Two steps of insulin receptor internalization depend on different domains of the beta-subunit [published erratum appears in J Cell Biol 1993 Nov;123(4):1047]

The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin- coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.     

The journey of the insulin receptor into the cell: from cellular biology to pathophysiology

Summary and conclusions The data that we have reviewed indicate that insulin binds to a specific cell-surface receptor. The complex then becomes involved in a series of steps which lead the insulin-receptor complex to be internalized and rapidly delivered to endosomes. From this sorting station, the hormone is targeted to lysosomes to be degraded while the receptor is recycled back to the cell surface. This sequence of events presents two degrees of ligand specificity: (a) The first step is ligand-dependent and requires insulin-induced receptor phosphorylation of specific tyrosine residues. It consists in the surface redistribution of the receptor from microvilli where it preferentially localizes in its unoccupied form. (b) The second step is more general and consists in the association with clathrincoated pits which represents the internalization gate common to many receptors.This sequence of events participates in the regulation of the biological action of the hormone and can thus be implicated in the pathophysiology of diabetes mellitus and various extreme insulin resistance syndromes, including type A extreme insulin resistance, leprechaunism, and Rabson-Mendehall syndrome. Alterations of the internalization process can result either from intrinsic abnormalities of the receptor or from more general alteration of the plasma membrane or of the cell metabolism. Type I diabetes is an example of the latter possibility, since general impairment of endocytosis could contribute to extracellular matrix accumulation and to an increase in blood cholesterol. Thus, better characterization of the molecular and cellular biology of the insulin receptor and of its journey inside the cell definitely leads to better understanding of disease states, including diabetes.Presented at the XXXV Symposium of the Society for Histochemistry, 2 October 1993, Gargellen, Austria     

Internalization of the human insulin receptor. The insulin-independent pathway.

Internalization of the human insulin receptor requires the activation by insulin of the intrinsic kinase of the receptor. However, even in the absence of kinase activation, insulin receptors slowly enter the cells. In the present study, we addressed the question of this insulin-independent pathway of internalization. To that end, we traced insulin receptor internalization with a monoclonal antibody (mAb 83-14) directed against the alpha-subunit of the human insulin receptor. Internalization of this antibody was followed in Chinese hamster ovary (CHO) cells transfected with either normal (CHO.HIRC2) or kinase-deficient (CHO.A1018) human insulin receptors. The internalization rate of 125I-mAb 83-14 was comparable in CHO cells expressing kinase-active or kinase-inactive receptors and was similar to that observed for 125I-insulin in CHO.A1018 cells. Moreover, in CHO.HIRC2 cells, the internalization of 125I-mAb 83-14 was identical with that of its 125I-Fab fragments. Thus, mAb 83-14 represents an appropriate tool to study the constitutive internalization of the insulin receptor. Internalization of insulin receptors tagged with 125I-mAb 83-14 was unaffected by cytochalasin B, which excluded a macropinocytotic process. By contrast, internalization was sensitive to hypertonia, which abrogates clathrin-coated pits-mediated endocytosis. The implication of clathrin-coated pits in this internalization process was directly demonstrated by quantitative electron microscopic autoradiography, which showed that 125I-mAb 83-14 present on the nonvillous domain of the cell surface preferentially associate with clathrin-coated pits at all time points.     

Absence of down-regulation of the insulin receptor by insulin. A possible mechanism of insulin resistance in the rat.

Insulin resistance occurs in rat adipocytes during pregnancy and lactation despite increased or normal insulin binding respectively; this suggests that a post-receptor defect exists. The possibility has been examined that, although insulin binding occurs normally, internalization of insulin or its receptor may be impaired in these states. Insulin produced a dose-dependent reduction in the number of insulin receptors on adipocytes from virgin rats maintained in culture medium, probably due to internalization of the hormone-receptor complex. In contrast, adipocytes from pregnant and lactating rats did not exhibit this 'down-regulation' phenomenon. Down regulation was, however, apparent in all groups when the experiments were performed in Tris buffer (where receptor recycling is inhibited), suggesting that in pregnant and lactating rats insulin receptors are rapidly recycled back to the plasma membrane, whereas in virgin rats this recycling process is less effective. Internalization of insulin was also determined by using 125I-labelled insulin. Adipocytes from pregnant and lactating rats appeared to internalize similar amounts of insulin to virgin rats. In the presence of the lysosomal inhibitor chloroquine, adipocytes from pregnant rats internalized more insulin than virgin or lactating rats. These results suggest that adipocytes from pregnant and lactating rats internalize insulin and its receptor normally, whereas intracellular processing of the insulin receptor may differ from that in virgin rats. In addition the rate of lysosomal degradation of insulin may be altered in adipocytes from pregnant rats.     

