Recycling and resensitization of delta opioid receptors

Exposure to opioids results in the activation of opioid receptors; this is followed by receptor endocytosis. Previously, we showed that delta opioid receptors undergo rapid agonist-mediated internalization and that mutations in the C-tail result in a substantial loss of agonist-mediated internalization. In this study, we investigated the fate of receptors following rapid internalization. We found that the majority of the wild type receptors recycled back to the surface after acute agonist treatment. The kinetics of internalization and recycling of the receptor were virtually identical to the kinetics of internalization and recycling of the radiolabeled agonist. In contrast, the kinetics of internalization and recycling of a C-tail mutant receptor were substantially altered, suggesting an involvement of the C-tail in the recycling process. It is possible that in addition to agonist-mediated internalization, opioid receptors undergo constitutive, agonist-independent internalization. We directly examined this possibility using an antibody-prebinding assay. The wild type delta opioid receptors exhibited agonist-independent internalization via the clathrin-coated pit pathway. We also examined the role of receptor internalization and recycling in the modulation of its function by quantitating the level of opioid-stimulated phosphorylation of MAP kinase (MAPK) under conditions of receptor internalization and recycling. We found that agonist treatment caused a rapid increase in the level of phosphorylated MAPK that was rapidly desensitized. The removal of the agonist, which results in receptor recycling, led to the resensitization of the receptor, as evidenced by the agonist's ability to reinduce MAPK phosphorylation. Mutant receptors that underwent rapid recycling exhibited enhanced resensitization, suggesting a role for receptor recycling in the resensitization process. Taken together, these results indicate that agonist-mediated internalization and recycling modulate opioid receptor function and that the receptor C-tail plays an important role in both processes.     

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.     

Protein kinase C mediates phosphorylation, desensitization, and trafficking of the D2 dopamine receptor

Previously, D2 dopamine receptors (D2 DARs) have been shown to undergo G-protein-coupled receptor kinase phosphorylation in an agonist-specific fashion. We have now investigated the ability of the second messenger-activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC), to mediate phosphorylation and desensitization of the D2 DAR. HEK293T cells were transiently transfected with the D2 DAR and then treated with intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D2 DAR, suggesting that PKA does not phosphorylate the D2 DAR in HEK293T cells. In contrast, cellular treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in an approximately 3-fold increase in D2 DAR phosphorylation. The phosphorylation was specific for PKC as the PMA effect was mimicked by phorbol 12,13-dibutyrate, but not by 4alpha-phorbol 12,13-didecanoate, active and inactive, phorbol diesters, respectively. The PMA-mediated D2 DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II, and augmented by co-transfection with PKCbetaI. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation, suggesting that PKC is not involved in this response. PKC phosphorylation of the D2 DAR was found to promote receptor desensitization as reflected by a decrease in agonist potency for inhibiting cAMP accumulation. Most interestingly, PKC phosphorylation also promoted internalization of the D2 DAR through a beta-arrestin- and dynamin-dependent pathway, a response not usually associated with PKC phosphorylation of G-protein-coupled receptors. Site-directed mutagenesis experiments resulted in the identification of two domains of PKC phosphorylation sites within the third intracellular loop of the receptor. Both of these domains are involved in regulating sequestration of the D2 DAR, whereas only one domain is involved in receptor desensitization. These results indicate that PKC can mediate phosphorylation of the D2 DAR, resulting in both functional desensitization and receptor internalization.     

Dual regulation of EDG1/S1P(1) receptor phosphorylation and internalization by protein kinase C and G-protein-coupled receptor kinase 2.

