Compartmentalization and in vivo insulin-induced translocation of the insulin-signaling inhibitor Grb14 in rat liver

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1 Compartmentalization and in vivo insulin-induced translocation of the insulin-signaling inhibitor in rat liver Bernard Desbuquois 1,,Véronique Béréziat 3, François Authier, Jean Girard 1, and Anne-Françoise Burnol 1, 1 Institut Cochin, Université Paris Descartes, CNRS (UMR 81), France Inserm, U567, Paris, France 3 Centre de Recherche Saint-Antoine, UMR S893, Faculté de Médecine Pierre et Marie Curie, Paris, France Inserm, U756, Faculté de Pharmacie Paris 11, Châtenay-Malabry, France Keywords endocytosis; insulin receptor; liver; molecular adaptor; tyrosine kinase activity Correspondence B. Desbuquois, Département d Endocrinologie, Métabolisme, Cancer, Institut Cochin, rue du Faubourg Saint-Jacques, 751 Paris, France Fax: Tel: bernard.desbuquois@inserm.fr (Received 1 September 7, revised 9 April 8, accepted July 8) doi:1.1111/j x The molecular adaptor binds in vitro to the activated insulin receptor (IR) and inhibits IR signaling. In this study, we have used rat liver subcellular fractionation to analyze in vivo insulin effects on compartmentalization and IR phosphorylation and activity. In control rats, was recovered mainly in microsomal and cytosolic fractions, but was also detectable at low levels in plasma membrane and Golgi endosome fractions. Insulin injection led to a rapid and dose-dependent increase in content, first in the plasma membrane fraction, and then in the Golgi endosome fraction, which paralleled the increase in IR b-subunit tyrosine phosphorylation. Upon sustained in vivo IR tyrosine phosphorylation induced by high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous phosphatases, and in vivo phosphorylation of the IR induced by injection of bisperoxo(1,1 phenanthroline)oxovanadate, a phosphotyrosine phosphatase inhibitor, we observed a striking correlation between IR phosphorylation state and content in both the plasma membrane and Golgi endosome fractions. In addition, coimmunoprecipitation experiments provided evidence that was associated with phosphorylated IR b-subunit in these fractions. Altogether, these data support a model whereby insulin stimulates the recruitment of endogenous to the activated IR at the plasma membrane, and induces internalization of the IR complex in endosomes. Removal of from fractions of insulin-treated rats by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine kinase activity, indicating that endogenous exerts a negative feedback control on IR catalytic activity. This study thus demonstrates that is a physiological regulator of liver insulin signaling. is a member of the Grb7 Grb1 family of adaptor proteins, which lack intrinsic enzymatic activity and share a common multidomain structure. These adaptors bind to several receptor tyrosine kinases and signaling proteins, and are involved in the regulation of various processes, including cell growth and Abbreviations BPS, between plekstrin homology and SH; bpv(phen), bisperoxo(1,1-phenanthroline) oxovanadate; EGF, epidermal growth factor; ER, endoplasmic reticulum; GST, glutathione S-transferase; IR, insulin receptor; IRS-1, insulin receptor substrate-1; IRb, insulin receptor b-subunit; PDK-1, 3-phosphoinositide-dependent kinase-1; PI3-kinase, phosphoinositide-3-kinase; PIR, phosphorylated insulin receptorinteracting region; WGA, wheat germ agglutinin. FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS 363

2 Insulin-induced translocation of in rat liver B. Desbuquois et al. metabolism, apoptosis, and cell migration [1 5]., which is selectively expressed in insulin target tissues, interacts with the phosphorylated insulin receptor (IR) through a region called BPS [between plekstrin homology (PH) and SH] or phosphorylated insulin receptor-interacting region (PIR) [6] and inhibits the tyrosine kinase activity of the IR [7]. On the basis of the crystal structure of the tyrosine kinase domain of the IR in complex with the PIR BPS domain of, Depetris et al. [8] have shown that binds as a pseudosubstrate inhibitor to the peptide-binding groove of the kinase and thus acts as a selective inhibitor of insulin signaling. Consistent with this observation, overexpression of in CHO-IR cells impairs Akt and ERK insulin signaling pathways and inhibits distal effects of insulin on glycogen and DNA synthesis [6 9], and microinjection of into Xenopus laevis oocytes inhibits insulin-induced oocyte maturation [1]. Conversely, disruption of the gene in mice ameliorates glucose tolerance in vivo and insulin signaling in both liver and skeletal muscle [11]. However, although it improves the Akt insulin signaling pathway, depletion of by RNA interference in mouse primary cultured hepatocytes inhibits the stimulatory effect of insulin on glycogen synthesis and on glycolytic and lipogenic gene expression, suggesting that action on insulin signaling cannot be restricted to its inhibitory action on IR catalytic activity [1]. In addition to the IR, three partners of involved in insulin signaling have been identified: (a) protein kinase Cf interacting protein (ZIP), an adaptor protein that binds to the PIR domain of and mediates the assembly of a protein kinase Cf ZIP heterotrimer [1]; (b) insulin receptor substrate-1 (IRS-1), which binds through its phosphotyrosine-binding domain to an NPXY motif of in a phosphorylation-independent manner [13]; and (c) 3-phosphoinositide-dependent kinase-1 (PDK- 1), which binds constitutively to a PDK-1 consensus binding motif of [1]. Although lacking a hydrophobic transmembrane domain, like many signaling proteins is found in cells in both soluble and membrane-associated states. In DU 15 human prostate cancer cells, endogenous is predominantly associated with a lowdensity microsomal fraction, where it colocalizes with tankyrase [15]. In indirect immunofluorescence studies, is detected as a diffuse but also punctate cytoplasmic staining that is more concentrated around the nucleus, suggesting its association with the Golgi; a pool of also localizes at the plasma membrane [15]. In HEK 93 cells, epitope-tagged is mainly expressed in the cytosol in the resting state, but redistributes in part to the membrane fraction upon insulin stimulation [1]. In rat retina, endogenous shows a perinuclear and nuclear localization, consistent with the identification of a functional nuclear localization signal in the N-terminus [16]. However, unlike with other insulin signaling proteins, compartmentalization has not been characterized in physiological insulin target cells. In the present study, subcellular fractionation and western blotting procedures have been used