The NPEY sequence is not necessary for endocytosis and processing of insulin-receptor complexes.

The tetrameric amino acid sequence AsnProXTyr (NPXY), where X represents any amino acid, is conserved in the intracytoplasmic domains of several membrane proteins and has been postulated to play a role in receptor-mediated endocytosis. The human insulin receptor (hIR) contains a single copy of the sequence AsnProGluTyr (NPEY) in its intracytoplasmic domain. To determine if this putative consensus sequence is necessary for endocytic functions of hIR, we constructed a mutant receptor, hIR delta NPEY, that lacks NPEY sequence, stably expressed this mutant receptor in Chinese hamster ovary cells, and then studied its endocytic functions. When compared to wild type hIR similarly expressed in Chinese hamster ovary cells, the hIR delta NPEY mutant exhibited: 1) normal subunit organization and insulin binding affinity; 2) essentially normal internalization of covalent photoaffinity labeled insulin-receptor complexes; and 3) normal internalization of receptor-bound [125I]insulin as well as normal degradation and release of the internalized insulin. Therefore, we conclude that the NPEY sequence in the juxtamembrane domain of hIR is not necessary for its endocytic function.     

Regulation of transmembrane signaling by receptor phosphorylation

At least two major effects of receptor phosphorylation have been identified--regulation of receptor function, and regulation of receptor distribution. In many cases where phosphorylation directly alters the functions of receptors, this appears to be in a negative direction. Such decreases in receptor activity may reflect reduced ability to interact with biochemical effectors (e.g., the beta-adrenergic receptor, rhodopsin), reduced affinity for binding agonist ligands (EGF,IGF-I, insulin receptors) or reduced enzymatic activity (e.g., tyrosine kinase activity of the insulin or EGF receptor). In all instances, these negative modulations are associated with phosphorylation of serine and/or threonine residues of the receptor proteins. In contrast, the tyrosine kinase receptors also appear to be susceptible to positive modulation by phosphorylation. With these receptors, autophosphorylation of tyrosine residues may lead to enhanced protein-tyrosine kinase activity of the receptors and increased receptor function. In addition, the subcellular distribution of a receptor may be regulated by its phosphorylation status (e.g., the beta-adrenergic receptor, receptors for insulin, EGF, IGF-II, and transferrin). The emerging paradigm is that receptor phosphorylation may in some way promote receptor internalization into sequestered compartments where dephosphorylation occurs. The molecular and cellular mechanisms involved in translating changes in receptor phosphorylation into changes in receptor distribution remain to be elucidated. Moreover, the biological role of receptor internalization may be quite varied. Thus, in the case of the beta-adrenergic receptor, it may serve primarily as a mechanism for bringing the phosphorylated receptors into contact with intracellular phosphatases that dephosphorylate and resensitize it. By contrast, for the transferrin receptor and other receptors involved in receptor-mediated endocytosis, the internalization presumably functions to carry some specific ligand or metabolite into the cell. The role of phosphorylation in regulating receptor function dramatically extends the range of regulatory control of this important covalent modification.     

Ligand-independent internalization and recycling of the insulin receptor. Effects of chronic treatment of 3T3-C2 fibroblasts with insulin and dexamethasone.

Upon binding insulin at the plasma membrane, the insulin receptor internalizes into the endosomal compartment of the cell with a half-time of approximately 10 min. Our earlier work demonstrated that receptor inactivation (loss of insulin binding capacity) is a regulated process. Long term treatment of cultured cells with insulin or the glucocorticoid dexamethasone increases or decreases, respectively, the rate constant for insulin receptor inactivation (Knutson, V. P., Ronnett, G. V., and Lane, M. D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2822-2826). In these studies, monolayer cultures of 3T3-C2 fibroblasts were chronically treated with insulin or dexamethasone. Subsequently, the surface receptors were labeled with the photoactivatable cross-linking agent 125I-labeled 2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionate -insulin. Following equilibration of the radiolabeled receptor between the plasma membrane and internal pools, the steady-state rate constant for receptor recycling was determined by quantitating the rate at which internal radiolabeled receptor was inserted into the plasma membrane. The steady-state rate constant for this recycling process was the same in control, insulin-treated, or steroid-treated cells (t1/2 = 2h). In contrast, the rate constant for receptor internalization was regulated; the half-times were 10 h for control cells, 5 h for insulin-treated cells, and 19 h for dexamethasone-treated cells. These changes in rate constants for internalization and inactivation lead to changes in the relative numbers of receptor molecules undergoing recycling versus inactivation. Therefore, whereas the recycling of the insulin receptor is not a regulated process, the internalization of surface receptor in the absence of bound ligand is a metabolically controlled step in receptor processing.     