Here we demonstrate that phosphorylation of the sphingosine 1-phosphate (SSP) receptor "endothelial differentiation gene 1" (EDG1 or S1P(1)) receptor is increased in response to either SSP or phorbol 12-myristate 13-acetate (PMA) exposure but not lysophosphatidic acid. Phosphoamino acid analysis demonstrated that SSP stimulated the accumulation of phosphoserine and phosphothreonine but not phosphotyrosine. An inhibitor of PMA-stimulated EDG1 phosphorylation failed to block SSP-stimulated phosphorylation. Additionally, removal of 12 amino acids from the carboxyl terminus of EDG1 specifically reduced SSP- but not PMA-stimulated phosphorylation, suggesting that SSP and PMA increase EDG1 phosphorylation via distinct mechanisms. In vitro assays revealed that G-protein-coupled receptor kinase 2 may be at least partially responsible for SSP-stimulated EDG1 phosphorylation observed in intact cells. In addition, phosphorylation by PMA and SSP were associated with a loss of EDG1 from the cell surface by distinct mechanisms. Removal of 12 residues from the carboxyl terminus of EDG1 completely inhibited SSP-mediated internalization, suggesting that this domain dictates susceptibility to receptor internalization while retaining sensitivity to SSP-stimulated phosphorylation. Thus, we conclude that (a) EDG1 phosphorylation and internalization are controlled via independent mechanisms by agonist occupation of the receptor and protein kinase C activation, and (b) although determinants within the receptor's carboxyl-terminal tail conferring EDG1 sensitivity to agonist-mediated internalization and G-protein-coupled receptor kinase phosphorylation exhibit a degree of overlap, the two phenomena are separable.     

Trafficking of M(2) muscarinic acetylcholine receptors

Internalization is an important mechanism regulating the agonist-dependent responses of G-protein-coupled receptors. The internalization of the M(2) muscarinic cholinergic receptors (mAChR) in HEK293 cells has been demonstrated to occur by an unknown mechanism that is independent of arrestins and dynamin. In this study we examined various aspects of the trafficking of the M(2) mAChR in HEK293 cells to characterize this unknown pathway of internalization. Internalization of the M(2) mAChR was rapid and extensive, but prolonged incubation with agonist did not lead to appreciable down-regulation (a decrease in total receptor number) of the receptors. Recovery of M(2) mAChRs to the cell surface following agonist-mediated internalization was a very slow process that contained protein synthesis-dependent and -independent components. The protein synthesis-dependent component of the recovery of receptors to the cell surface did not appear to reflect a requirement for synthesis of new receptors, as no changes in total receptor number were observed either in the presence or absence of cycloheximide. Phosphorylation of the M(2) mAChR did not appear to influence the rate or extent of the recovery of receptors to the cell surface, as the recovery of a phosphorylation-deficient mutant M(2) mAChR, the N,C(Ala-8) mutant, was similar to the recovery of the wild type M(2) mAChR. Finally, the constitutive, nonagonist-dependent internalization and recycling of the M(2) mAChR was very slow and also contained protein synthesis-dependent and -independent components, suggesting that a similar pathway controls the recovery from agonist-dependent and -independent internalization. Overall, these data demonstrated a variety of previously unappreciated facets involved in the regulation of M(2) mAChRs.     

D2-class dopamine receptor inhibition of NMDA currents in prefrontal cortical neurons is platelet-derived growth factor receptor-dependent

NMDA receptor function is modulated by both G-protein-coupled receptors and receptor tyrosine kinases. In acutely isolated rat hippocampal neurons, direct activation of the platelet-derived growth factor (PDGF) receptor or transactivation of the PDGF receptor by D4 dopamine receptors inhibits NMDA-evoked currents in a phospholipase C (PLC)-dependent manner. We have investigated further the ability of D2-class dopamine receptors to modulate NMDA-evoked currents in isolated rat prefrontal cortex (PFC). We have demonstrated that, similar to isolated hippocampal neurons, the application of PDGF-BB or quinpirole to isolated PFC neurons induces a slow-onset and long-lasting inhibition of NMDA-evoked currents. However, in contrast to hippocampal neurons, the inhibition of NMDA-evoked currents by quinpirole in PFC neurons is dependent upon D2/3, rather than D4, dopamine receptors. In PFC slices, application of both PDGF-BB and quinpirole induced a phosphorylation of the PDGF receptor at the PLCgamma binding and activation site, Tyr1021. The PDGF receptor kinase inhibitor, tyrphostin A9, and the D2/3 dopamine receptor antagonist, raclopride, inhibited quinpirole-induced Tyr1021 phosphorylation. These finding suggest that quinpirole treatment inhibits NMDAR signaling via PDGF receptor transactivation in both the hippocampus and the PFC, and that the effects of quinpirole in these regions are mediated by D4 and D2/3 dopamine receptors, respectively.     