to assess the compartmentalization of in rat liver, an organ that expresses both the IR and at a high level, and where insulin-induced phosphorylation, activation and internalization of the IR have been previously documented [17 19]. Our results show that, in the basal state, is localized mainly in highdensity microsomal elements and cytosol. Upon insulin stimulation, is rapidly and dose-dependently translocated first to plasma membranes and then to endosomes, in which it associates with phosphorylated IR. These results suggest that is recruited by the activated IR at the plasma membrane and then undergoes internalization as a complex with the IR. In addition, our results provide the first evidence for an in vivo inhibitory effect of endogenously recruited on IR catalytic activity in both compartments. Results Subcellular distribution of and the IR in liver from control and insulin-injected rats The subcellular distribution of liver immunoreactive was examined using preparative and analytical fractionation and compared to that of the IR. Upon differential centrifugation (Fig. 1A), was detectable as a major protein of 6 kda in both particulate and soluble fractions. A minor component of slightly reduced electrophoretic mobility, which may represent a phosphorylated form of [1], was also observed. Under basal conditions, content was three-fold to four-fold higher in the light mitochondrial microsomal and cytosolic fractions than in the nuclear and mitochondrial fractions (P <.1). Analysis of separate light mitochondrial and microsomal fractions showed that content was about 1-fold higher in the microsomal fraction than in the light mitochondrial fraction (results not shown). In recovery studies, the microsomal and cytosolic fractions accounted respectively for about % and 9% of total contained in the whole homogenate. was present at relatively low levels in the plasma membrane and Golgi endosome fractions, with 36 FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS

3 B. Desbuquois et al. Insulin-induced translocation of in rat liver A ins ins H N M LP S PM GE B ins ins EEA1 Na + K + ATPase Calnexin number: ins control ins min ins 5 min Fraction number H N M LP S PM GE Fig. 1. Comparative expression of and IR proteins in liver subcellular fractions of control and insulin-injected rats. (A) Liver homogenates (H) prepared from five control ()ins) and five insulin-injected (+ins, min postinjection) rats were fractionated into nuclear (N), mitochondrial (M), light mitochondrial microsomal (LP), cytosolic (S), plasma membrane (PM) and Golgi endosome (GE) fractions as described in Experimental procedures. Aliquots (1 lg of protein) were analyzed by western blotting using antibodies against, IRb and phosphotyrosine to detect phosphorylated IRb (p-irb) as indicated. Upper panel: representative immunoblots. No signal was detected on an anti-phosphotyrosine immunoblot performed in the absence of insulin. Lower panel: quantitation of and IRb signals by scanning densitometry, with results expressed as percentage of signal intensity in the homogenate (mean ± SEM of five determinations on fractions originating from separate livers). (B) Liver microsomal fractions prepared from three control and six insulin-treated rats ( and 5 min postinjection, three rats per time point) were subjected to centrifugation through a continuous sucrose density gradient. Fourteen subfractions with densities increasing linearly from 1.65 (fraction 1) to gæml )1 (fraction 1) were collected, and aliquots (1 lg of protein) were analyzed by western blotting using antibodies directed against, IRb, EEA1 (endosomal marker), Na + K + -ATPase (plasma membrane marker) and calnexin (ER marker) as indicated. Top: representative immunoblots in control ()ins) and insulin-injected (+ins, 5 min postinjection) rats. Bottom: quantitation of and IRb signals, with results expressed as percentage of maximum (mean ± SEM of three determinations on microsomal fractions originating from separate livers). Significant differences between control and insulin-treated liver fractions [ min in (A) and 5 min in (B)] using the LSD test are indicated as follows: P <.5; P <.1; P <.1. recoveries of about 1% and.%, respectively. The subcellular distribution of clearly differed from that of the IR, which was detected only in particulate fractions with a marked enrichment in the plasma membrane and Golgi endosome fractions, and, to a lesser extent, in the light mitochondrial microsomal fraction, in agreement with a previous report [17]. Two minutes after insulin injection, consistent with the insulin-induced internalization and phosphorylation of the IR [17 19], IR content decreased in the plasma membrane fraction and reciprocally increased in the light mitochondrial microsomal and Golgi endosome fractions, and IR b-subunit (IRb) phosphorylation was detected in each of these fractions. Concomitantly, a significant increase in content was observed in the plasma membrane and Golgi endosome fractions (twofold and 1-fold increase respectively, P <.1) and to a lesser extent in the light mitochondrial microsomal fraction (3% increase, P <.5). As the content in crude FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS 365

4 Insulin-induced translocation of in rat liver B. Desbuquois et al. homogenates was unchanged, these results suggest a translocation of cytosolic to IR-enriched compartments. However, the increased recovery of in IR-enriched fractions did not exceed 5% of the total liver pool, explaining why no decrease in cytosolic could be detected. To further characterize the distribution of membraneassociated and IR under basal and insulinstimulated conditions, the microsomal fraction, which contained the majority of both proteins, was subjected to analytical density gradient centrifugation. The distribution of and IRb was analyzed and compared to that of three organelle markers: EEA1 (endosomes); Na + K + -ATPase (plasma membrane); and calnexin [endoplasmic reticulum (ER)] (Fig. 1B). In control rats, was expressed predominantly in the high-density region of the gradient (subfractions 8 1; density range, gæml )1 ), as was calnexin. In contrast, IRb was expressed mainly at intermediate densities (subfractions 6 1; density range, gæml )1 ), similarly to Na + K + -ATPase, and to a lesser extent at low densities (subfractions 5; density range, gæml )1 ), coinciding with EEA1. Insulin treatment caused a shift in the distribution of both and IRb towards lower densities, which was more pronounced at 5 min than at min. The shift in distribution resulted from an increased content at low and intermediate densities (subfractions 3 8; density range, gæml )1 ), whereas the shift in IRb distribution involved both