The CEACAM1 transmembrane domain, but not the cytoplasmic domain, directs internalization of human pathogens via membrane microdomains

Several bacterial pathogens exploit carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to promote attachment and uptake into eukaryotic host cells. The widely expressed isoform CEACAM1 is involved in cell–cell adhesion, regulation of cell proliferation, insulin homeostasis, and neo-angiogenesis, processes that depend on the cytoplasmic domain of CEACAM1. By analysing the molecular requirements for CEACAM1-mediated internalization of bacteria, we surprisingly find that the CEACAM1 cytoplasmic domain is completely obsolete for bacterial uptake. Accordingly, CEACAM1-4L as well as a CEACAM1 mutant with a complete deletion of the cytoplasmic domain (CEACAM1 ΔCT) promote equivalent internalization of several human pathogens. CEACAM1-4L- and CEACAM1 ΔCT-mediated uptake proceeds in the presence of inhibitors of actin microfilament dynamics, which is in contrast to CEACAM3-mediated internalization. Bacteria-engaged CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3, localize to a gangliosid GM1- and GPI-anchored protein-containing portion of the plasma membrane. In addition, interference with cholesterol-rich membrane microdomains severely blocks bacterial uptake via CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3. Similar to GPI-anchored CEACAM6, both CEACAM1-4L as well as CEACAM1 ΔCT partition into a low-density, Triton-insoluble membrane fraction upon receptor clustering, whereas CEACAM3 is not detected in this fraction. Bacterial uptake by truncated CEACAM1 or chimeric CEACAM1/CEACAM3 molecules reveals that the transmembrane domain of CEACAM1 is responsible for its association with membrane microdomains. Together, these data argue for a functional role of lipid rafts in CEACAM1-mediated endocytosis that is promoted by the transmembrane domain of the receptor and that might be relevant for CEACAM1 function in physiologic settings.     

Mutation of tyrosine residues 1162 and 1163 of the insulin receptor affects hormone and receptor internalization

Insulin internalization and degradation, insulin receptor internalization and recycling, as well as long term receptor down-regulation were comparatively studied in Chinese hamster ovary (CHO) cell lines, either parental or expressing the wild-type human insulin receptor (CHO.R) or a mutated receptor in which the tyrosine residues in positions 1162 and 1163 were replaced by phenylalanines (CHO.Y2). The two transfected cell lines presented very similar binding characteristics, and their pulse labeling with [35S]methionine revealed that the receptors were processed normally. As expected, the mutation of these twin tyrosines resulted in a defective insulin stimulation of both receptor kinase activity and glycogen synthesis. We now present evidence that compared to CHO.R cells, which efficiently internalized and degraded insulin, CHO.Y2 cells exhibited a marked defect in hormone internalization, leading to impaired insulin degradation. Moreover, the mutated receptors were found to be less effective than the wild-type receptors in transducing the hormone signal for receptor internalization, whereas the process of receptor recycling after internalization seemed not to be altered. In parental CHO cells, insulin induced long term receptor down-regulation, but was totally ineffective in both transfected cell lines. These results reveal that the tyrosines 1162 and 1163 in the kinase regulatory domain of the receptor beta-subunit play a pivotal role in insulin and receptor internalization.     

Defective internalization of insulin and its receptor in cells expressing mutated insulin receptors lacking kinase activity

The internalization and degradation of insulin was assessed in Chinese hamster ovary cell lines expressing either the wild-type receptor or mutated receptors lacking kinase activity. The mutated receptors included receptors which differed from the wild-type receptor by a single amino acid (substitution of an arginine for lysine at position 1030, a site critical for ATP binding) as well as receptors which had a deletion of 112 amino acids at the carboxyl terminus. Cells expressing mutated receptors lacking kinase activity were found to internalize and degrade insulin at about half the rate of cells expressing wild-type receptors with kinase activity. Moreover, insulin was found incapable of inducing the internalization of the mutated receptors, whereas it could stimulate the internalization of the wild-type receptor. Finally, the constitutive rate of receptor internalization was found to be the same for the mutant and wild-type receptors. These results implicate the intrinsic tyrosine-specific kinase activity of the insulin receptor in the ligand-induced, but not the constitutive, internalization of this receptor.     