The G-protein-coupled receptor kinase GRK4 mediates homologous desensitization of metabotropic glutamate receptor 1.

G-protein-coupled receptor kinases (GRKs) are involved in the regulation of many G-protein-coupled receptors. As opposed to the other GRKs, such as rhodopsin kinase (GRK1) or beta-adrenergic receptor kinase (beta ARK, GRK2), no receptor substrate for GRK4 has been so far identified. Here we show that GRK4 is expressed in cerebellar Purkinje cells, where it regulates mGlu(1) metabotropic glutamate receptors, as indicated by the following: 1) When coexpressed in heterologous cells (HEK293), mGlu(1) receptor signaling was desensitized by GRK4 in an agonist-dependent manner (homologous desensitization). 2) In transfected HEK293 and in cultured Purkinje cells, the exposure to glutamate agonists induced internalization of the receptor and redistribution of GRK4. There was a substantial colocalization of the receptor and kinase both under basal condition and after internalization. 3) Kinase activity was necessary for desensitizing mGlu(1a) receptor and agonist-dependent phosphorylation of this receptor was also documented. 4) Antisense treatment of cultured Purkinje cells, which significantly reduced the levels of GRK4 expression, induced a marked modification of the mGlu(1)-mediated functional response, consistent with an impaired receptor desensitization. The critical role for GRK4 in regulating mGlu(1) receptors implicates a major involvement of this kinase in the physiology of Purkinje cell and in motor learning.     

Mu-opioid receptor desensitization: role of receptor phosphorylation,internalization, and representation

It is generally accepted that the internalization and desensitization of mu-opioid receptor (MOR) involves receptor phosphorylation and beta-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser363 (MOR363D), was found to undergo phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced phosphorylation sites, did not exhibit detectable agonist-induced phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of beta-arrestin 2 when compared with wild type MOR, but the level of receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type receptors recycled back to membrane after agonist-induced internalization, which contributed to the receptor resensitization and thus partially reversed the receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the receptor internalization and the recycling of internalized receptor to cell surface in an active state. Taken together, our data indicated that receptor phosphorylation is not absolutely required in the internalization, but receptor phosphorylation and subsequent beta-arrestin recruitment play important roles in the resensitization of internalized receptors.     

Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2

beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.     

Kinetics of tyrosine phosphorylation and internalization of human EGF receptors overexpressed in NIH 3T3 fibroblasts

Binding of epidermal growth factor (EGF) to cells rapidly induces tyrosine phosphorylation of its receptor which is followed by its internalization and dephosphorylation. The kinetics of these processes differs widely in time from minutes to hours according to cell types. In this paper we analyzed EGF receptor phosphorylation and down-regulation in NIH 3T3 cells transfected with the recombinant hEGF-R cDNA which express 4 X 10(5) receptors/cell. In the presence of EGF receptor phosphorylation reached a maximum after 1 min and was then maintained for about 1 h, while during this time the number of EGF-binding sites was reduced to 40% of the initial number. Detailed analysis of the fate of a population of receptors previously activated and autophosphorylated at 4 degrees C, after warming to 37 degrees C in the absence of the ligand, showed that internalization of the cell surface-associated EGF and dephosphorylation of the receptor were rapid (t1/2 15 min) and followed a similar kinetics. Our data indicate that at any given time only a fraction of the total cell surface receptors is phosphorylated on tyrosine and that dephosphorylation occurs at the cell surface or very rapidly after internalization. In addition the data also suggest that a certain recycling of previously internalized receptors may occur in these cells during EGF treatment.     

