suckle-weigh, was similar in TG and CON as was litter weight gain. Milk nutrient composition was not significantly affected by IGF over-expression. Thus, mammary specific transgenic over-expression of IGF-I significantly increased milk IGF-I and IGFBP content, but did not impact lactation performance in swine.     

Small-molecule inhibition and activation-loop trans-phosphorylation of the IGF1 receptor

The insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase (RTK) that has a critical role in mitogenic signalling during embryogenesis and an antiapoptotic role in the survival and progression of many human tumours. Here, we present the crystal structure of the tyrosine kinase domain of IGF1R (IGF1RK), in its unphosphorylated state, in complex with a novel compound, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which we show is a potent inhibitor of both the unphosphorylated (basal) and phosphorylated (activated) states of the kinase. PQIP interacts with residues in the ATP-binding pocket and in the activation loop, which confers specificity for IGF1RK and the highly related insulin receptor (IR) kinase. In this crystal structure, the IGF1RK active site is occupied by Tyr1135 from the activation loop of an symmetry (two-fold)-related molecule. This dimeric arrangement affords, for the first time, a visualization of the initial trans-phosphorylation event in the activation loop of an RTK, and provides a molecular rationale for a naturally occurring mutation in the activation loop of the IR that causes type II diabetes mellitus.     

Insulin receptor structure and its implications for the IGF-1 receptor

The insulin receptor (isoforms IR-A and IR-B) and the type-I insulin-like growth factor receptor (IGF-1R) are homologous, multi-domain tyrosine kinases that bind insulin and IGF-1 with differing specificity. IR is involved in metabolic regulation and IGF-1R in normal growth and development. IR-A also binds IGF-2 with an affinity comparable to IGF-1R and, like the latter, is implicated in a range of cancers. The recent structure of the IR ectodomain dimer explains many features of ligand-receptor binding and provides insight into the structure of the intact ligand-binding site in both receptors. The structures of the L1-CR-L2 fragments of IR and IGF-1R reveal major differences in the regions that govern ligand specificity. The IR ectodomain X-ray structure raises doubts about that obtained by STEM reconstruction.     

Expression and regulation of mRNAs for insulin-like growth factor-I receptor and LH receptor in corpora lutea

Relationship between insulin-like growth factor-I receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF2 inhibited expression of IGF-IR and LHR. PGE2 negated the inhibiting effects of PGF2. These data suggest that IGF-I may be involved in regulating CL function, and maintaining CL structure through changes in expression of its receptors. Inhibited expression of IGF-IR by PGF2 may be part of mechanisms for regression of CL.     

The transmembrane domain directs TLR9 to intracellular compartments that contain TLR3

Toll-like receptors (TLRs) are type I transmembrane (TM) proteins indispensable for sensing microbial and viral infection. Despite their conserved primary structures, some TLRs that detect pathogen-derived nucleic acids (TLR3, TLR7, TLR8, and TLR9) are retained in the cytoplasm. The intracellular localization of TLR9 is important for its ability to discriminate self- and non-self DNA, but the mechanism by which it is retained in the cytoplasm is unclear. In the present study, we found that the TM domain of TLR9 directs its intracellular localization. The TM domain of TLR9 also targets CD25, a heterologous type I TM protein, to intracellular compartments that contain TLR9. We also found that TLR9 generally co-localizes with TLR3, although its linker region, not its TM domain, directs intracellular localization of TLR3. These data demonstrate that the TM domain of TLR9 is a critical regulatory element that targets TLR9 to its intracellular location.