An arginine to cysteine(252) mutation in insulin receptors from a patient with severe insulin resistance inhibits receptor internalisation but preserves signalling events.

AIMS/HYPOTHESIS: We examined the properties of a mutant insulin receptor (IR) with an Arg(252) to Cys (IR(R252C)) substitution in the alpha-subunit originally identified in a patient with extreme insulin resistance and acanthosis nigricans. METHODS: We studied IR cell biology and signalling pathways in Chinese Hamster Ovary cells overexpressing this IR(R252C). RESULTS: Our investigation showed an impairment in insulin binding to IR(R252C) related mostly to a reduced affinity of the receptor for insulin and to a reduced rate of IR(R252C) maturation; an inhibition of IR(R252C)-mediated endocytosis resulting in a decreased insulin degradation and insulin-induced receptor down-regulation; a maintenance of IR(R252C) on microvilli even in the presence of insulin; a similar autophosphorylation of mutant IR(R252C) followed by IRS 1/IRS 2 phosphorylation, p85 association with IRS 1 and IRS 2 and Akt phosphorylation similar to those observed in cells expressing wild type IR (IRwt); and finally, a reduced insulin-induced Shc phosphorylation accompanied by decreased ERK1/2 phosphorylation and activity and of thymidine incorporation into DNA in cells expressing IR(R252C) as compared to cells expressing IRwt. CONCLUSION/INTERPRETATION: These observations suggest that: parameters other than tyrosine kinase activation participate in or control the first steps of IR internalisation or both; IR-mediated IRS 1/2 phosphorylation can be achieved from the cell surface and microvilli in particular; Shc phosphorylation and its subsequent signalling pathway might require IR internalisation; defective IR endocytosis correlates with an enhancement of some biological responses to insulin and attenuation of others.

Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells.

Cyclic AMP (cAMP) stimulates the transport of Na(+) and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct cells. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of (86)Rb(+) uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degrees C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

The HIV Nef protein alters Ca(2+) signaling in myelomonocytic cells through SH3-mediated protein-protein interactions.

Human immunodeficiency virus Nef plays an important role in AIDS pathogenesis. In addition to the well known down-regulation of cell surface receptors (CD4, MHCI), Nef is able to alter cellular signaling. Of particular interest for this study is the ability of Nef to bind with a very high affinity to SH3 domains of myelomonocyte-specific protein-tyrosine kinases of the Src family (Src-like PTK). We have therefore investigated Ca(2+) signaling in HL60 cells retrovirally transduced with wild type Nef or with a Nef mutant deficient in the SH3-interacting proline-rich motif (Nef((PXXP)4(-))). In differentiated HL60 cells, Nef markedly altered cellular Ca(2+) signaling; the amount of intracellularly stored Ca(2+) was increased, and as a consequence, store-operated Ca(2+)-influx was decreased. This effect was not observed in undifferentiated HL60 cells or in CEM T-lymphocytes and correlated with the differentiation-induced up-regulation of Src-like PTK. The Nef effect on Ca(2+) signaling depended entirely on the integrity of its PXXP motif. The Src-like PTK p56/59(hck) co-immunoprecipitated with both Nef and with the inositol 1,4,5-trisphosphate receptor, providing a possible mechanistic link between the viral protein and intracellular Ca(2+) stores of the host cell. Collectively, our results demonstrate that the human immunodeficiency virus 1 Nef protein manipulates intracellular Ca(2+) stores through SH3-mediated interactions in myelomonocytic cells.

Direct demonstration of the endocytic function of caveolae by a cell-free assay.

