Protein-tyrosine phosphatase 1B associates with insulin receptor and negatively regulates insulin signaling without receptor internalization

Phosphorylated platelet-derived growth factor (PDGF) receptor becomes internalized and then is dephosphorylated by protein-tyrosine phosphatase (PTP) 1B at the endoplasmic reticulum (ER). However, it remains unclear where PTP1B dephosphorylates insulin receptor and inhibits its activity. To clarify how and where PTP1B could interact with insulin receptor, we overexpressed a phosphatase-inactive mutant, PTP1BC/S, in 3T3-L1 adipocytes. Although PDGF receptor was maximally associated with PTP1BC/S at 30 min after PDGF stimulation, the maximal association of insulin receptor with PTP1BC/S was attained at 5 min after insulin stimulation. Furthermore, dansylcadaverine, a blocker of receptor internalization, inhibited this PDGF-induced association of PTP1BC/S with its receptor. However, dansylcadaverine did not affect the insulin-stimulated association of PTP1BC/S with insulin receptor, as well as dephosphorylation of insulin receptor by PTP1B. These results indicate that PTP1B might interact with insulin receptor and deactivate it without internalization. Finally, we overexpressed the wild-type and cytosolic-form of PTP1B to determine the role of ER-anchoring of PTP1B, and found that both inhibited insulin signaling equally. Thus, our data indicate that localization of PTP1B at the ER is not needed for insulin receptor dephosphorylation by PTP1B.     

RACK1 associates with muscarinic receptors and regulates M(2) receptor trafficking

Receptor internalization from the cell surface occurs through several mechanisms. Some of these mechanisms, such as clathrin coated pits, are well understood. The M(2) muscarinic acetylcholine receptor undergoes internalization via a poorly-defined clathrin-independent mechanism. We used isotope coded affinity tagging and mass spectrometry to identify the scaffolding protein, receptor for activated C kinase (RACK1) as a protein enriched in M(2)-immunoprecipitates from M(2)-expressing cells over those of non-M(2) expressing cells. Treatment of cells with the agonist carbachol disrupted the interaction of RACK1 with M(2). We further found that RACK1 overexpression inhibits the internalization and subsequent down regulation of the M(2) receptor in a receptor subtype-specific manner. Decreased RACK1 expression increases the rate of agonist internalization of the M(2) receptor, but decreases the extent of subsequent down-regulation. These results suggest that RACK1 may both interfere with agonist-induced sequestration and be required for subsequent targeting of internalized M(2) receptors to the degradative pathway.     

Dynamin and rab5 regulate GRK2-dependent internalization of dopamine D2 receptors.

Dopamine D2 receptors (D2Rs; short form, which is one of the alternative splicing variants) expressed in COS-7 cells are internalized in an agonist-dependent manner only when G protein-coupled receptor kinase 2 (GRK2) is coexpressed [Ito, K., Haga, T., Lameh, J. & Sadée, W., (1999) Eur. J. Biochem. 260, 112-119]. We have examined the effects of coexpression of dynamin, a small molecular mass GTP-binding protein, rab5A, and their mutants on the internalization of D2Rs in the presence of both dopamine (10 or 100 microM) and GRK2. The rate and extent of D2R internalization was increased or decreased by coexpression of dynamin I or a dominant-negative form of dynamin I (dynamin I K44E), respectively. The effects of coexpressing these two dynamins were more prominent at 10 microM dopamine than at 100 microM. In the presence of 10 microM dopamine, internalization of D2R was completely suppressed when dynamin I K44E was coexpressed, and the half-life (t 1/2) of D2R internalization decreased relative to cells not expressing dynamin from 82 to 29 min when dynamin I was coexpressed. Internalization of D2Rs was facilitated or suppressed by coexpression of a constitutively active form of rab5A (rab5A Q79L) or a dominant-negative form of rab5A (rab5A S34N), respectively. The t 1/2 of D2R internalization at 10 microM dopamine decreased from 82 to 16 min in cells coexpressing rab5A Q79L. The effect of coexpression of rab5A S34N was more apparent at 100 microM dopamine than at 10 microM; the t 1/2 of D2R internalization at 100 microM dopamine increased from 20 to 56 min and the proportion of internalized D2Rs after 120 min decreased from 53 to 28%. These results indicate that the internalization of D2Rs is dependent on the action of dynamin as well as GRK2, and is regulated by the action of rab5A.     

