Insulin and insulin-like growth factor II differentially regulate endocytic sorting and stability of insulin receptor isoform A

The insulin receptor isoform A (IR-A) binds both insulin and insulin-like growth factor (IGF)-II, although the affinity for IGF-II is 3-10-fold lower than insulin depending on a cell and tissue context. Notably, in mouse embryonic fibroblasts lacking the IGF-IR and expressing solely the IR-A (R-/IR-A), IGF-II is a more potent mitogen than insulin. As receptor endocytosis and degradation provide spatial and temporal regulation of signaling events, we hypothesized that insulin and IGF-II could affect IR-A biological responses by differentially regulating IR-A trafficking. Using R-/IR-A cells, we discovered that insulin evoked significant IR-A internalization, a process modestly affected by IGF-II. However, the differential internalization was not due to IR-A ubiquitination. Notably, prolonged stimulation of R-/IR-A cells with insulin, but not with IGF-II, targeted the receptor to a degradative pathway. Similarly, the docking protein insulin receptor substrate 1 (IRS-1) was down-regulated after prolonged insulin but not IGF-II exposure. Similar results were also obtained in experiments using [NMeTyr(B26)]-insulin, an insulin analog with IR-A binding affinity similar to IGF-II. Finally, we discovered that IR-A was internalized through clathrin-dependent and -independent pathways, which differentially regulated the activation of downstream effectors. Collectively, our results suggest that a lower affinity of IGF-II for the IR-A promotes lower IR-A phosphorylation and activation of early downstream effectors vis à vis insulin but may protect IR-A and IRS-1 from down-regulation thereby evoking sustained and robust mitogenic stimuli.     

Decorin binds myostatin and modulates its activity to muscle cells

Myostatin, a member of TGF-beta superfamily of growth factors, acts as a negative regulator of skeletal muscle mass. The mechanism whereby myostatin controls the proliferation and differentiation of myogenic cells is mostly clarified. However, the regulation of myostatin activity to myogenic cells after its secretion in the extracellular matrix (ECM) is still unknown. Decorin, a small leucine-rich proteoglycan, binds TGF-beta and regulates its activity in the ECM. Thus, we hypothesized that decorin could also bind to myostatin and participate in modulation of its activity to myogenic cells. In order to test the hypothesis, we investigated the interaction between myostatin and decorin by surface plasmon assay. Decorin interacted with mature myostatin in the presence of concentrations of Zn(2+) greater than 10microM, but not in the absence of Zn(2+). Kinetic analysis with a 1:1 binding model resulted in dissociation constants (K(D)) of 2.02x10(-8)M and 9.36x10(-9)M for decorin and the core protein of decorin, respectively. Removal of the glycosaminoglycan chain by chondroitinase ABC digestion did not affect binding, suggesting that decorin could bind to myostatin with its core protein. Furthermore, we demonstrated that immobilized decorin could rescue the inhibitory effect of myostatin on myoblast proliferation in vitro. These results suggest that decorin could trap myostatin and modulate its activity to myogenic cells in the ECM.     

Evolution of the insulin receptor family and receptor isoform expression in vertebrates

The molecular phylogeny of the vertebrate insulin receptor (IR) family was reconstructed under maximum likelihood (ML) to establish homologous relationships among its members. A sister group relationship between the orphan insulin-related receptor (IRR) and the insulin-like growth factor 1 receptor (IGF1R) to the exclusion of the IR obtained maximal bootstrap support. Although both IR and IGF1R were identified in all vertebrates, IRR could not be found in any teleost fish. The ancestral character states at each position of the receptor molecule were inferred for IR, IRR + IGF1R, and all 3 paralogous groups based on the recovered phylogeny using ML in order to determine those residues that could be important for the specific function of IR. For 18 residues, ancestral character state of IR was significantly distinct (probability >0.95) with respect to the corresponding inferred ancestral character states both of IRR + IGF1R and of all 3 vertebrate paralogs. Most of these IR distinct (shared derived) residues were located on the extracellular portion of the receptor (because this portion is larger and the rate of generation of IR shared derived sites is uniform along the receptor), suggesting that functional diversification during the evolutionary history of the family was largely generated modifying ligand affinity rather than signal transduction at the tyrosine kinase domain. In addition, 2 residues at positions 436 and 1095 of the human IR sequence were identified as radical cluster-specific sites in IRR + IGF1R. Both Ir and Irr have an extra exon (namely exon 11) with respect to Igf1r. We used the molecular phylogeny to infer the evolution of this additional exon. The Irr exon 11 can be traced back to amphibians, whereas we show that presence and alternative splicing of Ir exon 11 seems to be restricted exclusively to mammals. The highly divergent sequence of both exons and the reconstructed phylogeny of the vertebrate IR family strongly indicate that both exons were acquired independently by each paralog.     