The endocytic function of caveolae was challenged by taking advantage of a cell-free assay directly measuring the detachment of receptor-containing vesicles from isolated plasma membranes. Plasma membranes from cultured cells surface-labeled with 125I-cholera toxin (segregating in caveolae) were isolated as described previously. Following incubation of these labeled membranes in the presence of nucleotide(s) and cytosol, a significant proportion of the initially membrane-associated radioactivity was released into the incubation medium in sedimentable form (14*10(6 )g). Results of biochemical, morphological, and fractionation analysis of the material containing the released radioactivity directly demonstrated that caveolae are plasma membrane domains involved in an endocytic process and resulting in the formation of caveolae-derived vesicles. In addition, these studies allowed a direct comparison of caveolae- and clathrin-coated pit-mediated endocytosis and reveal that these two processes diverge in terms of kinetics, cytosol and nucleotide requirements as well as in terms of the density and size of the endocytic vesicles formed.

Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of beta-COP in endosomes.

The Nef protein of primate lentiviruses downregulates the cell surface expression of CD4 through a two-step process. First, Nef connects the cytoplasmic tail of CD4 with adaptor protein complexes (AP), thereby inducing the formation of CD4-specific clathrin-coated pits that rapidly endocytose the viral receptor. Second, Nef targets internalized CD4 molecules for degradation. Here we show that Nef accomplishes this second task by acting as a connector between CD4 and the beta subunit of COPI coatomers in endosomes. A sequence encompassing a critical acidic dipeptide, located nearby but distinct from the AP-binding determinant of HIV-1 Nef, is responsible for beta-COP recruitment and for routing to lysosomes. A novel class of endosomal sorting motif, based on acidic residues, is thus revealed, and beta-COP is identified as its downstream partner.

Sec24 proteins and sorting at the endoplasmic reticulum.

COPII proteins are necessary to generate secretory vesicles at the endoplasmic reticulum. In yeast, the Sec24p protein is the only COPII component in which two close orthologues have been identified. By using gene knock-out in yeast, we found that the absence of one of these Sec24 orthologues resulted in a selective secretion defect for a subset of proteins released into the medium. Data base searches revealed the existence of an entire family of Sec24-related proteins in humans, worms, flies, and plants. We identified and cloned two new human cDNAs encoding proteins homologous to yeast Sec24p, in addition to two human cDNAs already present within the data bases. The entire Sec24 family identified to date is characterized by clusters of highly conserved residues within the 2/3 carboxyl-terminal domain of all the proteins and a divergent amino terminus domain. Human (h) Sec24 orthologues co-immunoprecipitate with hSec23Ap and migrate as a complex by size exclusion chromatography. Immunofluorescence microscopy confirmed that these proteins co-localize with hSec23p and hSec13p. Together, our data suggest that in addition to its role in the shaping up of the vesicle, the Sec23-24p complex may be implicated in cargo selection and concentration.

CD14-dependent endotoxin internalization via a macropinocytic pathway.

Gram-negative bacterial endotoxin (a lipopolysaccharide (LPS)) specifically binds to CD14, a glycosylphosphatidyl inositol (GPI)-anchored surface myeloid glycoprotein. This interaction leads to cell activation, but it also promotes LPS internalization and detoxification. In this work, we investigated the route of LPS and CD14 internalization and the relevance of CD14 GPI anchor in the endocytic pathway. In promonocytic THP-1 cells transfected with a GPI or a chimeric integral form of CD14, we showed by differential buoyancy in sucrose density gradients that these two forms of CD14 were sorted to different plasma membrane subdomains. However, both forms of CD14 associated preferentially with the same surface microfilament-enriched microvilli or ruffles. Electron microscopic studies indicated that CD14 internalized via macropinocytosis, a process resembling that of phagocytosis, different from "classical" receptor-mediated endocytic pathways, such as clathrin-coated pits or caveolae. With cell warming, the CD14-enriched ruffles fused and formed large vesicles. Later, these vacuoles made stacks and condensed into phago-lysosomes. CD14 was specifically associated with all of these structures. Radiolabeled LPS internalization paralleled CD14 internalization. Confocal microscopic studies confirmed the co-localization of LPS and CD14 both at the cell surface and in endosomal compartments. The microfilament-disrupting, macropinocytosis blocking agent cytochalasin D inhibited LPS and CD14 internalization but did not prevent LPS-dependent activation, indicating that these two processes are dissociated.

Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the mu chain of adaptor complexes.

The Nef protein of primate lentiviruses down-regulates the cell surface expression of CD4 and probably MHC I by connecting these receptors with the endocytic machinery. Here, we reveal that Nef interacts with the mu chains of adaptor complexes, key components of clathrin-coated pits. For human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) Nef, this interaction occurs via tyrosine-based motifs reminiscent of endocytosis signals. Mutating these motifs prevents the binding of SIV Nef to the mu chain of plasma membrane adaptor complexes, abrogates its ability to induce CD4 internalization, suppresses the accelerated endocytosis of a chimeric integral membrane protein harboring Nef as its cytoplasmic domain and confers a dominant-negative phenotype to the viral protein. Taken together, these data identify mu adaptins as downstream mediators of the down-modulation of CD4, and possibly MHC I, by Nef.

Analysis of the juxtamembrane dileucine motif in the insulin receptor.

Dileucine-containing motifs are involved in trans-Golgi sorting, lysosomal targeting, and internalization. Previously, we have shown that the dileucine motif (EKITLL, residues 982-987) in the juxtamembrane region of the insulin receptor is involved in receptor internalization. Substitution of alanine residues for Leu986 and Leu987 led to a 3- to 5-fold decrease in the ability of the receptors to mediate insulin uptake. In the current study, we show that mutation of the same motif to Met986Ser987, the sequence found in the homologous position in the type I insulin-like growth factor receptor, did not affect insulin uptake. Therefore, we inquired whether the sequence EKITMS as an isolated motif could mediate the targeting of a reporter molecule to endosomes and then lysosomes, as was shown previously with the EKITLL motif of the normal receptor. Chimeric molecules containing Tac antigen fused to different hexapeptide sequences showed distinct patterns of subcellular localization by immunofluorescence microscopy. Tac-EKITLL and Tac-EKITAA were found predominantly in lysosomes and the plasma membrane, respectively. In contrast, Tac-EKITMS was found at the plasma membrane, in the trans-Golgi network, and in endosomes, but only small amounts were found in lysosomes. Thus, the dileucine motif (EKITLL) plays an important role in directing endocytosis of the intact insulin receptor and in mediating efficient endocytosis and lysosomal targeting as an isolated motif. Substitution of AA for LL inhibits endocytosis and lysosomal targeting in both systems. In contrast, substitution of MS for LL permits rapid endocytosis in the intact receptor, but mediates modest endocytosis and very little targeting to lysosomes as an isolated motif. Our observations support the idea that sorting signals are recognized at multiple steps in the cell, and that specific amino acid substitutions may differentially affect each of these sorting steps.

Cys860 in the extracellular domain of insulin receptor beta-subunit is critical for internalization and signal transduction.

The C860S mutation (IRC860S) in the extracellular domain of the insulin receptor beta-subunit has previously been shown to result in an inhibition of insulin receptor internalization. The present work aims at further dissecting the consequences of this mutation not only on insulin receptor internalization, but also on the signaling of the receptor. Following transfection of Chinese hamster ovary (CHO) cells with insulin receptors with the C860S mutation (CHO-IRC860S) and quantitative electron microscopic analysis of [125I]insulin localization in these cells, the inhibition of receptor internalization appears to be due to an inhibition of the lateral translocation of the receptor from microvilli to nonvillous domains of the cell surface. At 37 C, insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation is inhibited by 50% in CHO-IRC860S, whereas Shc phosphorylation remains unaffected. The inhibition of IRS-1 phosphorylation is still present when experiments are conducted at 4 C, a temperature at which insulin receptor internalization is prevented, suggesting that the defect in IRS-1 phosphorylation is not due to the reduced internalization of the receptor. In terms of biological effects, the mutation has negative consequences on insulin-stimulated c-fos expression and DNA synthesis as well as on glycogen synthase activity. Eventually, the events observed are specific for Cys860, as individual substitution of the two more proximal Cys residues (795 and 872) to Ser is not accompanied by any change in either insulin-induced insulin receptor internalization or IRS-1 phosphorylation. Thus, the present analysis of CHO-IRC860S 1) reveals that insulin receptor surface redistribution is not solely dependent on receptor autophosphorylation, 2) emphasizes that IRS-1 phosphorylation is not dependent on receptor internalization and can be triggered from microvilli, and 3) stresses divergent aspects between two of the major signaling pathways of the insulin receptor.