Grb2 regulates internalization of EGF receptors through clathrin-coated pits

The molecular mechanisms of clathrin-dependent internalization of epidermal growth factor receptor (EGFR) are not well understood and, in particular, the sequence motifs that mediate EGFR interactions with coated pits have not been mapped. We generated a panel of EGFR mutants and stably expressed these mutants in porcine aortic endothelial (PAE) cells. Interestingly, mutations of tyrosine phosphorylation sites 1068 and 1086 that interact with growth-factor-receptor-binding protein Grb2 completely abolished receptor internalization in PAE cells. Quantitative analysis of colocalization of EGF-rhodamine conjugate and coated pits labeled with yellow-fluorescent-protein-tagged beta2 subunit of clathrin adaptor complex AP-2 revealed that EGFR mutants lacking Grb2 binding sites do not efficiently enter coated pits. The depletion of Grb2 from PAE as well as HeLa cells expressing endogenous EGFRs by RNA interference substantially reduced the rate of EGFR internalization through clathrin-dependent pathway, thus providing the direct evidence for the important role of Grb2 in this process. Overexpression of Grb2 mutants, in which the SH3 domains were either deleted or inactivated by point mutations, significantly inhibited EGFR internalization in both PAE and HeLa cells. These findings indicate that Grb2, in addition to its key function in signaling through Ras, has a major regulatory role at the initial steps of EGFR internalization through clathrin-coated pits. Furthermore, the EGFR mutant lacking Grb2 binding sites did not efficiently recruit c-Cbl and was not polyubiquitinated. The data are consistent with the model whereby Grb2 participates in EGFR internalization through the recruitment of Cbl to the receptor, thus allowing proper ubiquitylation of EGFR and/or associated proteins at the plasma membrane.     

Distinct regulation of internalization and mitogen-activated protein kinase activation by two isoforms of the dopamine D2 receptor

Two isoforms of the dopamine D2 receptor, D2L (long) and D2S (short), differ by the insertion of a 29-amino acid specific to D2L within the putative third intracellular loop of the receptor. Here, we examined D2 receptor-mediated MAPK activation in association with receptor internalization. Overexpression of beta-arrestin 1 and 2 increased the D2S-mediated activation of MAPK, whereas it did not affect the activation of MAPK by D2L. Expression of a dominant negative beta-arrestin 2 (319-418) mutant and of a dominant negative dynamin I (K44A) mutant inhibited the activation of MAPK by D2S, but not the activation of MAPK by D2L. Treatment with inhibitors of internalization, i.e. concanavalin A and monodansylcadaverin, blocked D2S-mediated MAPK activation but not D2L-mediated activation. By confocal microscopy, we observed beta-arrestin 1 and 2, translocated to the plasma membrane and colocalized with D2L and D2S receptors upon stimulation with dopamine, and this was followed by the translocation of receptors into endocytic vesicles. Moreover, the expression of the beta-arrestin 2 (319-418) mutant blocked the internalization of both D2L and D2S. In addition, although K44A dynamin mutant expression did not alter D2L internalization, it completely blocked the internalization of D2S. The stimulation of D2L induces activation of MAPK via transactivation of the platelet-derived growth factor receptor, whereas D2S does not. Taken together, these data suggest that D2L activates MAPK signaling by mobilizing the growth factor receptor, platelet-derived growth factor receptor, whereas D2S appears to activate MAPK signaling by mobilizing clathrin-mediated endocytosis in a beta-arrestin/dynamin-dependent manner.     

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors

D₂ and D₃ dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D₂ receptor couples only to inhibitory G proteins, D₃ receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D₂ and D₃ receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D₂ receptor couple to Gi protein in a pattern characteristic of D₂ receptor. On the other hand chimeras containing a third cytoplasmic loop of D₃ receptor have coupling characteristics like those of D₃ receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D₂ and D₃ to G proteins.     