Protein kinase A modulates the activity of a major human isoform of ABCG1

ABCG1 is an ABC half-transporter that exports cholesterol from cells to HDL. This study set out to investigate differences in posttranslational processing of two human ABCG1 protein isoforms, termed ABCG1(+12) and ABCG1(- 12), that differ by the presence or absence of a 12 amino acid peptide. ABCG1(+12) is expressed in human cells and tissues, but not in mice. We identified two protein kinase A (PKA) consensus sites in ABCG1(+12), absent from ABCG1(- 12). Inhibition of PKA with either of two structurally unrelated inhibitors resulted in a dose-dependent increase in cholesterol export from cells expressing ABCG1(+12), whereas ABCG1(- 12)-expressing cells were unaffected. This was associated with stabilization of the ABCG1(+12) protein, and ABCG1(+12)-S389 was necessary to mediate these effects. Mutation of this serine to aspartic acid, simulating a constitutively phosphorylated state, resulted in accelerated degradation of ABCG1(+12) and reduced cholesterol export. Engineering an equivalent PKA site into ABCG1(- 12) rendered this isoform responsive to PKA inhibition, confirming the relevance of this sequence. Together, these results demonstrate an additional level of complexity to the posttranslational control of this human ABCG1 isoform that is absent from ABCG1(- 12) and the murine ABCG1 homolog.     

Varying brain insulin concentrations differentially regulate the fetal brain insulin receptor

We investigated the downregulating effect of varying states (physiologic and pharmacologic) of systemic and intracranial hyperinsulinism on the 28 to 30 day fetal rabbit brain insulin receptor. Alloxan-induced maternal diabetes (n = 5) produced mild fetal hyperinsulinemia (D) (plasma insulin concentrations = 59.80 +/- 8.10 microU/ml, control = 26.25 +/- 3.70; p less than 0.01), whereas systemic administration (IMI) of 1.0 U (n = 4) and 2.0 U (n = 4) of insulin to the fetus resulted in moderate (103.13 +/- 34.63 microU/ml) and severe (288.3 +/- 51 microU/ml) fetal hyperinsulinemia respectively. All three states of systemic hyperinsulinemia neither altered the fetal brain insulin content nor the brain insulin receptor number and affinity. 0.01 U (n = 4) of intracranial insulin administration (ICI) increased the brain insulin content four-fold (p less than 0.01) but did not alter the brain insulin receptor number or affinity. 0.1 (n = 5) and 2.0 U (n = 7) of intracranial insulin increased the brain insulin content to supraphysiologic concentrations (p less than 0.01) and decreased the fetal brain insulin receptor number (p less than 0.01), the affinity remaining constant. We conclude that 1) regardless of the ability of insulin to cross the blood brain barrier, the downregulation of the brain insulin receptor is insulin dose-dependent and 2) the downregulation of the fetal brain insulin receptor is not a physiologic but a pharmacologic effect of insulin.     

Alternatively spliced FGFR-1 isoform signaling differentially modulates endothelial cell responses to peroxynitrite

Mounting experimental evidence has suggested that the trophic environment of cells in culture is an important determinant of their vulnerability to the cytotoxic effects of reactive oxidants such as peroxynitrite (ONOO(-)). However, acidic fibroblast growth factor (FGF-1)-induced signaling renders some cells more sensitive and others resistant to the cytotoxic effects of ONOO(-). To determine whether alternatively spliced fibroblast growth factor receptor (FGFR-1) isoforms are responsible for this differential response, we have stably transfected FGFR-negative rat brain-derived resistant vessel endothelial cells (RVEC) with human cDNA sequences encoding either FGFR-1 alpha or FGFR-1 beta. FGF-1 treatment of RVEC(R-1 alpha) transfectants enhanced ONOO(-)-mediated cell death in a manner dependent upon FGFR-1 tyrosine kinase, MEK/Erk 1/2 kinase, and p38 MAP kinase activities and independent of Src-family kinase (SFK) activity. FGF-1 treatment of RVEC(R-1 beta) transfectants inhibited the cytotoxic effects of ONOO(-) in a manner dependent upon FGFR-1 tyrosine kinase, MEK/Erk 1/2 kinase, and SFK activities and independent of p38 MAP kinase activity. FGF-1-induced preactivation of both FGFR-1 tyrosine and Erk 1/2 kinases was detected in both RVEC(R-1 alpha) and RVEC(R-1 beta) transfectants. FGF-1-induced preactivation of p38 MAPK was restricted to RVEC(R-1 alpha) transfectants, whereas, ligand-induced preactivation of SFK was limited to RVEC(R-1 beta) transfectants. Collectively, these results both reemphasize the role of extracellular trophic factors and their receptor-mediated signaling pathways during cellular responses to oxidant stress and provide a first indication that the alternatively spliced FGFR-1 isoforms induce differential signal transduction pathways.     

