Replacement of threonine 394 by alanine facilitates internalization and resensitization of the rat mu opioid receptor

Signaling of G protein-coupled receptors is terminated by phosphorylation of intracellular serine and threonine residues. Resensitization of these receptors requires internalization and subsequent dephosphorylation. We have recently shown that the resensitization rate of the rat micro opioid receptor (MOR) isoforms MOR1 and MOR1B is mainly determined by the amino acid composition of their alternatively spliced C-terminal tails. Upon agonist stimulation, MOR1B passes through an accelerated cycle of receptor endocytosis and reactivation, which in turn promotes a greater resistance to agonist-induced desensitization, as compared with MOR1. Given the fact that MOR1B lacks only one putative phosphorylation site (T394 of MOR1), we replaced this threonine by an alanine and stably expressed the wild-type MOR1 and its T394A mutant in mouse neuroblastoma Neuro2a cells. We show that during prolonged [D-Ala2, MePhe4, Gly5-ol]enkephalin exposure (5 h), the T394A receptor mutant desensitized at a slower rate than MOR1. In contrast, T394A is more rapidly removed from the cell surface than MOR1, as determined by flow cytometry using epitope-tagged receptors. This fast internalization was followed by immediate resensitization of T394A during 20 min of agonist removal while the wild-type MOR1 remained inactive. Similar to MOR1B, rapid internalization and reactivation of T394A may explain its delayed desensitization. These findings suggest that T394 represents a negative regulatory signal for MOR1 internalization. Furthermore, phosphorylation of this threonine residue may influence the time course of micro opioid receptor resensitization.     

Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors

We compared the desensitization of neurokinin1 and neurokinin2 (NK1 and NK2) receptors expressed in Chinese hamster ovary cells to substance P and neurokinin A, respectively. Substance P and neurokinin A stimulated a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) for both receptors, which was due to release of Ca2+ from intracellular stores. This was followed by a plateau in [Ca2+]i, which was due to influx of extracellular Ca2+, and was more sustained for the NK2 receptor. When Ca2+ was present in the extracellular solution, the Ca2+ response of the NK1 receptor, but not the NK2 receptor, rapidly desensitized and slowly resensitized to two exposures to agonist. In contrast, the [Ca2+]i response, measured in Ca2+-free solution, and inositol triphosphate generation desensitized and resensitized similarly for the NK1 and NK2 receptors. Thus, differences in desensitization between the NK1 receptor and the NK2 receptor may be related to differences in entry of extracellular Ca2+. We compared endocytosis of the NK1 and NK2 receptors to determine whether disparities could account for differences in desensitization. Fluorescent and radiolabeled substance P and neurokinin A were internalized similarly by cells expressing NK1 and NK2 receptors. Thus, disparities in internalization cannot account for differences in desensitization. We used inhibitors to examine the contribution of endocytosis, recycling, and phosphatases to desensitization and resensitization of the NK1 receptor. Desensitization did not require endocytosis. However, resensitization required endocytosis, recycling, and phosphatase activity. This suggests that the NK1 receptor desensitizes by phosphorylation and resensitizes by dephosphorylation in endosomes and recycling.     

Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics

The mammalian gonadotropin-releasing hormone receptor (GnRH-R) is the only G-protein-coupled receptor (GPCR) in which the intracellular C-terminal tail is completely absent. In contrast to other GPCRs, the GnRH-R does not show rapid desensitization of total inositol (IP) production, and the rates of internalization are exceptionally slow. We investigated whether the incorporation of a cytoplasmic tail into the C terminus of the GnRH-R affects desensitization events and receptor internalization rates. A GnRH-R/TRH-R chimera was created where the intracellular tail of the rat thyrotropin-releasing hormone receptor (TRH-R) was engineered into the C terminus of the rat GnRH-R. Three different rat GnRH-R cDNA stop codon mutations (one for each reading frame) were also made. The GnRH-stimulated IP production of the wild-type rat GnRH-R expressed in either COS-7 or HEK 293 cells did not desensitize even after prolonged stimulation with GnRH. In contrast, the catfish GnRH-R (which does possess an intracellular tail) and the TRH-R rapidly (<10 min) desensitized following agonist stimulation. The GnRH-R/TRH-R chimera also desensitized following treatment with GnRH, resembling the pattern shown by the TRH-R and the catfish GnRH-R. Two of the stop codon mutants did not show desensitization of IP production, and the third mutant with the longest tail was not functional. Internalization experiments showed that the rat GnRH-R had the slowest endocytosis and recycling rates compared with the TRH-R, the catfish GnRH-R, and the chimeric GnRH/TRH-R. This study demonstrates that the addition of a functional intracellular C-terminal tail to the GnRH-R produces rapid desensitization of IP production and significantly increases internalization rates.     