Regulation of phosphorylation of the GluR1 AMPA receptor by dopamine D2 receptors

In the striatum, stimulation of dopamine D2 receptors results in attenuation of glutamate responses. This effect is exerted in large part via negative regulation of AMPA glutamate receptors. Phosphorylation of the GluR1 subunit of the AMPA receptor has been proposed to play a critical role in the modulation of glutamate transmission, in striatal medium spiny neurons. Here, we have examined the effects of blockade of dopamine D2-like receptors on the phosphorylation of GluR1 at the cAMP-dependent protein kinase (PKA) site, Ser845, and at the protein kinase C and calcium/calmodulin-dependent protein kinase II site, Ser831. Administration of haloperidol, an antipsychotic drug with dopamine D2 receptor antagonistic properties, increases the phosphorylation of GluR1 at Ser845, without affecting phosphorylation at Ser831. The same effect is observed using eticlopride, a selective dopamine D2 receptor antagonist. In contrast, administration of the dopamine D2-like agonist, quinpirole, decreases GluR1 phosphorylation at Ser845. The increase in Ser845 phosphorylation produced by haloperidol is abolished in dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) knockout mice, or in mice in which the PKA phosphorylation site on DARPP-32 (i.e. Thr34) has been mutated (Thr34-->Ala mutant mice), and requires tonic activation of adenosine A2A receptors. These results demonstrate that dopamine D2 antagonists increase GluR1 phosphorylation at Ser845 by removing the inhibitory tone exerted by dopamine D2 receptors on the PKA/DARPP-32 cascade.     

Differential regulation of the dopamine D2 and D3 receptors by G protein-coupled receptor kinases and beta-arrestins

The D(2) and D(3) receptors (D(2)R and D(3)R), which are potential targets for antipsychotic drugs, have a similar structural architecture and signaling pathway. Furthermore, in some brain regions they are expressed in the same cells, suggesting that differences between the two receptors might lie in other properties such as their regulation. In this study we investigated, using COS-7 and HEK-293 cells, the mechanism underlying the intracellular trafficking of the D(2)R and D(3)R. Activation of D(2)R caused G protein-coupled receptor kinase-dependent receptor phosphorylation, a robust translocation of beta-arrestin to the cell membrane, and profound receptor internalization. The internalization of the D(2)R was dynamin-dependent, suggesting that a clathrin-coated endocytic pathway is involved. In addition, the D(2)R, upon agonist-mediated internalization, localized to intracellular compartments distinct from those utilized by the beta(2)-adrenergic receptor. However, in the case of the D(3)R, only subtle agonist-mediated receptor phosphorylation, beta-arrestin translocation to the plasma membrane, and receptor internalization were observed. Interchange of the second and third intracellular loops of the D(2)R and D(3)R reversed their phenotypes, implicating these regions in the regulatory properties of the two receptors. Our studies thus indicate that functional distinctions between the D(2)R and D(3)R may be found in their desensitization and cellular trafficking properties. The differences in their regulatory properties suggest that they have distinct physiological roles in the brain.     

Annexin V associates with the IFN-gamma receptor and regulates IFN-gamma signaling

Many of the biological activities of IFN-gamma are mediated through the IFN-gammaR3-linked Jak-Stat1alpha pathway. However, regulation of IFN-gamma signaling is not fully understood, and not all responses to IFN-gamma are Stat1alpha dependent. To identify novel elements involved in IFN-gamma cell regulation, the cytoplasmic domain of the R2 subunit of the human IFN-gammaR was used as bait in a yeast two-hybrid screen of a human monocyte cDNA library. This identified annexin A5 (AxV) as a putative IFN-gammaR binding protein. The interaction was confirmed in pull-down experiments in which a GST-R2 cytoplasmic domain fusion protein was incubated with macrophage lysates. Furthermore, immunoprecipitation using anti-IFN-gammaR2 Abs showed that AxV interacted with IFN-gammaR2 to form a stable complex following incubation of cells with IFN-gamma. In 293T cells with reduced expression of AxV, brought about by small interfering RNA targeting, activation of Jak2 and Stat1alpha in response to IFN-gamma was enhanced. Inhibition of cell proliferation, a hallmark of the IFN-gamma response, also was potentiated in HeLa cells treated with small interfering RNA directed at AxV. Taken together, these results suggest that through an inducible association with the R2 subunit of the IFN-gammaR, AxV modulates cellular responses to IFN-gamma by modulating signaling through the Jak-Stat1 pathway.     

Polypyrimidine tract-binding protein 1 regulates the alternative splicing of dopamine receptor D(2)

Dopamine receptor D(2) (DRD2) has two splicing isoforms, a long form (D2L) and short form (D2S), which have distinct functions in the dopaminergic system. However, the regulatory mechanism of the alternative splicing of DRD2 is unknown. In this study, we examined which splicing factors regulate the expression of D2L and D2S by over-expressing several RNA-binding proteins in HEK293 cells. In a cellular splicing assay, the over-expression of polypyrimidine tract-binding protein 1 (PTBP1) reduced the expression of D2S, whereas the knockdown of PTBP1 increased the expression of D2S. We also identified the regions of DRD2 that are responsive to PTBP1 using heterologous minigenes and deletion mutants. Our results indicate that PTBP1 regulates the alternative splicing of DRD2. Considering that DRD2 inhibits cAMP-dependent protein kinase A, which modulates the intracellular localization of PTBP1, PTBP1 may contribute to the autoregulation of DRD2 by regulating the expression of its isoforms.     