a decreased content at intermediate and high densities (subfractions 8 1; density range, gæml )1 ) and an increased content at low densities (subfractions 5; density range, gæml )1 ). Insulin treatment also resulted in an increased content of phosphorylated IRb, the distribution of which was superimposable on that of IRb (data not shown). Taken together, these results extend those obtained with preparative procedures and confirm that, upon insulin-induced IR internalization and activation, associates in part with plasma membranes and endosomes. Time course and dose-dependence of the insulin effect on content in plasma membrane and endosomal liver fractions In time-course studies (Fig. A), content in the plasma membrane fraction reached a maximum (three A ins (min).5 5 PM GE B ins ( µg) : PM GE p p Fig.. Time course and dose-dependence of in vivo insulin effects on, phosphorylated IRb (p-irb) and IRb content in liver plasma membrane (PM) and Golgi endosome (GE) fractions. PM and GE fractions were prepared from livers of rats studied at the indicated time points after injection of 3 lg of insulin (four to six rats per time point) (A), or studied min after injection of the indicated dose of insulin (two rats per dose) (B). Aliquots (1 lg of protein) were analyzed by western blotting using antibodies against, phosphotyrosine and IRb, as indicated. The figure shows representative immunoblots and quantitation of protein signals by scanning densitometry, with results expressed as fold change relative to basal value [mean ± SEM of four to six determinations for (A) or two determinations for (B), on fractions originating from separate livers]. Significant changes relative to control (no insulin) using the LSD test are indicated as follows: P <.5; P <.1; P < FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS

5 B. Desbuquois et al. Insulin-induced translocation of in rat liver times basal level) as early as 3 s after insulin injection, and subsequently declined to almost basal values within 3 min, whereas content in the Golgi endosome fraction was maximal at min (about six times basal level) and declined more slowly, remaining elevated at 6 min. The insulin-induced increase in content in the plasma membrane and Golgi endosome fractions was significantly correlated with the increase in phosphorylated IRb content in the same fractions (plasma membrane, r =.7, P <.1; Golgi endosome, r =.6, P <.1). When normalized to IRb content, insulin-induced changes in content were similar in the two fractions (twofold to threefold maximal increase), as were the changes in phosphorylated IRb content (1 -fold maximal increase). These findings suggest that the ability of phosphorylated IRb to recruit is the same in the plasma membrane and Golgi endosome fractions. In dose response studies (Fig. B), the insulininduced increase in content was detectable for 3 lg of insulin in the plasma membrane fraction and.3 lg in the Golgi endosome fraction, and was maximal for 3 lg in both fractions, again in good agreement with the increase in phosphorylated IRb content. Functional relationships between membrane association of and IR tyrosine phosphorylation The parallel increase in content and phosphorylated IRb content in the plasma membrane and Golgi endosome fractions of insulin-treated rats suggested that IRb phosphorylation state was involved in the association of with these compartments. To gain further insight into the functional relationship between these two events, we used three complementary approaches. First, we assessed the response of endosomal to [GluA13,GluB1]insulin and [HisA8,HisB,GluB1,HisB7]insulin, two high-affinity insulin analogs that were previously reported to induce prolonged tyrosine phosphorylation of the endosomal IR [], administered in vivo. As shown in Fig. 3, these analogs also induced a more sustained association of with the Golgi endosome fraction, which paralleled their effects on phosphorylated IRb content. Second, we examined the ability of associated with the Golgi endosome fraction to dissociate upon incubation at 37 C, conditions under which the phosphorylated IR has been shown to be rapidly dephosphorylated by endogenous phosphatases [1]. As shown in Fig., incubation of Golgi endosome fractions from insulin-treated rats indeed resulted in a progressive, time-dependent dephosphorylation of min : A3 H WT Fig. 3. Time course of in vivo effects of human insulin analogs on and phosphorylated IRb (p-irb) content in liver Golgi endosome fractions. Golgi endosome fractions were isolated from livers of rats studied at the indicated time points (one rat per time point) after injection of 3 lg of [GluA13,GluB1]insulin (A3), [HisA8,HisB,GluB1,HisB7]insulin (H) or wild-type human insulin (WT). Aliquots (3 lg protein) were analyzed by western blotting using antibodies against and phosphotyrosine. phosphorylated IRb. Concurrently, content decreased in sedimented membranes, while remaining unchanged in total incubation mixtures (data not shown), indicating a dissociation of membrane-bound. Membrane-bound and phosphorylated IRb were significantly correlated (r =.56; P <.5). Similar results were obtained using the plasma membrane fraction (data not shown). Importantly, both processes were almost fully inhibited by bisperoxo(1,1-phenanthroline) oxovanadate [bpv(phen)], a potent inhibitor of endosomal phosphotyrosine phosphatases. Finally, we examined the response of endosomal to bpv(phen) administered in vivo; this was previously shown to increase IRb phosphorylation [] and to prevent the dephosphorylation of activated IRb that occurs shortly after insulin injection [3]. Our results confirm these observations, and further show that the changes in phosphorylated IRb content induced by bpv(phen) in the Golgi endosome fraction are accompanied by somewhat parallel changes in content (Fig. 5). When injected alone, bpv(phen) led, at 15 and 5 min postinjection, to a 1 16-fold increase in phosphorylated IRb content and to a nearly two-fold increase in content in the Golgi endosome fraction. When injected 15 min prior to an inframaximal dose of insulin, bpv(phen) FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS 367