Bombesin receptor subtype-3 modulates plasma insulin concentration

Mice lacking a functional bombesin receptor subtype-3 (BRS-3) develop mild obesity. However, the origin of obesity in BRS-3 knockout (KO) mice remains unclear. We used a strain-crossing strategy to investigate the physiological role of the BRS-3 pathway. We crossed female heterozygous BRS-3 KO mice (X-/X) and male KK-Ay mice (Ay/+) to obtain BRS-3 KO/KK-Ay hybrid animals. In X-/Y:Ay/+ mice, plasma insulin concentrations were significantly higher, and on the oral glucose tolerance test, the additional secretion of insulin was impaired compared to other genotypes. Our results indicate that the BRS-3 pathway contributes to the regulation of plasma insulin concentrations.     

The sterol-binding antibiotic nystatin differentially modulates ligand binding of the bovine hippocampal serotonin1A receptor

We have monitored the ligand binding of the bovine hippocampal 5-HT1A receptor following treatment with the sterol-binding antifungal antibiotic nystatin. Nystatin considerably inhibits the specific binding of the antagonist to 5-HT1A receptors in a concentration-dependent manner. However, the specific agonist binding does not show significant changes. Fluorescence polarization measurements of membrane probes incorporated at different locations in the membrane revealed a substantial decrease in the membrane order in the interior of the bilayer. Experiments with cholesterol-depleted membranes indicate that the action of nystatin is mediated through membrane cholesterol. These results represent the first report on the effect of a cholesterol-perturbing agent on the ligand-binding activity of this important neurotransmitter receptor.     

The specific protein phosphatase inhibitor okadaic acid differentially modulates insulin action.

The pleiotropic nature of insulin action suggests diverse mechanisms of signal transduction for the hormone. The specific protein phosphatase inhibitor, okadaic acid, is utilized to differentiate metabolic pathways that may be regulated by phosphorylation or dephosphorylation of key enzymes. In H-35 hepatoma cells, okadaic acid inhibits insulin-stimulated glycogen synthesis with an IC50 of 400 nM. In contrast, activation of lipogenesis by insulin is inhibited with an IC50 of 50 nM okadaic acid. The toxin also inhibits stimulation of lipogenesis in these cells by the insulin-sensitive inositol glycan enzyme modulator. In isolated rat adipocytes, insulin-stimulated lipogenesis is also inhibited by okadaic acid with an IC50 of approximately 1,700 nM. The antilipolytic effect of insulin in these cells is more sensitive to okadaic acid, exhibiting an IC50 of 150 nM. Maximal activation of lipogenesis by insulin is dramatically reduced by okadaic acid with no effect on the concentration required for half-maximal activation, whereas the sensitivity of insulin-induced antilipolysis is attenuated by okadaic acid, with no apparent reduction in the maximal effect of the hormone. Taken together, these data suggest that specific phosphatases may be differentially involved in some of the metabolic pathways regulated by insulin.     

Proteolytic activity in the insulin receptor

When a highly purified preparation of rat liver insulin receptor is incubated with trypsin, the receptor develops hydrolytic activity towards N alpha-benzoyl-L-arginine ethyl ester, N alpha-p-tosyl-L-arginine methyl ester, and N alpha-benzoyl-DL-arginine-p-nitroanilide, (compounds which are synthetic substrates of trypsin). The activity toward N alpha-benzoyl-DL-arginine-p-nitroanilide is inhibited by soybean trypsin inhibitor but not by N alpha-p-tosyl-L-lysil chloromethyl ketone. These data are consistent with the presence of proteolytic activity in the insulin receptor specific for the bonds whose carbonyl functions are provided by arginine but not by lysine. Furthermore we found that the esterase activity toward N alpha-benzoyl-L-arginine ethyl ester in the presence of trypsin is enhanced by insulin, and that the concentration of insulin that produced the half maximum stimulation is of the same magnitude as the dissociation constant for the soluble receptor. These data suggest that the insulin receptor is a zymogen, activated by trypsin, and based on its specific activity, may be the protease which releases a peptide chemical mediator of intracellular action of insulin.     

