Intracellular localization of phosphatidylinositide 3-kinase and insulin receptor substrate-1 in adipocytes: potential involvement of a membrane skeleton

Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1-PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells.     

Interaction of insulin receptor substrate-1 with the sigma3A subunit of the adaptor protein complex-3 in cultured adipocytes

Signaling through the insulin receptor tyrosine kinase involves its autophosphorylation in response to insulin and the subsequent tyrosine phosphorylation of substrate proteins such as insulin receptor substrate-1 (IRS-1). In basal 3T3-L1 adipocytes, IRS-1 is predominantly membrane-bound, and this localization may be important in targeting downstream signaling elements that mediate insulin action. Since IRS-1 localization to membranes may occur through its association with specific membrane proteins, a 3T3-F442A adipocyte cDNA expression library was screened with non-tyrosine-phosphorylated, baculovirus-expressed IRS-1 in order to identify potential IRS-1 receptors. A cDNA clone that encodes sigma3A, a small subunit of the AP-3 adaptor protein complex, was demonstrated to bind IRS-1 utilizing this cloning strategy. The specific interaction between IRS-1 and sigma3A was further verified by in vitro binding studies employing baculovirus-expressed IRS-1 and a glutathione S-transferase (GST)-sigma3A fusion protein. IRS-1 and sigma3A were found to co-fractionate in a detergent-resistant population of low density membranes isolated from basal 3T3-L1 adipocytes. Importantly, the addition of exogenous purified GST-sigma3A to low density membranes caused the release of virtually all of the IRS-1 bound to these membranes, while GST alone had no effect. These results are consistent with the hypothesis that sigma3A serves as an IRS-1 receptor that may dictate the subcellular localization and the signaling functions of IRS-1.     

Insulin receptor substrate-2 binds to the insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591-786

We compared the interaction between the insulin receptor (IR) and the IR substrate (IRS) proteins IRS-1 and IRS-2) using the yeast two-hybrid system. Both IRS proteins interact specifically with the cytoplasmic portion of the IR and the related insulin-like growth factor-I receptor, and these interactions require receptor tyrosine kinase activity. Alignment of IRS-1 and IRS-2 revealed two conserved domains at the NH2 terminus, called IH1PH and IH2PTB, which resemble a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, respectively. The IH2PTB binds to the phosphorylated NPXY motif (Tyr-960) in the activated insulin receptor, providing a specific mechanism for the interaction between the receptor and IRS-1. Although the IH2PTB of IRS-2 also interacts with the NPEY motif of the insulin receptor, it is not essential for the interaction between the insulin receptor and IRS-2 in the yeast two-hybrid system. IRS-2 contains another interaction domain between residues 591 and 786, which is absent in IRS-1. This IRS-2-specific domain is independent of the IH2PTB and does not require the NPEY motif; however, it requires a functional insulin receptor kinase and the presence of three tyrosine phosphorylation sites in the regulatory loop (Tyr-1146, Tyr-1150, and Tyr-1151). Importantly, this novel domain mediates the association between IRS-2 and insulin receptor lacking the NPXY motif and may provide a mechanism by which the stoichiometry of regulatory loop autophosphorylation enhances IRS-2 phosphorylation.     

Proliferation of human and mouse astrocytes in vitro: signalling through the protein kinase C pathway

While several mitogens for astrocytes have been described, the signal transduction pathway(s) that mediates their proliferative effect remains unclear; in this report, a major role for the protein kinase C (PKC) system is suggested by several lines of evidence. Firstly, biologically active phorbol esters, 4 beta-phorbol-12,13-dibutyrate and phorbol-12-myristate-13-acetate, increase the proliferation of astrocytes as determined by [3H]thymidine incorporation or bromodeoxyuridine immunofluorescence; this effect is not reproduced by a phorbol ester that binds to PKC but does not activate it (4 alpha-phorbol-12,13-didecanoate). Secondly, 2 relatively selective inhibitors of PKC, H7 and staurosporine, attenuate the basal rate of proliferation of astrocytes in concentrations that were not cytotoxic to cells. Thirdly, mitogen-enhanced proliferation of astrocytes can be blocked by PKC inhibitors; this is observed for all astrocyte mitogens tested. Fourthly, measurements of PKC enzyme activity in astrocytes in response to serum-mitogenic factors, or to staurosporine, revealed a statistically significant correlation with proliferation rate. The mediation by PKC is not dependent on species- or age factors, since neonatal mouse or adult human astrocytes gave comparable results. The results have relevance to normal development and reactive gliosis post-injury, 2 conditions where astrocytes undergo proliferation, and to glioma growth.     