6 Insulin-induced translocation of in rat liver B. Desbuquois et al. bpv A bpv (min) p bpv (min) B bp V min ins (min) ins (min) 5 15 Fig.. In vitro dissociation of membrane-bound upon dephosphorylation of in vivo activated IRs in liver Golgi endosome fractions. Golgi endosome fractions from five insulin-injected rats ( min postinjection) were incubated at 37 C for the indicated times in the absence or presence of bpv(phen) (. mm), and then subjected to high-speed centrifugation as described in Experimental procedures. Total incubation mixtures and resuspended pellets were analyzed by western blotting using antibodies against phosphotyrosine and as indicated. Incubation did not affect the intensity of signals in total incubation mixtures (data not shown). Top: blots representative of five experiments carried out on Golgi endosome fractions originating from separate livers. Bottom: quantitation of (gray bars) and phosphorylated IRb (p-irb) signals (white bars) in the absence of bpv(phen), with results expressed as percentage of the time value (mean ± SEM of five determinations). Both membrane-bound and p-ir show a significant, time-dependent decrease according to ANOVA (P <.1). prevented the decrease in phosphorylated IRb and content that occurred between and 15 min after insulin injection. A significant correlation was observed between phosphorylated IRb and (r =.81, P <.1). Comparable effects of bpv(phen) treatment on IRb phosphorylation and content, albeit less marked, were also observed in the plasma membrane fraction of untreated and insulin-treated rats (data not shown). Taken together, these data strongly suggest that the phosphorylation status of IRb is implicated in the in vivo association of with IR-containing subcellular compartments. Fig. 5. In vivo effects of bpv(phen), alone or in association with insulin, on, phosphorylated IRb (p-irb) and IRb content in liver Golgi endosome fractions. Golgi endosome fractions were prepared from livers of rats studied at the indicated time points after injection of 1. lmol of bpv(phen) (A) (three to four rats per time point) or 3 lg of insulin (B). In (B), rats were pretreated or not with 1. lmol of bpv(phen) 15 min prior to insulin injection (two to three rats per time point and per condition). Fractions (1 lg of protein) were analyzed by western blotting using antibodies against, phosphotyrosine and IRb. The blots (left part) are representative of experiments carried out on Golgi endosome fractions originating from separate livers, and densitometric measurements of, p-irb and IRb signals are shown on the right. In (A), results are expressed as fold increase in (white bars) and p-irb (numbers under the blot) above the bpv(phen) control (mean ± SEM of the three or four determinations). In (B), results are expressed as fold increase above the insulin control, with (gray bars) or without (white bars) bpv(phen) pretreatment (mean ± SEM of the two or three determinations). A significant effect of bpv(phen) treatment using the LSD test is indicated as follows: P <.5; P <.1; P <.1. Nature of the association of with liver membrane fractions from control and insulin-treated rats As expected for a peripheral protein lacking a transmembrane domain, associated with the microsomal, plasma membrane and Golgi endosome fractions was partially extracted by treatment with KCl at concentrations above 1 m. On the basis of the comparative quantitation of sedimentable in untreated and KCl-treated fractions, the proportion of removed by m KCl was about 6 75% in 368 FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS

7 B. Desbuquois et al. Insulin-induced translocation of in rat liver fractions of control rats and 6% in fractions isolated shortly after (3 s to 5 min) insulin injection (data not shown). Under these conditions, at least 9% of the IR remained membrane-associated as assessed by both western blotting and insulin binding. NaCl, at 1 m, was less effective than KCl at the same concentration, and so was urea at m. On the other hand, treatment with.1 m Na CO 3 (ph 11.5) removed at least 6 7% of, again leaving the IR membrane-associated (data not shown). These findings indicate that recruited under insulin stimulation is tightly associated with membranes. To determine whether translocated to the plasma membrane and Golgi endosome fractions in insulin-treated rats interacts with the phosphorylated IR, fractions were solubilized with Triton X-1 and soluble extracts were subjected to immunoprecipitation using antibodies against IRb and, followed by western blotting using antibodies against and phosphotyrosine, respectively. As shown in Fig. 6, insulin treatment increased content in IR immunoprecipitates, and phosphorylated IRb content in immunoprecipitates, in a time-dependent manner. As in crude lysates, these changes were maximal at 3 s in the plasma membrane fraction and at min in the Golgi endosome fraction, and subsequently declined. These findings strongly favor a direct interaction between and the phosphorylated IRb induced by in vivo insulin stimulation. Furthermore, they suggest that recruited by the phosphorylated IR at the plasma membrane may undergo cointernalization along with the IR. Specificity of insulin-induced changes in content in liver subcellular fractions The IR also binds to Grb7 and Grb1, two adaptor proteins that are structurally related to, and besides the IR, the epidermal growth factor (EGF), fibroblast growth factor and platelet-derived growth factor receptors also can bind [5]. As liver expresses both Grb7 and the EGF receptor at high levels, we performed a comparative analysis, in time studies, of the in vivo effects of insulin and EGF on the contents of Grb7 and in the plasma membrane and Golgi endosome fractions. As shown in PM GE IP ins. (min) IP Fig. 6. Time course of in vivo insulin-induced coimmunoprecipitation of with phosphorylated IRb (p-irb). Plasma membrane (PM) and Golgi endosome (GE) fractions were prepared from livers of rats killed at the indicated time points after injection of 3 lg of insulin (two rats per time point). Following solubilization by Triton X-1, fractions were immunoprecipitated with monoclonal antibody against IRb or polyclonal antibodies against as indicated. Immunoprecipitates (IP) were analyzed by western blotting using antibodies against and phosphotyrosine, and polyclonal antibodies against IRb. Immunoblots are representative of two experiments, carried out on fractions originating from separate livers. Densitometric measurements of (white bars) and p-irb (gray bars) signals are expressed as percentage of maximum (mean ± SEM of these duplicate determinations). FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS 369