Electroconvulsive shock treatment differentially modulates cortical and subcortical endocannabinoid activity

Previous studies indicate that the endocannabinoid system is a potential target for the treatment of depression. To further examine this question we assessed the effects of electroconvulsive shock (ECS) treatment, both a single session and 10 daily sessions, on endocannabinoid content, CB(1) receptor binding parameters and CB(1) receptor-mediated [(35)S]GTPgammaS binding in the prefrontal cortex, hippocampus, hypothalamus and amygdala. A single ECS session resulted in a general reduction in the binding affinity of the CB(1) receptor in all brain regions examined, as well as reductions in N-arachidonylethanolamine (anandamide) content in the prefrontal cortex and the hippocampus, reduced hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) in the prefrontal cortex and an increase in the binding site density of the CB(1) receptor in the amygdala. Following 10 ECS sessions, all these effects subsided except for the reductions in anandamide content in the prefrontal cortex, which increased in magnitude, as well as the reductions in FAAH activity in the prefrontal cortex. Additionally, repeated ECS treatment resulted in a significant reduction in the binding site density of the CB(1) receptor in the prefrontal cortex, but did not alter CB(1) receptor-mediated [(35)S]GTPgammaS binding. Repeated ECS treatment also significantly enhanced the sensitivity of CB(1) receptor-mediated [(35)S]GTPgammaS binding in the amygdala. Collectively, these data demonstrate that ECS treatment results in a down-regulation of cortical and an up-regulation of subcortical endocannabinoid activity, illustrating the possibility that the role of the endocannabinoid system in affective illness may be both complex and regionally specific.     

Intrinsic kinase activity of the insulin receptor

Since the identification of the insulin receptor by insulin-binding activity almost two decades ago, our understanding of the structure and function of the insulin receptor has progressed tremendously. The importance of the intrinsic tyrosine protein kinase activity of the insulin receptor is implied by the fact that the insulin receptor belongs to a family of receptor tyrosine kinases which play a role in growth control, by experiments demonstrating the intimate association of normal kinase activity and insulin action, and by evidence that the intrinsic kinase activity can be regulated under certain conditions. There are still some major gaps in our knowledge concerning the structure/function of the insulin receptor such as how activation of the intrinsic kinase activity of the receptor leads to altered cellular physiology. The kinase may phosphorylate endogenous substrates or autophosphorylation may simply alter beta subunit conformation so it can then interact with an effector system (i.e. a serine kinase) directly, or indirectly through a G-protein. The truth may lie somewhere between these two pathways.     

p38 and p42/44 MAPKs differentially regulate progesterone receptor A and B isoform stabilization

Progesterone receptor (PR) isoforms (PRA and PRB) are implicated in the progression of breast cancers frequently associated with imbalanced PRA/PRB expression ratio. Antiprogestins represent potential antitumorigenic agents for such hormone-dependent cancers. To investigate the mechanism(s) controlling PR isoforms degradation/stability in the context of agonist and antagonist ligands, we used endometrial and mammary cancer cells stably expressing PRA and/or PRB. We found that the antiprogestin RU486 inhibited the agonist-induced turnover of PR isoforms through active mechanism(s) involving distinct MAPK-dependent phosphorylations. p42/44 MAPK activity inhibited proteasome-mediated degradation of RU486-bound PRB but not PRA in both cell lines. Ligand-induced PRB turnover required neosynthesis of a mandatory down-regulating partner whose interaction/function is negatively controlled by p42/44 MAPK. Such regulation strongly influenced expression of various endogenous PRB target genes in a selective manner, supporting functional relevance of the mechanism. Interestingly, in contrast to PRB, PRA stability was specifically increased by MAPK kinase kinase 1-induced p38 MAPK activation. Selective inhibition of p42/p44 or p38 activity resulted in opposite variations of the PRA/PRB expression ratio. Moreover, MAPK-dependent PR isoforms stability was independent of PR serine-294 phosphorylation previously proposed as a major sensor of PR down-regulation. In sum, we demonstrate that MAPK-mediated cell signaling differentially controls PRA/PRB expression ratio at posttranslational level through ligand-sensitive processes. Imbalance in PRA/PRB ratio frequently associated with carcinogenesis might be a direct consequence of disorders in MAPK signaling that might switch cellular responses to hormonal stimuli and contribute towards pathogenesis.     