Regulation of PTP-1 and insulin receptor kinase by fractions from cinnamon: implications for cinnamon regulation of insulin signalling

PURPOSE: We have previously reported incomplete cytotoxic responses of other human solid tumors (bladder, head and neck, ovarian and prostate) to paclitaxel. This finding is qualitatively different from the nearly complete response observed in monolayer cultures of human cancer cell lines. The present study examined the pharmacodynamics of paclitaxel in human breast tumors. METHODS: Three-dimensional histocultures of patient tumors were used. The cytostatic effect was evaluated by measurement of the inhibition of 48-h cumulative bromodeoxyuridine (BrdUrd) incorporation. The apoptotic effect was evaluated in terms of morphological changes and by in situ DNA end labeling. RESULTS: Paclitaxel produced partial cytostasis (approximately 30% maximum) and induced apoptosis (maximum apoptotic index of 3.3% to 29%) in all 15 tumors. More than 95% of apoptotic cells were BrdUrd labeled, but not all BrdUrd-labeled cells were apoptotic. The maximal apoptotic indices in the tumors were significantly correlated with the BrdUrd labeling index of untreated controls (r2 = 0.63, P < 0.01). The maximum apoptotic effect was observed at a tenfold lower drug concentration (0.1 microM) compared to the maximum cytostatic effect (1 microM). Neither of these effects was enhanced by increasing the drug concentration to 10 microM. CONCLUSIONS: The pharmacodynamics of paclitaxel in human breast tumors are comparable to those found in other human solid tumors. The labeling of apoptotic cells by BrdUrd and the correlation between the proliferation index and apoptosis suggest that drug-induced apoptosis is linked to cell proliferation and is completed after DNA synthesis. The finding that maximal cytostatic and apoptotic effects of paclitaxel were achieved at or below the clinically achievable concentration of 1 microM suggests further increasing the dose to elevate plasma concentration beyond 1 microM may not improve treatment outcome.     

Interaction of insulin receptor substrate-1 (IRS-1) with phosphatidylinositol 3-kinase: effect of substitution of serine for alanine in potential IRS-1 serine phosphorylation sites

Serine and threonine phosphorylation has been shown to down-regulate insulin signaling at multiple steps, including the receptor and downstream molecules such as insulin receptor substrate-1 (IRS-1). To further address the mechanism of this regulation at the level of IRS-1, we constructed a double serine mutant of IRS-1: S662A/S731A-IRS-1. The serines 662 and 731 mutated to alanine are surrounding tyrosines Y658 and Y727, respectively. These tyrosines are comprised in YXXM motifs, which are potential binding sites for the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase. In a first series of experiments using the yeast two-hybrid system, we show that IRS-1 interacts with p85alpha, and this interaction depends on tyrosine phosphorylation, as shown with the IRS-1 mutant F18 and 3Y-IRS-1. F18-IRS-1 contains 18 potential tyrosine phosphorylation sites mutated to phenylalanine; three of them, i.e. Y608, 628, and 658, which are potential binding sites for p85alpha, have been added back in the 3Y-IRS-1 mutant. The tyrosine phosphorylation of IRS-1, which is required for the interaction with p85alpha, is thought to occur via endogenous yeast kinases that phosphorylate IRS-1 at least on these PI 3-kinase-binding sites. Next, we show that not only p85alpha but also p55PIK, another regulatory subunit of PI 3-kinase, interacts with IRS-1 in yeast. Interestingly, for both regulatory subunits their interaction with IRS-1 is up-regulated by mutating serines 662 and 731 on IRS-1. In a previous study we found that insulin-stimulated PI 3-kinase activity was increased not only in the presence of S662A/S731A-IRS-1 but also under resting conditions compared with the activity seen with WT-IRS-1. Here we demonstrate in 293-EBNA cells overexpressing S662A/S731A-IRS-1 that insulin-stimulated protein kinase B activity is not augmented, whereas without insulin treatment, basal activity is increased compared with that in cells overexpressing wild-type IRS-1. In conclusion, we have shown that 1) potential serine phosphorylation sites on IRS-1, which are adjacent to YXXM binding motifs for PI 3-kinase, negatively regulate binding of IRS-1 to PI 3-kinase regulatory subunits; and 2) these modulations affect protein kinase B activity.     