The effect of inhibitors of receptor internalization on the desensitization and resensitization of three Gs-coupled receptor responses

Many G protein-coupled receptors (GPCRs) are known to internalize following agonist exposure, however the relative importance of this mechanism for the desensitization and resensitization of different GPCRs is unclear. In the present study, we have pretreated NG108-15 cells with hypertonic sucrose or concanavalin A (con A), to investigate the effects of these inhibitors of internalization on the agonist-induced desensitization and subsequent resensitization of three Gs-coupled receptor responses. Incubation of cells with sucrose or con A did not affect subsequent acute agonist stimulation of the A2A adenosine receptor or the agonist-induced desensitization of this receptor response. However, the resensitization of the A2A adenosine receptor response following agonist removal was abolished in the presence of sucrose or con A. Sucrose or con A treatment affected neither the desensitization nor resensitization of IP-prostanoid receptor responsiveness. On the other hand con A but not sucrose reduced the agonist-induced desensitization of secretin receptor responsiveness. However, secretin receptor responsiveness did not resensitize within the time period studied whether or not inhibitors of internalization were present. These results indicate that receptor internalization appears to subserve different functions for different GPCRs.     

Carboxyl-terminal splicing of the rat mu opioid receptor modulates agonist-mediated internalization and receptor resensitization

The rat mu opioid receptor is alternatively spliced into two isoforms (MOR1 and MOR1B) which differ in length and amino acid composition at the carboxyl terminus. When stably expressed in HEK 293 cells, both splice variants bind the mu receptor agonist [D-Ala2,N-Me-Phe4,-Gly-ol5]enkephalin (DAMGO) with similar affinity and exhibit functional coupling to adenylyl cyclase with similar efficiency. However, the shorter isoform, MOR1B, desensitized at a slower rate during prolonged DAMGO exposure (4 h) but resensitized at a faster rate than MOR1 during agonist withdrawal (20 min). Immunocytochemical analysis revealed that DAMGO-induced internalization of MOR1B proceeded much faster than that of MOR1 followed by rapid recycling of the receptor to the cell surface. In addition, the greater resistance of MOR1B to homologous desensitization compared with MOR1 as well as MOR1B resensitization was abolished when receptor reactivation/recycling was blocked with monensin, an inhibitor of endosomal acidification. It is concluded that the sequence at the cytoplasmic tail of MOR1B facilitates clathrin-coated vesicle-mediated endocytosis which, in turn, promotes accelerated receptor reactivation. Taken together, our findings suggest that carboxyl-terminal splicing of the rat mu opioid receptor modulates agonist-induced internalization and receptor resensitization.     

Internalization and homologous desensitization of the GLP-1 receptor depend on phosphorylation of the receptor carboxyl tail at the same three sites

Homologous desensitization and internalization of the GLP-1 receptor correlate with phosphorylation of the receptor in a 33-amino acid segment of the cytoplasmic tail. Here, we identify the sites of phosphorylation as being three serine doublets located at positions 441/442, 444/445, and 451/452. The role of phosphorylation on homologous desensitization was assessed after stable expression in fibroblasts of the wild type or of mutant receptors in which phosphorylation sites were changed in various combinations to alanines. We showed that desensitization, as measured by a decrease in the maximal production of cAMP after a first exposure of the cells to GLP-1, was strictly dependent on phosphorylation. Furthermore, the number of phosphorylation sites correlated with the extent of desensitization with no, intermediate, or maximal desensitization observed in the presence of one, two, or three phosphorylation sites, respectively. Internalization of the receptor-ligand complex was assessed by measuring the rate of internalization of bound [125I]GLP-1 or the redistribution of the receptor to an endosomal compartment after agonist binding. Our data demonstrate that internalization was prevented in the absence of receptor phosphorylation and that intermediate rates of endocytosis were obtained with receptors containing one or two phosphorylation sites. Thus, homologous desensitization and internalization require phosphorylation of the receptor at the same three sites. However, the differential quantitative impairment of these two processes in the single and double mutants suggests different molecular mechanisms controlling desensitization and internalization.     

Using green fluorescent protein to understand the mechanisms of G-protein-coupled receptor regulation

G protein-coupled receptor (GPCR) activation is followed rapidly by adaptive changes that serve to diminish the responsiveness of a cell to further stimulation. This process, termed desensitization, is the consequence of receptor phosphorylation, arrestin binding, sequestration and down-regulation. GPCR phosphorylation is initiated within seconds to minutes of receptor activation and is mediated by both second messenger-dependent protein kinases and receptor-specific G protein-coupled receptor kinases (GRKs). Desensitization in response to GRK-mediated phosphorylation involves the binding of arrestin proteins that serve to sterically uncouple the receptor from its G protein. GPCR sequestration, the endocytosis of receptors to endosomes, not only contributes to the temporal desensitization of GPCRs, but plays a critical role in GPCR resensitization. GPCR down-regulation, a loss of the total cellular complement of receptors, is the consequence of both increased lysosomal degradation and decreased mRNA synthesis of GPCRs. While each of these agonist-mediated desensitization processes are initiated within a temporally dissociable time frame, recent data suggest that they are intimately related to one another. The use of green fluorescent protein from the jellyfish Aqueora victoria as an epitope tag with intrinsic fluorescence has facilitated our understanding of the relative relationship between GRK phosphorylation, arrestin binding, receptor sequestration and down-regulation.     