Inverse agonist-induced signaling and down-regulation of the platelet-activating factor receptor

Platelet-activating factor (PAF) is a potent phospholipid mediator involved in several diseases such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G-protein-coupled receptor family. Following stimulation, PAFR becomes rapidly desensitized; this refractory state is dependent on PAFR phosphorylation, internalization and down-regulation. In this report, we show that the PAFR inverse agonist, WEB2086, can induce phosphorylation and down-regulation of PAFR. Using selective inhibitors, we determined that the agonist, PAF, and WEB2086 could induce phosphorylation of PAFR by PKC. Moreover, dominant-negative (DN) mutant of PKC isoforms beta inhibited WEB2086-stimulated PAFR phosphorylation, whereas PAF-stimulated phosphorylation was inhibited by DN PKCalpha and delta. WEB2086 also induced PAFR down-regulation which could be blocked by PKC inhibitors and by DN PKCbeta. WEB2086-induced down-regulation was dynamin-dependent but arrestin-independent. Unlike PAF, WEB2086-stimulated intracellular trafficking of PAFR was independent of Rab5. Specific inhibitors of lysosomal proteases and of proteasomes were both effective in reducing WEB2086-induced PAFR down-regulation, indicating the importance of receptor targeting to both lysosomes and proteasomes in long-term cell desensitization to WEB2086. These results indicate that although both agonists and inverse agonists induce receptor PAFR down-regulation, this may be accomplished through different signal transduction and trafficking pathways.     

Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.     

C-terminal splice variants of the mouse mu-opioid receptor differ in morphine-induced internalization and receptor resensitization

The main analgesic effects of the opioid alkaloid morphine are mediated by the mu-opioid receptor. In contrast to endogenous opioid peptides, morphine activates the mu-opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse mu-opioid receptor (MOR1) have been identified. In the present study, we show that these receptors differ substantially in their agonist-selective membrane trafficking. MOR1 and MOR1C stably expressed in human embryonic kidney 293 cells exhibited phosphorylation, internalization, and down-regulation in the presence of the opioid peptide [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) but not in response to morphine. In contrast, MOR1D and MOR1E exhibited robust phosphorylation, internalization, and down-regulation in response to both DAMGO and morphine. DAMGO elicited a similar desensitization (during an 8-h exposure) and resensitization (during a 50-min drug-free interval) of all four mu-receptor splice variants. After morphine treatment, however, MOR1 and MOR1C showed a faster desensitization and no resensitization as compared with MOR1D and MOR1E. These results strongly reinforce the hypothesis that receptor phosphorylation and internalization are required for opioid receptor reactivation thus counteracting agonist-induced desensitization. Our findings also suggest a mechanism by which cell- and tissue-specific C-terminal splicing of the mu-opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.     

Lysophosphatidic acid modulates c-Met redistribution and hepatocyte growth factor/c-Met signaling in human bronchial epithelial cells through PKC delta and E-cadherin

Previously we demonstrated that ligation of lysophosphatidic acid (LPA) to G protein-coupled LPA receptors induces transactivation of receptor tyrosine kinases (RTKs), such as platelet-derived growth factor receptor beta (PDGF-Rbeta) and epidermal growth factor receptor (EGF-R), in primary cultures of human bronchial epithelial cells (HBEpCs). Here we examined the role of LPA on c-Met redistribution and modulation of hepatocyte growth factor (HGF)/c-Met pathways in HBEpCs. Treatment of HBEpCs with LPA-induced c-Met serine phosphorylation and redistribution to plasma membrane, while treatment with HGF-induced c-Met internalization. Pretreatment with LPA reversed HGF-induced c-Met internalization. Overexpression of dominant negative (Dn)-PKC delta or pretreatment with Rottlerin or Pertussis toxin (PTx) attenuated LPA-induced c-Met serine phosphorylation and redistribution. Co-immnuoprecipitation and immunocytochemistry showed that E-cadherin interacted with c-Met in HBEpCs. LPA treatment induced E-cadherin and c-Met complex redistribution to plasma membranes. Overexpression of Dn-PKC delta attenuated LPA-induced E-cadherin redistribution and E-cadherin siRNA attenuated LPA-induced c-Met redistribution to plasma membrane. Furthermore, pretreatment of LPA attenuated HGF-induced c-Met tyrosine phosphorylation and downstream signaling, such as Akt kinase phosphorylation and cell motility. These results demonstrate that LPA regulates c-Met function through PKC delta and E-cadherin in HBEpCs, suggesting an alternate function of the cross-talk between G-protein-coupled receptors (GPCRs) and RTKs in HBEpCs.     