Soluble form of complement C3b/C4b receptor (CR1) results from a proteolytic cleavage in the C-terminal region of CR1 transmembrane domain.

The complement C3b/C4b receptor (CR1) is an integral protein, anchored in the plasma membrane through a hydrophobic domain of 25 amino acids, but is also found in the plasma in soluble form (sCR1). A recombinant, soluble form of CR1 has been demonstrated to reduce complement-dependent tissue injury in animal models of ischaemia/reperfusion. In view of the important pathophysiological relevance of sCR1, we have investigated the mechanisms governing CR1 release by using various mutated and chimaeric receptors transiently expressed in COS cells. Pulse-chase experiments revealed that (1) sCR1 is produced by a proteolytic process, (2) the cleavage site lies within the C-terminus of CR1 transmembrane domain, (3) the proteolytic process involves a fully glycosylated CR1 form and (4) this process takes place in late secretory vesicles or at the plasma membrane.

Nef-mediated clathrin-coated pit formation.

The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.

Dual role of a dileucine motif in insulin receptor endocytosis.

Two leucines (Leu986 and Leu987) have recently been shown to take part in the control of human insulin receptor (HIR) internalization (Renfrew-Haft, C., Klausner, R. D., and Taylor, S. I. (1994) J. Biol. Chem. 269, 26286-26294). The aim of the present study was to further investigate the exact mechanism of this control process. Constitutive and insulin-induced HIR internalizations were studied biochemically and morphologically in NIH 3T3 cells overexpressing either a double alanine (amino acid residues 986-987) mutant HIR (HIR AA1) or HIR truncated at either amino acid residue 981 (HIR Delta981) or 1000 (HIR Delta1000). Data collected indicate that: (a) the three mutant HIR show a reduced association with microvilli as compared with HIR wild-type; (b) the two receptors containing the dileucine motif (HIR WT and HIR Delta1000) show the highest propensity to associate with clathrin-coated pits, independently of kinase activation; (c) the two receptors lacking the dileucine motif but containing two tyrosine-based motifs, previously described as participating in clathrin-coated pit segregation, associate with these surface domains with a lower affinity than the two others, (d) in the presence of the kinase domain, an unmasking of the tyrosine-based motifs mediated by kinase activation is required. These results indicate that the dileucine motif is not sufficient by itself, but participates in anchoring HIR on microvilli and that another sequence, located downstream from position 1000 is crucial for this event. This dileucine motif also plays a role in HIR segregation in clathrin-coated pits. This latter function is additive with that of the tyrosine-based motifs but the role of the dileucine motif predominates. Eventually, the clathrin-coated pit anchoring function of the dileucine motif is independent of receptor kinase activation in contrast to the tyrosine-based motifs.

Second-messenger regulation of receptor association with clathrin-coated pits: a novel and selective mechanism in the control of CD4 endocytosis.