Fibronectin receptor modulates cyclin-dependent kinase activity.

The high affinity fibronectin receptor (FNR) is expressed by hematopoietic cells, fibroblasts, and proliferating epidermal cells. Expression of this integrin is altered by chemical and viral transformation, suggesting that FNR dysfunction may play a role in growth control. This study demonstrates that exposing FA-K562 cells to glycine-arginine-glycine-aspartate-serine (GRGDS), a peptide ligand of the FNR, specifically stimulates p34/cdc2- and cyclin A-associated kinase activities. This occurs within 2 h of peptide addition. The 110-kDa form of the retinoblastoma protein appears within 3 h of GRGDS addition, consistent with activation of a G1/S kinase. DNA staining profiles demonstrate that GRGDS induces cell cycle progression within 24 h. Increased anchorage-independent growth is subsequently observed in GRGDS-treated FA-K562 cells. The control peptide, GRGES, which cannot bind the FNR, has none of these effects. This demonstrates that an extracellular integrin ligand can regulate cell proliferation. Furthermore, these results suggest that integrins link the extracellular environment and intracellular growth regulators.     

Cholesterol modulates glycolipid conformation and receptor activity

We document a new dimension of surface recognition in which communication is controlled through the collective behavior of lipids. Membrane cholesterol induces a tilt in glycolipid receptor headgroup, resulting in loss of access for ligand binding. This property appears to organize erythrocyte blood group presentation and glycolipid receptor function during the activation of sperm fertility, suggesting that lipid 'allostery' is a means to regulate membrane recognition processes.     

Binding thermodynamics and intrinsic activity of adenosine A1 receptor ligands

A thermodynamic analysis of the binding to rat cortex adenosine A1 receptors of 5'-deoxyribose-N6-cyclopentyladenosine (full agonist) and several 8-alkylamino homologues of N6-cyclopentyladenosine (partial agonists) was performed. The intrinsic activity of the compounds was also evaluated by measuring the inhibition of forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (c-AMP) levels in isolated epididymal rat adipocytes. Standard free energy (deltaG), enthalpy (deltaH ) and entropy (deltaS ) of the binding equilibrium were determined by affinity measurements carried out at different temperatures (0, 10, 20, 25, 30 degrees C). Affinity constants of drug-receptor interactions were obtained by displacement experiments in the presence of 1nM [3H]N6-cyclohexyladenosine. Levels of c-AMP were evaluated by performing competitive binding assays. As the affinity of the ligands was found to increase with temperature enhancement, the binding of full and partial agonists is therefore totally entropy-driven. Standard entropy values of a wide series of adenosine derivatives, including the compounds under examination, are strictly correlated to those of intrinsic activity. Similarly, deltaS values appear correlated to the in vivo ability of the adenosine derivatives to inhibit rat heart rate. Thermodymanic data of adenosine A1 receptor ligands are proposed as an indicator of their pharmacodynamics.     

A novel murine Staufen isoform modulates the RNA content of Staufen complexes

Mouse Staufen (mStau) is a double-stranded RNA-binding protein associated with polysomes and the rough endoplasmic reticulum (RER). We describe a novel endogenous isoform of mStau (termed mStau(i)) which has an insertion of six amino acids within dsRBD3, the major double-stranded RNA (dsRNA)-binding domain. With a structural change of the RNA-binding domain, this conserved and widely distributed isoform showed strongly impaired dsRNA-binding ability. In transfected cells, mStau(i) exhibited the same tubulovesicular distribution (RER) as mStau when weakly expressed; however, when overexpressed, mStau(i) was found in large cytoplasmic granules. Markers of the RER colocalized with mStau(i)-containing granules, showing that overexpressed mStau(i) could still be associated with the RER. Cotransfection of mStau(i) with mStau relocalized overexpressed mStau(i) to the reticular RER, suggesting that they can form a complex on the RER and that a balance between these isoforms is important to achieve proper localization. Coimmunoprecipitation demonstrated that the two mStau isoforms are components of the same complex in vivo. Analysis of the immunoprecipitates showed that mStau is a component of an RNA-protein complex and that the association with mStau(i) drastically reduces the RNA content of the complex. We propose that this new isoform, by forming a multiple-isoform complex, regulates the amount of RNA in mStau complexes in mammalian cells.     

Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.

Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.