Differential regulation of insulin receptor substrate-2 and mitogen-activated protein kinase tyrosine phosphorylation by phosphatidylinositol 3-kinase inhibitors in SH-SY5Y human neuroblastoma cells

Insulin-like growth factor I (IGF-I) is a potent neurotropic factor promoting the differentiation and survival of neuronal cells. SH-SY5Y human neuroblastoma cells are a well characterized in vitro model of nervous system growth. We report here that IGF-I stimulated the tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and insulin receptor substrate-2 (IRS-2) in a time- and concentration-dependent manner. These cells lacked IRS-1. After being tyrosine phosphorylated, IRS-2 associated transiently with downstream signaling molecules, including phosphatidylinositol 3-kinase (PI 3-K) and Grb2. Treatment of the cells with PI 3-K inhibitors (wortmannin and LY294002) increased IGF-I-induced tyrosine phosphorylation of IRS-2. We also observed a concomitant increase in the mobility of IRS-2, suggesting that PI 3-K mediates or is required for IRS-2 serine/threonine phosphorylation, and that this phosphorylation inhibits IRS-2 tyrosine phosphorylation. Treatment with PI 3-K inhibitors induced an increased association of IRS-2 with Grb2, probably as a result of the increased IRS-2 tyrosine phosphorylation. However, even though the PI 3-K inhibitors enhanced the association of Grb2 with IRS-2, these compounds suppressed IGF-I-induced mitogen-activated protein kinase activation and neurite outgrowth. Together, these results indicate that although PI 3-K participates in a negative regulation of IRS-2 tyrosine phosphorylation, its activity is required for IGF-IR-mediated mitogen-activated protein kinase activation and neurite outgrowth.     

A new perspective on cannabinoid signalling: complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain

CB1-type cannabinoid receptors in the brain mediate effects of the drug cannabis. Anandamide and sn-2 arachidonylglycerol (2-AG) are putative endogenous ligands for CB1 receptors, but it is not known which cells in the brain produce these molecules. Recently, an enzyme which catalyses hydrolysis of anandamide and 2-AG, known as fatty acid amide hydrolase (FAAH), was identified in mammals. Here we have analysed the distribution of FAAH in rat brain and compared its cellular localization with CB1-type cannabinoid receptors using immunocytochemistry. High concentrations of FAAH activity were detected in the cerebellum, hippocampus and neocortex, regions of the rat brain which are enriched with cannabinoid receptors. Immunocytochemical analysis of these brain regions revealed a complementary pattern of FAAH and CB1 expression with CB1 immunoreactivity occurring in fibres surrounding FAAH-immunoreactive cell bodies and/or dendrites. In the cerebellum, FAAH was expressed in the cell bodies of Purkinje cells and CB1 was expressed in the axons of granule cells and basket cells, neurons which are presynaptic to Purkinje cells. The close correspondence in the distribution of FAAH and CB1 in rat brain and the complementary pattern of FAAH and CB1 expression at the cellular level provides important new evidence that FAAH may participate in cannabinoid signalling mechanisms of the brain.     

N-Methyl-D-aspartate inhibits apoptosis through activation of phosphatidylinositol 3-kinase in cerebellar granule neurons. A role for insulin receptor substrate-1 in the neurotrophic action of n-methyl-D-aspartate and its inhibition by ethanol

Primary cultured rat cerebellar granule neurons underwent apoptosis when switched from medium containing 25 mM K+ to one containing 5 mM K+. N-methyl-D-aspartate (NMDA) protected granule neurons from apoptosis in medium containing 5 mM K+. Inhibition of apoptosis by NMDA was blocked by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but it was unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. The antiapoptotic action of NMDA was associated with an increase in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), an increase in the binding of the regulatory subunit of PI 3-kinase to IRS-1, and a stimulation of PI 3-kinase activity. In the absence of extracellular Ca2+, NMDA was unable to prevent apoptosis or to phosphorylate IRS-1 and activate PI 3-kinase. Significant inhibition of NMDA-mediated neuronal survival by ethanol (10-15%) was observed at 1 mM, and inhibition was half-maximal at 45-50 mM. Inhibition of neuronal survival by ethanol corresponded with a marked reduction in the capacity of NMDA to increase the concentration of intracellular Ca2+, phosphorylate IRS-1, and activate PI 3-kinase. These data demonstrate that the neurotrophic action of NMDA and its inhibition by ethanol are mediated by alterations in the activity of a PI 3-kinase-dependent antiapoptotic signaling pathway.     