Adaptor protein complex-2 (AP-2) and epsin-1 mediate protease-activated receptor-1 internalization via phosphorylation- and ubiquitination-dependent sorting signals

Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs.     

Insulin receptor internalization and recycling: mechanism and significance

Using a 125I-photoreactive insulin analogue that can be covalently coupled to its receptor we have shown that in rat hepatocytes the insulin receptor is concomitantly internalized with the labeled hormone and afterwards is progressively recycled back to the cell surface. In the course of the internalization process the insulin-receptor complex associates with clear vesicles and later on with lysosomes from which it is recycled through clear vesicles. On the basis of these observations it is suggested that modulation of the rates of internalization and of recycling of the insulin receptor can regulate the number of available surface insulin receptors. This hypothesis is supported by the results of experiments showing that monensin, an inhibitor of receptor recycling enhances insulin induced loss of its own surface receptors (down regulation) in U-937 monocytes.     

Characterization of the carboxyl-terminal domain of the rat glucose-dependent insulinotropic polypeptide (GIP) receptor. A role for serines 426 and 427 in regulating the rate of internalization.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion. In non-insulin-dependent diabetes mellitus insulin responses to GIP are blunted, possibly due to altered signal transduction or reduced receptor number. Site-directed mutagenesis was used to construct truncated GIP receptors to study the importance of the carboxyl-terminal tail (CT) in binding, signaling, and receptor internalization. Receptors truncated at amino acids 425, 418, and 405, expressed in COS-7 or CHO-K1 cells, exhibited similar binding to wild type receptors. GIP-dependent cAMP production with the 405 mutant was decreased in COS-7 cells. Maximal cAMP production in CHO-K1 cells was reduced with all truncated forms. Binding was undetectable with a receptor truncated at amino acid 400; increasing tail length by adding 5 alanines restored binding and signaling. Mutants produced by alanine scanning of residues 394-401, adjacent to transmembrane domain 7, were all functional. CT truncation by 30 or more amino acids, mutation of serines 426/427, singly or combined, or complete CT serine knockout all reduced receptor internalization rate. The majority of the GIP receptor CT is therefore not required for signaling, a minimum chain length of approximately 405 amino acids is needed for receptor expression, and serines 426 and 427 are important for regulating rate of receptor internalization.     

Cis- and trans-acting functions required for endocytosis of the yeast pheromone receptors

The Saccharomyces cerevisiae a-factor receptor (STE3) is subject to two modes of endocytosis: a constitutive process that occurs in the absence of ligand and a regulated process that is triggered by binding of ligand. Both processes result in delivery of the receptor to the vacuole for degradation. Receptor mutants deleted for part of the COOH- terminal cytoplasmic domain are disabled for constitutive, but not ligand-dependent internalization. Trans-acting mutants that impair constitutive endocytosis have been isolated. One of these, ren1-1, is blocked at a late step in the endocytic pathway, as receptor accumulates in a prevacuolar endosome-like compartment. REN1 is identical to VPS2, a gene required for delivery of newly synthesized vacuolar enzymes to the vacuole. Based on this identity, we suggest a model in which the transport pathways to the vacuole--the endocytic pathway and the vacuolar biogenesis pathway--merge at an intermediate endocytic compartment. As receptor also accumulates at the surface of ren1 cells, receptor may recycle from the putative endosome to the surface, or REN1 may also be required to carry out an early step in endocytosis.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by β-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.     

Human insulin receptor juxtamembrane domain independent insulin signaling

The exon 16-encoded juxtamembrane (JM) domain of human insulin receptor (hIR) harbors the NPEY motif which couples the insulin-activated hIR kinase to downstream signal transduction molecules. We sought to determine if signal transduction requires the entire exon 16-encoded 22-amino acid JM domain. Transfected CHO cells were generated stably expressing either the wild-type hIR (hIR-WT) or two mutant hIRs (hIRDeltaEx16 in which the JM domain was deleted, and hIRrosJM in which the deleted segment was replaced by the corresponding domain of v-ros protein). The mutant hIRDeltaEx16 and hIRrosJM exhibited similar insulin-binding as the hIRWT. Insulin internalization and insulin dose-response experiments toward activation of downstream signal transduction molecules demonstrated that: i) the presence of intact hIR-JM domain which harbors the NPEY motif is essential for Shc phosphorylation but not for IRS-1 phosphorylation; ii) insulin signal transduction can occur independent of the JM domain of hIR and without participation of the NPEY motif; iii) engagement of this putative alternative downstream signal transduction is Shc independent and is dependent on insulin concentration; and iv) insulin internalization does not necessarily require the hIR specific aa sequence of the JM domain which can be partially substituted by the JM domain of the v-ros tyrosine kinase.     