CD4, a member of the immunoglobulin superfamily, is not only expressed in T4 helper lymphocytes but also in myeloid cells. Receptor-mediated endocytosis plays a crucial role in the regulation of surface expression of adhesion molecules such as CD4. In T lymphocytes p56lck, a CD4-associated tyrosine kinase, prevents CD4 internalization, but in myeloid cells p56lck is not expressed and CD4 is constitutively internalized. In this study, we have investigated the role of cyclic AMP (cAMP) in the regulation of CD4 endocytosis in the myeloid cell line HL-60. Elevations of cellular cAMP were elicited by 1) cholera toxin, 2) pertussis toxin, 3) forskolin and IBMX, 4) NaF, or 5) the physiological receptor agonist prostaglandin E1. All five interventions led to an inhibition of CD4 internalization. Increased cAMP levels did not inhibit endocytosis per se, because internalization of insulin receptors and transferrin receptors and fluid phase endocytosis were either unchanged or slightly enhanced. The mechanism of cAMP inhibition was further analyzed at the ultrastructural level. CD4 internalization, followed either by quantitative electron microscopy autoradiography or by immunogold labeling, showed a rapid and temperature-dependent association of CD4 with clathrin-coated pits in control cells. This association was markedly inhibited in cells with elevated cAMP levels. Thus these findings suggest a second-messenger regulation of CD4 internalization through an inhibition of CD4 association with clathrin-coated pits in p56lck-negative cells.

The HIV-1 Nef protein acts as a connector with sorting pathways in the Golgi and at the plasma membrane.

The HIV Nef protein down-regulates the cell surface expression of CD4 and of MHC I at least in part through accelerated endocytosis. To investigate further the mechanism of this effect, we created chimeric integral membrane proteins comprising the extracellular and transmembrane regions of CD4 or CD8 and Nef as the cytoplasmic domain. These fusion molecules could down-modulate CD4 in trans in a dileucine-dependent manner. Furthermore, in spite of lacking receptor-derived internalization signals, the Nef-containing chimeras underwent both Golgi retention and rapid endocytosis via clathrin-coated pits. Taken together, these data suggest that Nef down-regulates CD4 and probably MHC I by physically connecting these receptors with sorting pathways in the Golgi and at the plasma membrane.

Direct measurement of clathrin-coated vesicle formation using a cell-free assay.

Factors controlling the last stages of clathrin-coated vesicle formation were investigated using an assay allowing direct measurement of the detachment of these vesicles from the plasma membrane. Plasma membranes from cultured cells surface-labelled with 125I-alpha2-macroglobulin (a ligand that preferentially associates with clathrin-coated pits) were isolated by sonication of cells attached to a poly-L-lysine-coated substratum and incubated in the presence of nucleotide(s) +/- cytosol. A significant proportion of the membrane-associated radioactivity was released into the incubation medium in sedimentable form (14x10(6)g). The nucleotide and ligand specificities of this process together with the results of a series of biochemical, morphological and gradient analyses, led to the conclusion that measurement of the released sedimentable radioactivity provides a direct estimate of the formation of clathrin-coated vesicles from clathrin-coated pits. A morphological analysis of quick-frozen replicas of these membranes indicated that only the last stages of clathrin-coated vesicle formation were studied in the assay. Taking advantage of this cell-free system, we demonstrate that membrane-associated cytosolic factors and GTP-binding proteins, noteably dynamin, play a crucial role. Moreover, although these events can occur in the absence of ATP and Ca2+, optimal conditions for the formation of clathrin-coated vesicles require the presence of ATP, GTP and cytosol.

Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

The N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) increases the lateral diffusion of complement receptor 1 (CR1/CD35) in human neutrophils; a causative role for oxidative metabolites?

The effects of the N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) on the lateral mobility of the complement receptor type 1 (CR1/CD35) in glass-adherent human neutrophils were investigated, using fluorescence recovery after photobleaching (FRAP) and confocal microscopy (CSLM). It was found that addition of 0.1-1 microM fMLF increased the diffusion constant (D) of CR1/CD35 to 167-228% of controls. No effect was observed on the receptor distribution or the mobile fraction of receptors. The effect of fMLF on the lateral diffusion of CR1/CD35 could be totally inhibited by addition of pertussis toxon (PD, 250 ng/ml) or of the free radical scavenger enzymes superoxide dismutase (SOD, 2000 U/ml) and catalase (CAT, 200 U/ml), added together the results show that oxidative metabolites produced by neutrophils in response to fMLF can modulate CR1/CD35 diffusion, and indicate a regulatory role for oxygen radicals in phagocytosis.