Comparison of the signaling abilities of the cytoplasmic domains of the insulin receptor and the insulin receptor-related receptor in 3T3-L1 adipocytes

In the present work a chimeric receptor containing the intracellular domain of the insulin receptor-related receptor (IRR) and the extracellular domain of the colony stimulating factor-1 (CSF-1) receptor was expressed in 3T3-L1 adipocytes and compared with the parallel chimeric receptor containing the cytoplasmic domain of the insulin receptor (IR). Both chimeric receptors exhibited CSF-stimulated tyrosine kinase activity when assayed in vitro after in vivo activation comparable to that of the endogenous IR present in these cells. No cross-activation of the expressed chimeric and endogenous receptors was observed. The cytoplasmic domain of the IRR was found to 1) mediate activation of the Ser/Thr kinase Akt/PKB, 2) stimulate glucose uptake, 3) inhibit lipolysis, and 4) stimulate glycogen synthase, all with a potency comparable to those of the expressed CSF-1R/IR chimera and the endogenous insulin receptors. These results indicate that despite the extensive differences in sequence between the cytoplasmic domains of the IRR and IR, the elements required for insulin-specific responses have been conserved in this distinct member of the insulin receptor family.     

Fatty acid ethyl esters and HepG2 cells: intracellular synthesis and release from the cells

Fatty acid ethyl esters (FAEE), esterification products of fatty acid and ethanol, have been implicated as mediators of ethanol-induced organ damage. To understand the molecular and cellular events in FAEE synthesis and secretion, we developed a system in which HepG2 cells synthesize and release FAEE into the culture medium upon incubation with ethanol. The synthesis of FAEE was observed within 5 min of the addition of ethanol, with a plateau for FAEE synthesis after 2 h of incubation. It was also observed that FAEE are synthesized by both a microsomal FAEE synthase, which preferentially uses fatty acyl-CoA as a substrate, and a cytosolic FAEE synthase, which accepts both unesterified fatty acid and fatty acyl-CoA as substrates with a slight preference for fatty acyl-CoA. Although the kinetics of cellular FAEE synthesis await further characterization, the intracellular fatty acid substrate appears to be derived principally from glycerolipids and other esters. FAEE were released into the culture medium by a mechanism independent of the vesicular transport pathway. Lipoprotein particles and albumin were found to be carriers of FAEE after FAEE secretion from the cell.     

Growth hormone stimulates tyrosine phosphorylation of JAK2 and STAT5, but not insulin receptor substrate-1 or SHC proteins in liver and skeletal muscle of normal rats in vivo

GH has been shown to stimulate tyrosine phosphorylation of JAK2, several STAT proteins, insulin receptor substrate-1 (IRS-1), and SHC proteins in cultured cells. The goal of this study was to determine GH effects on protein tyrosine phosphorylation in liver and skeletal muscle of normal rats in vivo. Nonfasted male Sprague-Dawley rats (225-250 g) were injected with GH iv, and tissues were obtained after 5, 15, 30, or 60 min. At a maximally effective GH dose (1.5 mg/kg body weight), phosphotyrosine antibody immunoblots demonstrated marked stimulation of the tyrosine phosphorylation of JAK2 (maximal at 5 min) and a 95,000 Mr protein (maximal at 15 min) in both liver and skeletal muscle. The 95,000 Mr protein was recognized and immunodepleted by STAT5 antibody, but not by other STAT protein antibodies. Although basal tyrosine phosphorylation of IRS-1 and SHC was evident, GH did not stimulate tyrosine phosphorylation of either of these proteins in liver or skeletal muscle. In conclusion, GH stimulates the tyrosine phosphorylation of JAK2 and STAT5, but not IRS-1, SHC, or other STAT proteins in liver and skeletal muscle of normal rats. These results differ from findings in cultured cells and support the concept that selectivity for tyrosine kinase substrates is an important determinant of postreceptor signaling specificity in vivo.     

Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein

Fatty acid binding proteins (FABPs) are small cytoplasmic proteins that are expressed in a highly tissue-specific manner and bind to fatty acids such as oleic and retinoic acid. Mice with a null mutation in aP2, the gene encoding the adipocyte FABP, were developmentally and metabolically normal. The aP2-deficient mice developed dietary obesity but, unlike control mice, they did not develop insulin resistance or diabetes. Also unlike their obese wild-type counterparts, obese aP2-/- animals failed to express in adipose tissue tumor necrosis factor-alpha (TNF-alpha), a molecule implicated in obesity-related insulin resistance. These results indicate that aP2 is central to the pathway that links obesity to insulin resistance, possibly by linking fatty acid metabolism to expression of TNF-alpha.     

Protein kinase C isotypes theta, delta and eta in human lymphocytes: differential responses to signalling through the T-cell receptor and phorbol esters

CD-1 mice received daily subcutaneous injections of either cocaine (20 mg/kg or 40 mg/kg) or saline solution (0.9% NaCl) from postnatal days 2 to 15. Pups were tested on days 16-17 for learning and 24-h retention of a passive avoidance task, where entering a dark compartment was punished with a mild foot shock. Locomotor activity and general behaviour in an open field arena were assessed on day 21, following administration of either the muscarinic blocker scopolamine (0.8 mg/kg) or saline solution. In addition, immunostaining for the enzyme choline acetyltransferase (ChAT) was measured in different basal forebrain areas (medial septum, striatum, and nucleus basalis) on day 30. Cocaine treatment failed to affect either learning or retention capabilities. Nonetheless, neophobic behaviour during the learning session was enhanced in control nonpunished mice exposed to the 20-mg/kg dose. In the open field test, although baseline activity levels were unaffected by cocaine exposure, the 40-mg/kg cocaine-treated pups showed decreased sensitivity to the hyperkinetic effects of scopolamine. ChAT immunocytochemistry revealed a significant reduction of the number of ChAT-immunopositive neurons in the nucleus basalis but not in the other cholinergic basal forebrain regions.     

Human insulin receptor substrate-1 gene (IRS1): chromosomal localization to 2q35-q36.1 and identification of a simple tandem repeat DNA polymorphism

The protein designated as insulin receptor substrate-1 (IRS-1) is a major substrate for the insulin receptor tyrosine kinase. Since post-receptor defects in the insulin signalling pathway are a common feature of Type 2 (non-insulin-dependent) diabetes mellitus, we have cloned the human IRS-1 gene in order to study the role of genetic variation in this gene in the pathogenesis of diabetes mellitus. As a first step in these studies, we have mapped the IRS-1 gene to chromosome 2, bands q35-q36.1 and identified a simple tandem repeat DNA polymorphism in this gene that will be useful for genetic studies.     

Atopic sensitization during the first year of life in relation to long chain polyunsaturated fatty acid levels in human milk

The influence of low (0.39-1.1%), medium (4.25%) and high (7.1-32.5%) fat levels in fish on radiation inactivation of four food-borne pathogens was investigated. Cells of Listeria monocytogenes 036, Yersinia enterocolitica F5692, Bacillus cereus and Salmonella typhimurium at logarithmic phase were inoculated in 10% fish homogenates and subjected to gamma irradiation at ice temperature (0-1 degree C) with doses ranging from 0.05 to 0.8 kGy. The radiation survival curves of L. monocytogenes and B. cereus were characterized by shoulders, while a tailing effect was depicted by cells of Y. enterocolitica and B. cereus. The D10 values in kGy calculated on the exponential part of the curve ranged from 0.2 to 0.3, 0.15 to 0.25, 0.1 to 0.15 and 0.09 to 0.1 for L. monocytogenes 036, B. cereus, Salm. typhimurium and Y. enterocolitica F5692, respectively. This order (D10) of radiation resistance of each organism was not affected by the fat content of the fish. Inoculated pack studies carried out separately with each pathogen in fatty (Indian sardine, 7.1%) and lean (Golden anchovy, 0.39%) fish showed no difference in their survival after exposure to 1 kGy and 3 kGy doses, which corroborated the above observation. The practical significance of these results in the application of the technology is discussed.     