Glycoprotein nature of D2 dopamine receptors

The glycoprotein nature of the ligand binding subunit of photoaffinity-labeled striatal D2 receptors was investigated. Upon photolysis, [125I]N-azidophenethylspiperone covalently incorporated into a major band of Mr 94000 with an appropriate pharmacological profile for D2 receptors as assessed by autoradiography following SDS-polyacrylamide gel electrophoresis. The exoglycosidase, neuraminidase, altered the electrophoretic mobility of the 94 kDa labeled band to 54 kDa with a slight modification in the binding affinity of [3H]spiperone. Endoglycosidase treatment (glycopeptidase-F) produced a further increase in the mobility of the 94 kDa peptide to approximately 43 kDa. A smaller specifically photolabeled D2 receptor peptide of 34 kDa does not contain terminal sialic acid but is an N-linked glycoprotein as assessed by lectin affinity chromatography and susceptibility to digestion by glycopeptidase-F to a peptide of approximately 23 kDa.     

Irreversible interaction of beta-haloalkylamine derivatives with dopamine D1 and D2 receptors

The interaction of beta-haloalkylamine derivatives of dopamine agonists and antagonists with 3H-spiperone binding (D2 sites) and 3H-flupenthixol binding (D1 sites) was studied. N-chloroethyl derivatives of phenothiazines and thioxanthenes were potent inhibitors of the binding of both ligands. The in vitro inhibition of binding produced by these compounds was irreversible. The drugs were however only weakly active in vivo. The results suggest that beta-haloalkylamine derivatives of neuroleptics may be useful compounds for studying dopamine receptors in vitro.     

TTP specifically regulates the internalization of the transferrin receptor

Different plasma membrane receptors are internalized through saturable/noncompetitive pathways, suggesting cargo-specific regulation. Here, we report that TTP (SH3BP4), a SH3-containing protein, specifically regulates the internalization of the transferrin receptor (TfR). TTP interacts with endocytic proteins, including clathrin, dynamin, and the TfR, and localizes selectively to TfR-containing coated-pits (CCP) and -vesicles (CCV). Overexpression of TTP specifically inhibits TfR internalization, and causes the formation of morphologically aberrant CCP, which are probably fission impaired. This effect is mediated by the SH3 of TTP, which can bind to dynamin, and it is rescued by overexpression of dynamin. Functional ablation of TTP causes a reduction in TfR internalization, and reduced cargo loading and size of TfR-CCV. Tyrosine phosphorylation of either TTP or dynamin prevents their interaction, pointing to a possible mechanism of exclusion of TTP from some CCP. Thus, TTP might represent one of the long sought for molecules that allow cargo-specific control of clathrin endocytosis.     

Mechanisms of inverse agonism of antipsychotic drugs at the D(2) dopamine receptor: use of a mutant D(2) dopamine receptor that adopts the activated conformation

The antipsychotic drugs have been shown to be inverse agonists at the D(2) dopamine receptor. We have examined the mechanism of this inverse agonism by making mutations in residue T343 in the base of the sixth transmembrane spanning region of the receptor. T343R, T343S and T343K mutant D(2) dopamine receptors were made and the T343R mutant characterized in detail. The T343R mutant D(2) dopamine receptor exhibits properties of a receptor that resides more in the activated state, namely increased agonist binding affinity (independent of G-protein coupling and dependent on agonist efficacy), increased agonist potency in functional tests (adenylyl cyclase inhibition) and increased inverse agonist effects. The binding of agonists to the mutant receptor also shows sensitivity to sodium ions, unlike the native receptor, so that isomerization of the receptor to its inactive state may be driven by sodium ions. The binding of inverse agonists to the receptor is, however, unaffected by the mutation. We conclude that inverse agonism at this receptor is not achieved by the inverse agonist binding preferentially to the non-activated state of the receptor over the activated state. Rather the inverse agonist appears to bind to all forms of the receptor but then renders the receptor inactive.     