Molecular and cellular mechanisms governing the ligand-specific and non-specific steps of insulin receptor internalization.

The surface events leading to insulin-induced internalization of its specific receptor can be subdivided in three major steps: the first step consists in the surface redistribution of the receptor from the villous to the non-villous region of the cell surface, it is ligand-specific, depends on kinase activation and phosphorylation of tyrosines 1146, 1150 and 1151, and consists in the relief of a constraint immobilizing the receptor on microvilli; the second step is characterized by the shift of the insulin-receptor complex in the plane of the membrane allowing it to get access to the nonvillous domain of the cell surface where internalization gates (clathrin-coated pits) are located; this stage is controlled, at least in part, by the transmembrane domain of the molecule and its flanking amino acids; the third step corresponds to the segregation of the insulin-receptor complex in clathrin-coated pits, this step is relatively non-specific and is governed by well defined signal sequences present in the juxtamembrane domain of the cytoplasmic segment of the b-subunit. These surface events are then automatically followed by the entry of the insulin receptor inside the cells through the formation of clathrin-coated vesicles, in its subsequent association with endosomes which acidic pH allows insulin dissociation from its receptor and the sorting of the receptor and the hormone in different directions: insulin is targetted to lysosomes to be degraded while the receptor is recycled back to the cell surface to be reused. This complex process does not seem to be involved in the transmission of the biological signal of the hormone. Nevertheless, it is initiated and controlled by insulin and results in the intracellular degradation of insulin and in the modulation of the number of surface insulin receptors. Thus, even if it does not directly participate in insulin signaling, insulin receptor internalization plays a crucial role in the control of insulin action.

Insulin-induced translocation of Na+-K+-ATPase subunits to the plasma membrane is muscle fiber type specific.

We have previously shown that an acute insulin treatment induces redistribution of the alpha 2- and beta 1- isoforms of the Na+-K+-ATPase from intracellular membranes to plasma membranes detected on subcellular fractionation of mixed muscles and immunoblotting with isoform-specific antibodies (H. S. Hundal et al. J. Biol. Chem. 267: 5040-5043, 1992). In the present study we give both biochemical and morphological evidence that this insulin effect is operative in muscles composed mostly of oxidative (red) fibers but not in muscles composed mostly of glycolytic (white) fibers. The redistribution of the Na+-K+-ATPase alpha 2- and beta 1-isoforms after insulin injection was detected in membranes isolated from and muscles (soleus, red gastrocnemius, red rectus femoris, and red vastus lateralis) but not in membranes from white muscles (white gastrocnemius, tensor fasciae latae, white rectus femoris, and white vastus lateralis). After insulin injection, the potassium-dependent 3-O-methylfluorescein phosphatase activity of the enzyme was higher by 22% in the plasma membrane-enriched fraction and lower by 15% in the internal membrane fraction isolated from red but not from white muscles. Quantitative immunoelectron microscopy of ultrathin muscle cryosections showed that in vivo insulin stimulation augmented the density of Na+-K+-ATPase alpha 2- and beta 1- isoforms at the plasma membrane of soleus muscle by 80 and 124%, respectively, with no change in white gastrocnemius muscle. The effect of insulin to increase the content of Na+-K+-ATPase alpha 2- and beta 1-subunits in isolated plasma membranes was still observed when glycemia was prevented from dropping by using hyperinsulinemic-euglycemic clamps. We conclude that the insulin-induced redistribution of the alpha 2- and beta 1-isoforms of the Na+-K+-ATPase from an intracellular pool to the plasma membrane in restricted to oxidative fiber-type skeletal muscles. This may be related to the selective expression of beta 1-subunits in these fibers and implies that the beta 2-subunit, typical of glycolytic muscles, does not sustain translocation of alpha 2 beta 2-complexes.