Phosphorylation and regulation of a Gq/11-coupled receptor by casein kinase 1alpha

Agonist-mediated receptor phosphorylation by one or more of the members of the G-protein receptor kinase (GRK) family is an established model for G-protein-coupled receptor (GPCR) phosphorylation resulting in receptor desensitization. Our recent studies have, however, suggested that an alternative route to GPCR phosphorylation may be an operation involving casein kinase 1alpha (CK1alpha). In the current study we investigate the involvement of CK1alpha in the phosphorylation of the human m3-muscarinic receptor in intact cells. We show that expression of a catalytically inactive mutant of CK1alpha, designed to act in a dominant negative manner, inhibits agonist-mediated receptor phosphorylation by approximately 40% in COS-7 and HEK-293 cells. Furthermore, we present evidence that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor (Ser(345)-Leu(463)) is an inhibitor of CK1alpha due to its ability to both act as a pseudo-substrate for CK1alpha and form a high affinity complex with CK1alpha. Expression of this peptide was able to reduce both basal and agonist-mediated m3-muscarinic receptor phosphorylation in intact cells. These results support the notion that CK1alpha is able to mediate GPCR phosphorylation in an agonist-dependent manner and that this may provide a novel mechanism for GPCR phosphorylation. The functional role of phosphorylation was investigated using a mutant of the m3-muscarinic receptor that showed an approximately 80% reduction in agonist-mediated phosphorylation. Surprisingly, this mutant underwent agonist-mediated desensitization suggesting that, unlike many GPCRs, desensitization of the m3-muscarinic receptor is not mediated by receptor phosphorylation. The inositol (1,4, 5)-trisphosphate response did, however, appear to be dramatically potentiated in the phosphorylation-deficient mutant indicating that phosphorylation may instead control the magnitude of the initial inositol phosphate response.     

Phosphorylation of the surface transferrin receptor stimulates receptor internalization in HL60 leukemic cells

The transferrin receptor is a target protein for phosphorylation by activated intracellular protein kinase C (May, W. S., Sahyoun, N., Jacobs, S., Wolf, M., and Cuatrecasas, P. (1985) J. Biol. Chem. 260, 9419-9426). Recently we reported that the potent tumor-promoting agent phorbol diester or a synthetic diacylglycerol could mediate rapid down-regulation of the surface transferrin receptor in association with receptor phosphorylation in HL60 leukemic cells and suggested that this phosphorylation may provide a signal for receptor internalization. In this communication we have tested experimentally the predictions generated by the hypothesis that receptor phosphorylation may play such a role in the intracellular cycling of the transferrin receptor. Results indicate that phorbol diester-stimulated phosphorylation occurs stoichiometrically only on the surface-oriented receptor and precedes internalization. Using a specific inhibitor of protein kinase C, it was found that both phorbol diester-mediated receptor phosphorylation and down-regulation could be antagonized. While the mechanism of internalization of the phosphorylated receptor is not clear, phorbol diester treatment significantly increases the rate constant for endocytosis from 0.183 to 0.462 min-1, while inhibiting only slightly the rate constant for exocytosis of the internalized receptor from 0.113 to 0.079 min-1. Thus, we conclude that phorbol diester treatment affects intracellular cycling of receptors and establishes a new steady state distribution of surface and intracellular receptors. These data support a role for receptor phosphorylation as a trigger for internalization primarily by stimulating the process of transferrin receptor endocytosis while affecting the subsequent exocytosis of the receptor cycling only slightly.     