Internalization and degradation of insulin by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells

Chinese hamster ovary cell lines expressing either the wild-type human insulin receptor or a hybrid molecule in which the tyrosine kinase domain of the insulin receptor is replaced with that of the oncogene, v-ros were examined for their ability to internalize and degrade insulin. Cells expressing the hybrid receptor were found to internalize and degrade insulin at approximately half the rate of cells expressing the native insulin receptor. Moreover, insulin was incapable of inducing the internalization of the cell-surface hybrid molecule. In contrast, the constitutive rate of receptor internalization was found to be the same for the hybrid and wild-type receptors. These results obtained were similar to those with cells expressing either wild-type or mutant receptors lacking kinase activity. In conclusion, the substitution of the specificity of tyrosine kinase of the insulin receptor with that of the v-ros oncogene product results in defective internalization and degradation of insulin, and loss of ligand-induced receptor internalization.     

Extracellular matrix proteins modulate endocytosis of the insulin receptor

Internalization of the insulin receptor (IR) is a highly regulated multi-step process whose underlying molecular basis is not fully understood. Here we undertook to study the role of extracellular matrix (ECM) proteins in the modulation of IR internalization. Employing Chinese hamster ovary cells that overexpress IR (CHO-T cells), our results indicate that IR internalization proceeds unaffected even when Tyr phosphorylation of IR substrates, such as IRS-1, is impaired (e.g. in CHO-T cells overexpressing IRS-1 whose pleckstrin-homology domain has been deleted or in CHO-T cells that overexpress the PH/PTB domain of IRS-1). In contrast, IR internalization is affected by the context of the ECM proteins to which the cells adhere. Hence, IR internalization was inhibited 40-60% in CHO-T cells adherent onto galectin-8 (an ECM protein and an integrin ligand of the galectin family) when compared with cells adherent onto fibronectin, collagen, or laminin. Cells adherent to galectin-8 manifested a unique cytoskeletal organization, which involved formation of cortical actin and generation of F-actin microspikes that contrasted with the prominent stress-fibers formed when cells adhered to fibronectin. To better establish a role for actin filament organization in IR endocytosis, this process was assayed in CHO-T cells (adherent onto fibronectin), whose actin filaments were disrupted upon treatment with latrunculin B. Latrunculin B did not affect insulin-induced Tyr phosphorylation of IR or its ability to phosphorylate its substrates; still, a 30-50% reduction in the rate of IR internalization was observed in cells treated with latrunculin B. Treatment of cells with nocodazole, which disrupts formation of microtubules, did not affect IR internalization. These results indicate that proper actin, but not microtubular, organization is a critical requirement for IR internalization and suggest that integrin-mediated signaling pathways emitted upon cell adhesion to different extracellular matrices and the altered cytoskeletal organizations generated thereof affect the itinerary of the insulin receptor.     

Ligand-induced internalization and increased cell calcium are mediated via distinct structural elements in the carboxyl terminus of the epidermal growth factor receptor.

Signals that can mediate ligand-induced receptor internalization and calcium regulation are present in a 48-amino acid "calcium-internalization" domain in the C' terminus of the epidermal growth factor (EGF) receptor. The basis of calcium and internalization regulation signalled by this 48-amino acid sequence was analyzed using deletion and substitution mutant receptors. Cells expressing truncated receptors containing either the NH2- or COOH-terminal portion of the 48-residue domain displayed high affinity EGF-dependent endocytosis and receptor down-regulation. These endocytosis-competent EGF receptor mutants that lacked any autophosphorylation site were unable to increase the concentration of intracellular calcium. To investigate the role of self-phosphorylation in EGF-induced calcium mobilization, phenylalanine was substituted for the single autophosphorylated tyrosine residue in this region of an internalization-competent truncated receptor. The receptor-mediated calcium response was abolished, while ligand-dependent receptor internalization was unimpaired. These results demonstrate that EGF-dependent receptor endocytosis and calcium mobilization are separate events. Tyrosine self-phosphorylation is required for increased [Ca2+]i, while structural features distinct from autophosphorylation are required for receptor internalization.