8 Insulin-induced translocation of in rat liver B. Desbuquois et al. A ins (min): Grb7 B 3 1 EGF (min) : Grb7 PM PM GE GE brane and Golgi endosome fractions, except for for a three-fold increase at 15 min (P <.1) in the Golgi endosome fraction. It did, however, significantly increase Grb7 content in these two fractions, with a maximal effect at.5 min in the plasma membrane fraction (3.5-fold increase, P <.1) and at 5 15 min in the Golgi endosome fraction (sixfold increase, P <.1) (Fig. 7B). These changes were temporally correlated with the increase in phosphorylated EGF receptor content in both the plasma membrane and Golgi endosome fractions and in EGF receptor content in the Golgi endosome fraction (Fig. 7C). Effect of insulin-induced association of on IR tyrosine kinase activity C 3 1 EGF (min) : p-egfr EGFR PM Fig. 7A, insulin induced an increase in Grb7 content that paralleled the increase in content, with a maximal effect at 3 s in the plasma membrane fraction and at min in the Golgi endosome fraction. The response of Grb7 to insulin was similar to that of in the plasma membrane fraction but somewhat greater in the Golgi endosome fraction. EGF did not significantly affect content in the plasma mem- 6 GE Fig. 7. Comparative in vivo effects of insulin and EGF on Grb7 and content in liver subcellular fractions. Plasma membrane (PM) and Golgi endosome (GE) fractions were isolated from livers of rats studied at the indicated time points after injection of 3 lg of insulin (A) (four rats per time point) or 1 lg of EGF (B, C) (three rats per time point). Aliquots (1 lg of protein) were analyzed by western blotting using antibodies against and Grb7 as indicated (A, B). Following EGF treatment, aliquots were also immunoblotted with antibodies against phosphotyrosine and EGF receptor (EGFR) (C). The blots are representative of experiments carried out on fractions originating from separate livers. Densitometric measurements of (white bars) and Grb7 (gray bars) signals are expressed as fold increase above control (mean ± SEM of four determinations for insulin and three determinations for EGF). See text for statistical analysis of insulin and EGF effects on Grb7 and content in the PM and GE fractions. p-egfr, phosphorylated EGFR. We have previously shown that glutathione S-transferase (GST) inhibits the in vitro tyrosine kinase activity of the recombinant IR as measured using poly(glu,tyr) as substrate [7]. Consistent with this observation, GST also inhibited the ability of the endogenous liver IR partially purified from a microsomal fraction to phosphorylate RCAM-lysozyme, a high-affinity substrate of the receptor (Fig. 8). The inhibitory effect of GST on insulin-stimulated RCAM-lysozyme phosphorylation by the IR was dose-dependent, being detectable at.15 lgæml )1 (1.5 nm) and almost complete at 5 lgæml )1 (5 nm). Inhibition by exogenous of the IR activated in vitro suggested that removal of endogenous from the IR activated in vivo would elicit an opposite effect. To address this question, we first assessed the ability of insulin administered in vivo to increase the level of RCAM-lysozyme in cell fractions, and then assayed the fractions of insulin-injected rats for, IR and phosphorylated IR content and RCAM-lysozyme phosphorylation following treatment or no treatment with m KCl. As shown in Fig. 9A, intact Golgi endosome fractions isolated min after insulin injection displayed a four-fold increase (.3 ±.3, mean ± SEM, n = ) in the extent of RCAM-lysozyme phosphorylation relative to control fractions. KCl treatment of the Golgi endosome fractions from insulin-injected rats affected neither the content nor the tyrosine phosphorylation state of the IR, but led to a 6% decrease in content (Fig. 9B). Removal of from these fractions led to a nearly two-fold concomitant increase in the extent of RCAM-lysozyme phosphorylation (Fig. 9C). KCl treatment of the plasma membrane and microsomal fractions isolated and 5 min after insulin injection induced a similar increase in the extent of RCAM-lysozyme phosphoryla- 37 FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS

9 B. Desbuquois et al. Insulin-induced translocation of in rat liver A p-rcam-l p-rcam-l Time (min) : GST- : B p-rcam-l tion by subsequently prepared wheat germ agglutinin (WGA)-purified insulin receptors (data not shown). Altogether, these results suggest that endogenously recruited by the phosphorylated IR after in vivo insulin stimulation exerts an inhibitory action on IR catalytic activity. Discussion 1 1 ins. Insulin signaling proteins in adipocytes [ 7] and liver [8,9] have been shown to be compartmentalized and to undergo activation and or redistribution to specific subcellular compartments in response to insulin. In liver, plasma membranes and endosomes are major sites to which IRS-1, phosphoinositide-3-kinase (PI3-kinase) and Akt1 redistribute upon in vivo insulin stimulation, and where IRS-1 and PI3-kinase interact µg GST- GS T Fig. 8. In vitro effects of GST on the tyrosine kinase activity of liver IRs. IRs were partially purified from a liver microsomal fraction by adsorption on WGA Sepharose, and examined for their ability to phosphorylate RCAM-lysozyme as described in Experimental procedures. (A) Time course of RCAM-lysozyme phosphorylation in the presence (+ins) or absence ()ins) of.5 lm insulin, and in the presence (+) or absence ()) of 5 lgæml )1 GST. (B) Dosedependent effect of GST on insulin-stimulated RCAM-lysozyme phosphorylation ( min incubation of the receptor with RCAM-lysozyme). The blots in (A) and (B) are representative of two experiments on separate microsomal fractions, and the densitometric measurements in (B) are expressed as percentage of the value in the absence of GST (mean ± SEM of two determinations). p-rcam-l, phosphorylated RCAM-lysozyme. with phosphorylated IRs [8,9]. Our results extend these observations to the molecular adaptor, thus reinforcing its role as an insulin signaling protein. Specifically, we show that following insulin treatment, endogenous undergoes a time-dependent and reversible translocation to plasma membranes and endosomes, in which it is recruited by phosphorylated IRb. Furthermore, we present evidence that exerts a physiological negative feedback control on IR catalytic activity in these compartments. Under basal conditions, liver was recovered mainly in the cytosolic and microsomal fractions, and about 8% of microsomal was recovered in high-density subfractions, as was calnexin, a marker of the ER. Although final evidence that a pool of is localized in the ER awaits morphological confirmation, this localization is not unprecedented. Many peripheral membrane proteins, including signaling proteins such as Shc [3], mammalian target of rapamycin [31] and tyrosine phosphatase PTP-1B [3], have been shown to be localized on the cytosolic surface of the ER membrane [33]. Localization of to the ER may involve the interaction of its PH domain with membrane phosphoinositides, possibly phosphatidylinositol,5-bisphosphate, which was ultrastructurally identified in intracellular membranes [3], and shown to bind to in an insulin-independent manner [16]. The changes in the subcellular distribution of induced by insulin are consistent with a model whereby cytosolic is recruited to