beta-adrenergic mechanisms in cardiac diseases: a perspective

Several lines of evidence show that neurohumoral systems, especially those involving catecholamines, play a crucial role in cardiac diseases. Changes in the beta-adrenergic receptor (beta-AR) system such as receptor down-regulation, uncoupling from G-proteins, receptor internalization and receptor degradation may account for some of the abnormalities of contractile function in this disease. Increases in the level of inhibitory G-protein subunits also appears to be involved in attenuating the beta-AR signal. Finally beta-AR signalling is strongly regulated by members of the G-protein-coupled receptor kinase family (GRKs), the best known of which is beta-adrenergic receptor kinase 1 (beta-ARK1). beta-ARK1 mRNA, protein level and enzymatic activity is increased in heart disease, further contributing to an attenuation in beta-AR signalling. The combination of these negative alterations are presumably related to the contractile dysfunction seen in human heart disease. The combination of biochemical, physiological and molecular biological studies bearing on the normal function and regulation of these various molecules should provide strategies for elucidating the pharmacological basis of the regulation of myocardial contractility in the normal and failing heart.     

The molecular mechanisms of cellular tolerance to delta-opioid agonists. A minireview

Chronic treatment with deltaopioid agonists, similar to other agonist drugs, causes tolerance. Tolerance is a complex adaptation process that consists of multiple, cellular and neural-system adaptations. Cellular tolerance to delta-opioid agonists involves feedback-regulation of the function, concentration, and localization of the delta-opioid receptors (receptor desensitization) as well as of intracellular effectors (functional desensitization). We are using a recombinant Chinese hamster ovary cell line expressing the human delta-opioid receptors (hDOR/CHO) to investigate the molecular mechanisms of cellular tolerance. We found that the structurally distinct delta-opioid agonists mediate receptor down-regulation by different mechanisms. Thus, truncation of the last 35 C-terminal amino acids of the hDOR completely abolished DPDPE, but not SNC 80-mediated receptor down-regulation. In addition, down-regulation of the wild type-, and the truncated hDORs exhibited different inhibitor sensitivity-profile. Chronic delta-opioid agonist treatment also causes functional desensitization of forskolin-stimulated cAMP formation and cAMP overshoot in the hDOR/CHO cells. We have demonstrated that chronic SNC 80 treatment also causes concurrent phosphorylation of the adenylyl cyclase (AC) VI isoenzyme hDOR/CHO cells. Both AC superactivation and AC VI phosphorylation were SNC 80 dose-dependent, naltrindole-sensitive, and exhibited similar time course-, and protein kinase inhibitor-sensitivity profile. We hypothesize that phosphorylation of AC VI plays an important role in delta-opioid agonist-mediated AC superactivation in hDOR/CHO cells.     

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.     

Dynamic regulation, desensitization, and cross-talk in discrete subcellular microdomains during beta2-adrenoceptor and prostanoid receptor cAMP signaling

Dynamic and localized actions of cAMP are central to the generation of discrete cellular events in response to a range of G(s)-coupled receptor agonists. In the present study we have employed a cyclic nucleotide-gated channel sensor to report acute changes in cAMP in the restricted cellular microdomains adjacent to two different G(s)-coupled receptor pathways, beta(2)-adrenoceptors and prostanoid receptors that are expressed endogenously in HEK293 cells. We probed by either selective small interference RNA-mediated knockdown or dominant negative overexpression the contribution of key signaling components in the rapid attenuation of the local cAMP signaling and subsequent desensitization of each of these G-protein-coupled receptor signaling pathways immediately following receptor activation. Direct measurements of cAMP changes just beneath the plasma membrane of single HEK293 cells reveal novel insights into key regulatory roles provided by protein kinase A-RII, beta-arrestin2, cAMP phosphodiesterase-4D3, and cAMP phosphodiesterase-4D5. We provide new evidence for distinct modes of cAMP down-regulation in these two G(s)-linked pathways and show that these distinct G-protein-coupled receptor signaling systems are subject to unidirectional, heterologous desensitization that allows for limited cross-talk between distinct, dynamically regulated pools of cAMP.