Hierarchical phosphorylation of delta-opioid receptor regulates agonist-induced receptor desensitization and internalization

Treatment of HEK293 cells expressing the delta-opioid receptor with agonist [d-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr(358) and Ser(363), were phosphorylated, with Ser(363) being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr(358) and Ser(363), with Thr(358) being involved in the receptor internalization.     

Differential voltage-sensitivity of D2-like dopamine receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by transmembrane voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by autoreceptors and in synaptic plasticity. We have recently described the voltage-sensitivity of the dopamine D_2L receptor and we now extend our studies to include the other members of the D₂-like receptor subfamily; the D_2S, D₃, and D₄ dopamine receptors. Electrophysiological recordings were performed on Xenopus oocytes coexpressing human dopamine D_2S, D₃, or D₄ receptors with G protein-coupled potassium (GIRK) channels. Comparison of concentration-response relationships at −80 mV and at 0 mV for dopamine-mediated GIRK activation revealed significant rightward shifts for both D_2S and D₄ upon depolarization. In contrast, the concentration-response relationships for D₃-mediated GIRK activation were not appreciably different at the two voltages. Our findings provide new insight into the functional differences of these closely related receptors.     

The receptor tyrosine kinase Ror2 associates with and is activated by casein kinase Iepsilon

Ror2, a member of the mammalian Ror family of receptor tyrosine kinases, plays important roles in developmental morphogenesis, although the mechanism underlying activation of Ror2 remains largely elusive. We show that when expressed in mammalian cells, Ror2 associates with casein kinase Iepsilon (CKIepsilon), a crucial regulator of Wnt signaling. This association occurs primarily via the cytoplasmic C-terminal proline-rich domain of Ror2. We also show that Ror2 is phosphorylated by CKIepsilon on serine/threonine residues, in its C-terminal serine/threonine-rich 2 domain, resulting in autophosphorylation of Ror2 on tyrosine residues. Furthermore, it was found that association of Ror2 with CKIepsilon is required for its serine/threonine phosphorylation by CKIepsilon. Site-directed mutagenesis of tyrosine residues in Ror2 reveals that the sites of phosphorylation are contained among the five tyrosine residues in the proline-rich domain but not among the four tyrosine residues in the tyrosine kinase domain. Moreover, we show that in mammalian cells, CKIepsilon-mediated phosphorylation of Ror2 on serine/threonine and tyrosine residues is followed by the tyrosine phosphorylation of G protein-coupled receptor kinase 2, a kinase with a developmental expression pattern that is remarkably similar to that of Ror2. Intriguingly, a mutant of Ror2 lacking five tyrosine residues, including the autophosphorylation sites, fails to tyrosine phosphorylate G protein-coupled receptor kinase 2. This indicates that autophosphorylation of Ror2 is required for full activation of its tyrosine kinase activity. These findings demonstrate a novel role for CKIepsilon in the regulation of Ror2 tyrosine kinase.     

SKF83959 selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brain

Previously a distinct D1-like dopamine receptor (DAR) that selectively couples to phospholipase C/phosphatidylinositol (PLC/PI) was proposed. However, lack of a selective agonist has limited efforts aimed at characterizing this receptor. We characterized the in vitro and in vivo effects of SKF83959 in regulating PI metabolism. SKF83959 stimulates (EC50, 8 micro m) phosphatidylinositol 4,5-biphosphate hydrolysis in membranes of frontal cortex (FC) but not in membranes from PC12 cells expressing classical D1A DARs. Stimulation of FC PI metabolism was attenuated by the D1 antagonist, SCH23390, indicating that SKF83959 activates a D1-like DAR. The PI-linked DAR is located in hippocampus, cerebellum, striatum and FC. Most significantly, administration of SKF83959 induced accumulations of IP3 in striatum and hippocampus. In contrast to other D1 DAR agonists, SKF83959 did not increase cAMP production in brain or in D1A DAR-expressing PC12 cell membranes. However, SKF83959 inhibited cAMP elevation elicited by the D1A DAR agonist, SKF81297, indicating that the compound is an antagonist of the classical D1A DAR. Lastly, we demonstrated that SKF83959 enhances [35S]guanosine 5'-O-(3-thiotriphosphate) binding to membrane Galphaq and Galphai proteins, suggesting that PI stimulation is mediated by activation of these guanine nucleotide-binding regulatory proteins. Results indicate that SKF83959 is a selective agonist for the PI-linked D1-like DAR, providing a unique tool for investigating the functions of this brain D1 DAR subtype.