the phosphorylated IR at the plasma membrane and is then translocated to endosomes as a protein complex with the receptor. First, as expected from insulin-induced internalization of the IR, insulin led to an increase in level earlier in plasma membrane fractions than in endosomal fractions. Second, in kinetics and dose response studies with insulin and superactive insulin analogs, a striking correlation between the IR phosphorylation state and the content of in the plasma membrane and or endosomal fractions was observed. Third, a similar correlation was found upon in vitro dephosphorylation of the activated IR by endogenous phosphatases and, reciprocally, in vivo phosphorylation of the IR by bpv(phen), an inhibitor of tyrosine phosphatases. Finally, coimmunoprecipitation experiments showed that, following insulin treatment, was associated with the phosphorylated IR in both the plasma membrane and endosomal fractions. On the other hand, although at least part of the IR complex present in endosomes may derive from internalization of a complex formed at the plasma membrane, direct recruitment of cytosolic to activated IR internalized in the endosomes FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS 371

10 Insulin-induced translocation of in rat liver B. Desbuquois et al. A p -RCAM-L p-rcam-l B C min: p-rcam-l p-rcam-l min: ins AU p- KCl ins min: KCl + KCl AU KCl 3 + KCl + KCl min: 1 3 Fig. 9. Effect of recruited in vivo on IR kinase activity in the Golgi endosome fraction. (A) Golgi endosome fractions isolated from three control rats and four rats studied min after insulin injection were assayed for RCAM-lysozyme (RCAM-L) phosphorylation over an 8 3 min incubation. (B, C) Golgi endosome fractions from four insulin-injected rats ( min postinjection) were divided into two identical aliquots, one of which was treated with M KCl as described in Experimental procedures. Untreated and KCl-treated fractions were assayed for, IRb and phosphorylated IRb (p-irb) content (B) and for ability to phosphorylate RCAM-L (C). The figure shows representative immunoblots and results of densitometric measurements expressed as arbitrary units (AU) for phosphorylated RCAM-L (p-rcam-l) and percentage of control (no KCl treatment) for, IRb and p-irb [mean ± SEM of three or four determinations in (A), four to six determinations in (B), four determinations in (C)]. Significant effects of insulin in (A) and KCl in (B) and (C) are indicated as follows: P <.5; P <.1; P <.1. cannot be excluded. It is noteworthy that the recovery of phosphorylated IRb was about two to four times lower in immunoprecipitates than in IR immunoprecipitates (data not shown). As is less effectively immunoprecipitated by antibodies against than is the IR by antibodies to IR, these data would be compatible with a high proportion of phosphorylated receptors having bound to them. Structural studies of the kinase domain of the IR in complex with the PIR BPS of and of the SH domain have allowed us to propose a model for the IR interaction. The PIR BPS binds as a pseudosubstrate inhibitor in the substratebinding groove of the kinase, whereas the SH domain interacts with phosphorylated tyrosine residues of the IR kinase loop, which help position the PIR BPS and increase binding affinity [8]. Although the interaction of with the IR is probably the major determinant of the insulin-induced membrane translocation of, several lines of evidence suggest that the association of via its PH domain with locally produced phosphatidylinositol 3,,5-trisphosphate may also contribute to this process. First, insulin stimulates the association of the regulatory p85 subunit of PI3- kinase with both plasma membranes and endosomes [8]. Second, full-length, as well as its PH domain, bind D3 phosphoinositides in vitro in a protein lipid overlay assay, and in retina lysates is coimmunoprecipitated by antibodies to phosphatidylinositol 3,,5-trisphosphate in an insulin-dependent manner [16]. Finally, insulin-induced membrane translocation of epitope-tagged in HEK 93 cells is inhibited by wortmanin, a PI3-kinase inhibitor [1]. Previous studies have shown that the adaptor protein Grb7 interacts with the IR in two-hybrid, GST pull-down and coimmunoprecipitation assays [35]. On the other hand, although the EGF receptor binds both and Grb7 in cloner of receptor targets (CORT) and GST pull-down assays, neither endogenous in DU15 cells nor epitope-tagged in transfected HEK 93 cells is recruited by the EGF receptor [36]. Consistent with the association of Grb7 with the activated IR in crude liver lysates [35], insulin treatment led to an increase in Grb7 content in liver plasma membrane and endosomal fractions, the kinetics and extent of which were comparable to those observed with. These findings suggest that the relative affinities of Grb7 and for the IR are similarly increased by insulin, and that, like, Grb7 is recruited by the activated IR at the plasma membrane 37 FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS

11 B. Desbuquois et al. Insulin-induced translocation of in rat liver and undergoes internalization as a complex with the receptor. Like insulin, EGF induced an increased content of Grb7 in liver membrane fractions that was temporally correlated with the increased tyrosine phosphorylation of the EGF receptor. However, such treatment affected only marginally, suggesting a preferential association of the EGF receptor with Grb7, as previously documented [36]. Whether the membrane translocation of induced by EGF involves the direct interaction of this protein with the activated EGF receptor or the association of the PH domain with membrane phosphoinositides remains to be established. Inhibition by of IR catalytic activity was previously found using GST and IR purified from CHO-IR cells, and poly(glu,tyr) as a substrate [7]. The inhibitory effect of the GST fusion protein was confirmed in the present study using IR prepared from rat liver and RCAM-lysozyme as a substrate. Importantly, partial removal of endogenous by KCl treatment from the plasma membrane and endosomal fractions of insulin-treated rats led to a substantial increase in IR tyrosine kinase activity. This observation argues for a role of endogenous, physiologically recruited to the IR after in vivo insulin stimulation. Although KCl treatment removed Grb7 from cell fractions to the same extent as (data not shown), this is unlikely to contribute significantly to the increased kinase activity of the IR, as Grb7 is both less potent and less efficient than in its ability to inhibit in vitro activation of IR tyrosine kinase [7]. At the present time, we cannot assess whether the kinase activity of the IRs before KCl treatment represents residual