Species difference in the G protein selectivity of the human and bovine A1-adenosine receptor

The purified bovine brain A1-adenosine receptor has previously been shown to discriminate among closely related G protein alpha-subunits. To obtain analogous information for the human receptor, the cDNA coding for the human A1-adenosine receptor was inserted into a plasmid placing the synthesis of the receptor protein under the control of the MalE promoter. Following induction by maltose, active receptor accumulated in Escherichia coli membranes. Binding of the antagonist 8-[3H]cyclopentyl-1,3-dipropylxanthine to E. coli membranes (KD approximately 2 nM, Bmax approximately 0.2-0.4 pmol/mg) showed the appropriate pharmacological profile. Incubation of E. coli membranes with purified Go,i-reconstituted guanine nucleotide-sensitive high affinity binding of the agonist (-)[125I] N6-3-(iodo-4-hydroxyphenylisopropyl)adenosine to the receptor (KD approximately 1 nM). In the presence of purified beta gamma-subunit, the recombinant receptor interacted equally well with the recombinant G protein alpha-subunits Gi alpha-1, Gi alpha-2, Gi alpha-3; G(o) alpha displayed a lower affinity for the receptor while Gs alpha was inactive. Parallel experiments were carried out in bovine and human brain membranes pretreated with N-ethylmaleimide to inactivate the endogenous G(o)/Gi proteins; Gi alpha-3 was most potent in reconstituting 125I-HPIA binding to bovine membranes, while Gi alpha-1, Gi alpha-2, and G(o) alpha displayed similar affinities. However, in human membranes, Gi alpha-1, Gi alpha-2, and Gi alpha-3, were equipotent and high concentrations of G(o) alpha were required to promote 125I-HPIA binding. These observations show (i) that functional human A1-adenosine receptors were synthesized in E. coli; (ii) that the pattern of G protein coupling is identical for the recombinant human A1-receptor and its counterpart in the native membrane; (iii) and that species differences between bovine and human receptor exist not only in their pharmacological profile but also in their G protein specificity suggesting that species homologues of receptors may use different signaling mechanisms.     

A molecular basis for insulin resistance. Elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation

Tumor necrosis factor alpha (TNFalpha) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS-2 also interacts with the juxtamembrane (JM) domain (amino acids 943-984) but not with the carboxyl-terminal region (amino acids 1245-1331) of IR expressed in bacteria as His6 fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNFalpha, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNFalpha for periods as short as 30 min reversed its inhibitory effects on IR-IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20-60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNFalpha effects and the ability to mimic its action by exogenously added sphingomyelinase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNFalpha on insulin action.     

Alpha-1A adrenergic receptor stimulation with phenylephrine promotes arachidonic acid release by activation of phospholipase D in rat-1 fibroblasts: inhibition by protein kinase A

This study was conducted to determine the mechanism of arachidonic acid (AA) release elicited by phenylephrine (PHE) stimulation of alpha adrenergic receptor (AR), and its modulation by cyclic adenosine 3',5'-monophosphate (cAMP) in Rat-1 fibroblasts (R-1Fs) transfected with the alpha-1A, alpha-1B or alpha-1D AR. PHE increased AA release and also caused a marked accumulation of cAMP in R-1Fs expressing the alpha-1 AR subtypes, but not in those transfected with vector alone. PHE also enhanced phospholipase D (PLD), but not phospholipase A2 (PLA2) activity. The increase in PHE-induced AA release, PLD activity and cAMP accumulation differed among the various alpha AR subtypes with: alpha-1A > alpha-1B > alpha-1D AR. The effect of PHE to increase AA release was attenuated by C2-ceramide, an inhibitor of PLD; propranolol, a phosphatidate phosphohydrolase inhibitor; and RHC-80267, a diacylglycerol lipase inhibitor in R-1Fs expressing the alpha-1A AR. Forskolin, which activates adenylyl cyclase, increased cAMP accumulation and inhibited PHE-induced AA release and PLD activity in alpha-1A-AR-expressing R-1Fs. 8-(4-chlorophenyl-thio)-cAMP, a nonhydrolyzable analog of cAMP, also attenuated the rise in AA release and PLD activity elicited by PHE in these cells. In contrast, SQ 22536, an adenylyl cyclase inhibitor, and KT 5720, a protein kinase A inhibitor, increased PHE-induced AA release and PLD activity in R-1Fs expressing the alpha-1A AR. These data suggest that the alpha-1A, alpha-1B and alpha-1D ARs are coupled to PLD activation and cAMP accumulation. Moreover, PHE promotes AA release in R-1Fs expressing the alpha-1A AR through PLD activation. Furthermore, cAMP generated by alpha-1A AR stimulation acts as an inhibitory modulator of PLD activity and AA release via protein kinase A.