activity of receptors with bound to them, the activity of a fraction of receptors lacking bound, or a combination of both. The finding that, following insulin treatment, the majority of liver remains associated with the microsomal and cytosolic fractions suggests that, besides its ability to interact with the activated IR and to inhibit IR catalytic activity, may play additional roles in the liver. As a molecular adaptor, interacts constitutively in cells with several partners, including ZIP [1], PDK-1 [1], IRS-1 [13], and tankyrase [15]. The binding of ZIP to, by recruiting protein kinase Cf, enhances the serine phosphorylation of and its inhibitory effect on IR kinase and insulin signaling [1]. The interaction of with PDK-1, an upstream kinase that activates Akt in response to insulin, appears to be required for insulin-induced membrane translocation of PDK-1 and Akt activation in transfected HEK cells [1]. On the other hand, the functional significance of the interaction of with tankyrase, a poly(adpribose)polymerase closely related to tankyrase, is still unclear [15]. Conceivably, it could explain the colocalization of these two proteins in a low-density microsomal fraction in DU 15 prostate cancer cells and in transfected HEK 93 cells. Future identification of other partners should lead to interesting new information on the role of this adaptor protein. The endosomal colocalization and or cointernalization of the activated IR and raises the question as to whether could, directly or via its partners, regulate IR traffic and or degradation of the IR. However, despite a recent report showing the involvement of Grb1 in the ubiquitination and proteasomal degradation of the IR in transfected cells [37], there is so far no evidence to support a physiological role of Grb1 and in the regulation of IR degradation [5]. Additional studies are thus needed to determine whether is involved in insulin-induced endocytosis and or degradation of the IR. In summary, our results show that, whereas is located in the basal state mainly in high-density microsomal elements and the cytosol, upon insulin stimulation endogenous is in part translocated to plasma membranes and endosomes, where it is associated with phosphorylated IRb. Our data also suggest that recruited by the IR at the plasma membrane undergoes cointernalization with the receptor. Finally, evidence is presented that exerts negative feedback control on IR catalytic activity in these compartments, indicating that it is a physiological regulator of liver insulin signaling, and that it may represent an interesting molecule with which to regulate liver insulin action. Experimental procedures Materials Porcine insulin, recombinant human insulin, [GluA13, GluB1]insulin and [HisA8,HisB,GluB1,HisB7]insulin were gifts from Novo Nordisk A S (Bagsvaerd, Denmark). Mouse EGF (receptor grade) was from Collaborative Biomedical (Bedford, MA, USA). Monoclonal and polyclonal antibodies against IRb, polyclonal antibodies against EGF and polyclonal antibodies against Grb7 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal antibody against phosphotyrosine (clone G1) was from Upstate Biotechnology Incorporated (Lake Placid, NY, USA). Monoclonal antibody against EEA1 (clone 1) was from BD Transduction Laboratories (San Jose, CA, USA). Monoclonal antibody against Na + K + -ATPase a-subunit (clone M7-PB-E9) and polyclonal antibodies against calnex- FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS 373

12 Insulin-induced translocation of in rat liver B. Desbuquois et al. in were from Sigma-Aldrich (St Louis, MO, USA). GST and rabbit polyclonal antibodies against have been described previously [6]. Horseradish peroxidase-conjugated goat anti-(rabbit IgG) and goat anti-(mouse IgG) were from Biorad (Hercules, CA, USA). bpv(phen) was a gift from B. Posner (McGill University, Montreal, Quebec, Canada). Reduced and carboxyamidomethylated lysozyme was a gift from R. Kohanski (Mount Sinai School of Medicine, New York, NY, USA). [ 15 I]TyrA1 insulin, receptor grade, was from Perkin Elmer Life Sciences (Waltham, MA, USA). Nitrocellulose membranes were from BioRad and Schleicher and Schu ll (Du ren, Germany). The enhanced chemiluminescence detection kit was from Amersham-Pharmacia (Little Chalfont, UK) and Pierce Biotechnology Inc. (Rockford, IL, USA). WGA Sepharose 6MB (WGA Sepharose), protein G Sepharose, protein A Sepharose and other chemicals were purchased from Sigma-Aldrich. Animals and injections Animal studies were performed according to the French Guidelines for Use and Care of Experimental Animals. Male Sprague Dawley rats (body weight, 19 1 g) were obtained from Elevage Janvier (Le Genest Saint Isle, France). Animals were housed in an animal facility with 1 h light cycles, fed ad libitum, and fasted overnight (16 18 h) before study. After pentobarbital anesthesia, rats received an intravenous injection (penis vein) of insulin or insulin analogs (3 lg unless otherwise stated), EGF (1 lg) or bpv(phen) (1. lmol), diluted in.5 ml of NaCl P i containing.1% BSA. At the indicated times, the liver was rapidly excised through a median incision, and minced in ice-cold.5 m sucrose. Subcellular fractionation Livers were homogenized in.5 m sucrose containing.5 mm NaF, 1 mm Na 3 VO, 1 mm benzamidine and.5 mm phenylmethanesulfonyl fluoride (about 5 ml per gram of tissue) using a Dounce homogenizer (1 up-anddown strokes of the loose-fitting pestle). Following dilution to about 7 ml and removal of fibrous and undisrupted tissue by centrifugation at 5 g av for 5 min, the homogenate was subjected to differential centrifugation, generating nuclear (15 g av for 1 min), mitochondrial (3 g av for 1 min), light mitochondrial microsomal (15 g av for 5 min) and supernatant fractions. In some experiments, an additional centrifugation step was introduced after sedimentation of the mitochondrial fraction, generating separate light mitochondrial (5 g av for 1 min) and microsomal (15 g av for 5 min) fractions. Flotation through discontinuous sucrose density gradients was performed to isolate plasma membranes from the nuclear fraction (1..5 m sucrose interface) and Golgi endosomes from the light mitochondrial microsomal fraction (1.5 m sucrose interface), essentially as described in [38] and [18], respectively. Fractions were assayed for protein content according to the method of Lowry et al. [39], using BSA as standard. Measurement of organelle marker enzymes showed that enrichments of the plasma membrane fraction in 5 -nucleotidase and alkaline phosphodiesterase (plasma membrane markers) and of the Golgi endosome fraction in galactosyltransferase (Golgi marker), ATP-dependent acidification (endosomal marker) and in vivo internalized [ 15 I]insulin were as described in previous reports [17,38,,1]. Fractions were analyzed for, IRb and phosphorylated IRb by western blotting. In some experiments, the microsomal fraction was subfractionated by analytical centrifugation (85 g av for 15 h) through a linear sucrose density gradient ( gæml )1 ). Subfractions were assayed for density by refractometry and for content of the following organelle markers: calnexin (ER); Na + K + -ATPase (plasma membrane); and EEA1 (endosomes). Extraction and immunoprecipitation of membrane-associated and insulin receptor Subcellular fractions prepared as described above were resuspended in 5 mm Hepes buffer (ph 7.6) containing 1mm Na 3 VO,.5 mm NaF, 1 mm phenylmethanesulfonyl fluoride, 1 mm benzamidine, 1 lgæml )1 pepstatin A, lgæml )1 leupeptin, 5 lgæml )1 aprotinin, and, when indicated, 1 m KCl, 1 m NaCl, m urea or.1 m Na CO 3 (ph 11.5). After 6 75 min at C, suspensions were centrifuged at 1 g for 5 min. Pellets were resuspended into Laemmli sample buffer and analyzed for and IRb content by western blotting. For immunoprecipitation, cell fractions were resuspended in Hepes buffer containing 1 mm NaCl, phosphatase and protease inhibitors as indicated above, and.5 1% Triton X-1. After min at C, suspensions were centrifuged at 15 g for 6 min, and supernatants were incubated for 16 h at C with polyclonal antibodies against or monoclonal antibodies against IRb. Protein A agarose or protein G Sepharose, respectively, was then added, and after rotatory shaking for h, beads were sedimented and washed four times in 5 mm Hepes buffer, 1 mm NaCl, 1 mm Na 3 VO and.1% Triton X-1. Immunoprecipitated and IRb were analyzed by western blotting. In vitro dephosphorylation of IR phosphorylated in vivo Plasma membrane and Golgi endosome fractions of insulininjected rats ( min postinjection) prepared in the absence of phosphatase inhibitors were resuspended in 5 mm Hepes buffer (ph 7.6) containing.15 m NaCl, 1 mm EDTA, 1 mm dithiothreitol,.5 mm phenylmethanesulfonyl fluoride, 37 FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS

13 B. Desbuquois et al. Insulin-induced translocation of in rat liver. mgæml )1 BSA,.5 mgæml )1 bacitracin, 5 lgæml )1 aprotinin, 5 lgæml )1 leupeptin, and.5 lgæml )1 pepstatin A. After incubation at 37 C for 5 min in the absence or presence of. mm bpv(phen), suspensions were supplemented with KCl (.5 m final concentration) at C and immediately centrifuged at 15 g for min. The total incubation mixture and the pellet were analyzed for phosphorylated IRb and content by western blotting. IR purification and quantitation IR was partially purified from Triton X-1-soluble extracts of cell fractions by adsorption on WGA Sepharose. Extracts (.5 1 ml, 5 mg of protein) were incubated on a rotatory shaker with WGA Sepharose (1 : 1, v v) for h at C, and glycoproteins were eluted with.3 m N-acetylglucosamine in 5 mm Hepes buffer (ph 7.6),.1% Triton X-1, and. mm phenylmethanesulfonyl fluoride. IR contents in crude extracts and WGA eluates were quantitated by measurements of specific [ 15 I]insulin binding (. nm) as previously described [17]. Assay of IR tyrosine kinase activity This assay was based on the ability of IR to phosphorylate RCAM-lysozyme, a high-affinity substrate []. For assay of in vitro insulin-stimulated kinase activity, WGA eluates from a light mitochondrial microsomal fraction prepared from control rat liver (about fmol of insulin bound) were preincubated in the absence or presence of insulin (.5 lm) for 1 h at 3 C. A phosphorylation mix (5 mm ATP, 1mm CTP, mm MnCl,8mm MgCl ) was then added in the presence or absence of GST at the indicated concentration. After 3 min at 3 C, RCAM-lysozyme (6 lm) was added and incubations were allowed to proceed further for 1 min, until the reaction was stopped by the addition of Laemmli sample buffer. For assay of in vivo insulin-stimulated kinase activity, intact Golgi endosome fractions or WGA eluates of light mitochondrial microsomal and plasma membrane fractions prepared from insulin-injected rat liver and containing equal amounts of IR (.5 fmol of insulin bound) were incubated with the phosphorylation mix and RCAM-lysozyme added together at the same concentrations as above for 1 min at 3 C. RCAM-lysozyme phosphorylation was monitored by western blotting using antibody against phosphotyrosine. Western blotting Equal amounts of cell fraction proteins (1 lg unless otherwise indicated) were supplemented with Laemmli sample buffer, subjected to SDS PAGE analysis (15% acrylamide for RCAM-lysozyme, 8 1% acrylamide for other proteins), and immunodetected with the indicated antibodies. Immunoreactive bands were revealed using an enhanced chemiluminescence detection detection kit. The signals identified on the autoradiograms were quantitated by scanning densitometry using a chemi genius scan (Genesnap; Syngene, Cambridge, UK). Statistical analysis Values of densitometric measurements were expressed as means ± SEM and analyzed by ANOVA. When significant differences were detected, a posteriori comparisons between means were conducted using the Fisher LSD test. Correlation analysis were also performed between phosphorylated IR and. Calculations were carried out using the statview program. Acknowledgements This work was supported by the Ministe` re de la Recherche (ACI no ). We thank Dr Gillian Danielsen for her supply of superactive insulin analogs, Dr Barry Posner for his gift of bpv(phen), and Dr Ronald Kohanski for his gift of reduced and carboxyamidomethylated lysozyme. We also thank Dr Tarik Issad for critically reading the manuscript, and Dr Maria-Angeles Ventura for her help in statistical analysis. References 1 Cariou B, Bereziat V, Moncoq K, Kasus-Jacobi A, Perdereau D, Le Marcis V & Burnol AF () Regulation and functional roles of. Front Biosci 9, Riedel H () Grb1 exceeding the boundaries of a common signaling adapter. Front Biosci 9, Lim MA, Riedel H & Liu F () Grb1: more than a simple adaptor protein. Front Biosci 9, Shen TL & Guan JL () Grb7 in intracellular signaling and its role in cell regulation. Front Biosci 9, Holt LJ & Siddle K (5) Grb1 and : enigmatic regulators of insulin action and more? Biochem J 388, Kasus-Jacobi A, Perdereau D, Auzan C, Clauser E, Van Obberghen E, Mauvais-Jarvis F, Girard J & Burnol A-F (1998) Identification of the rat adapter as an inhibitor of insulin actions. J Biol Chem 73, Bereziat V, Kasus-Jacobi A, Perdereau D, Cariou B, Girard J & Burnol AF () Inhibition of insulin receptor catalytic activity by the molecular adapter. J Biol Chem 77, Depetris RS, Hu J, Gimpelevich I, Holt LJ, Daly RJ & Hubbard SR (5) Structural basis for inhibition of FEBS Journal 75 (8) ª 8 The Authors Journal compilation ª 8 FEBS 375