Role of the thrombin receptor's cytoplasmic tail in intracellular trafficking. Distinct determinants for agonist-triggered versus tonic internalization and intracellular localization

The G protein-coupled thrombin receptor is activated by an irreversible proteolytic mechanism and, perhaps as a result, exhibits an unusual trafficking pattern in the cell. Naive receptors tonically cycle between the cell surface and a protected intracellular pool, whereas receptors cleaved and activated at the cell surface internalize and move to lysosomes. Toward understanding how these trafficking events are regulated, we examined a series of receptor mutants. A receptor with alanine substitutions at all potential phosphorylation sites in the cytoplasmic tail failed to display agonist-triggered internalization but, like wild type receptor, displayed robust signaling, tonic cycling, and localization to both the cell surface and an intracellular pool. A truncation mutant that lacked most of the cytoplasmic tail also signaled robustly, lacked phosphorylation, and was defective in agonist-triggered internalization. However, in contrast to the specific phosphorylation site mutant, the truncation mutant did not display tonic cycling and localized exclusively to the cell surface. An analysis of a series of truncation mutants localized residues important for receptor trafficking to a 10-amino acid stretch in its cytoplasmic tail. These data suggest that phosphorylation may trigger internalization of activated thrombin receptors but that a second phosphorylation-independent signal mediates tonic internalization of naive receptors. They further suggest that maintenance of the intracellular pool of naive thrombin receptors requires tonic receptor internalization.     

Desensitization of the delta-opioid receptor correlates with its phosphorylation in SK-N-BE cells: involvement of a G protein-coupled receptor kinase

Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human delta-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human delta-opioid receptor, revealed that it corresponded to the delta-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the delta-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the delta-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn2+, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human delta-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.     

Identification of phosphorylation sites in the G protein-coupled receptor for parathyroid hormone. Receptor phosphorylation is not required for agonist-induced internalization

In some G protein-coupled receptors (GPCRs), agonist-dependent phosphorylation by specific GPCR kinases (GRKs) is an important mediator of receptor desensitization and endocytosis. Phosphorylation and the subsequent events that it triggers, such as arrestin binding, have been suggested to be regulatory mechanisms for a wide variety of GPCRs. In the present study, we investigated whether agonist-induced phosphorylation of the PTH receptor, a class II GPCR, also regulates receptor internalization. Upon agonist stimulation, the PTH receptor was exclusively phosphorylated on serine residues. Phosphoamino acid analysis of a number of receptor mutants in which individual serine residues had been replaced by threonine identified serine residues in positions 485, 486, and 489 of the cytoplasmic tail as sites of phosphorylation after agonist treatment. When serine residues at positions 483, 485, 486, 489, 495, and 498 were simultaneously replaced by alanine residues, the PTH receptor was no longer phosphorylated either basally or in response to PTH. The substitution of these serine residues by alanine affected neither the number of receptors expressed on the cell surface nor the ability of the receptor to signal via Gs. Overexpression of GRK2, but not GRK3, enhanced PTH-stimulated receptor phosphorylation, and this phosphorylation was abolished by alanine mutagenesis of residues 483, 485, 486, 489, 495, and 498. Thus, phosphorylation of the PTH receptor by the endogenous kinase in HEK-293 cells occurs on the same residues targeted by overexpressed GRK2. Strikingly, the rate and extent of PTH-stimulated internalization of mutated PTH receptors lacking phosphorylation sites were identical to that observed for the wild-type PTH receptor. Moreover, overexpressed GRK2, while enhancing the phosphorylation of the wild-type PTH receptor, had no affect on the rate or extent of receptor internalization in response to PTH. Thus, the agonist-occupied PTH receptor is phosphorylated by a kinase similar or identical to GRK2 in HEK-293 cells, but this phosphorylation is not requisite for efficient receptor endocytosis.     

Signal transduction, desensitization, and recovery of responses to thyrotropin-releasing hormone after inhibition of receptor internalization

Three independent methods were used to block internalization of the TRH receptor: cells were infected with vaccinia virus encoding a dominant negative dynamin, incubated in hypertonic sucrose, or stably transfected with a receptor lacking the C-terminal tail. Internalization was blocked in all three paradigms as judged by microscopy using a fluorescently labeled TRH agonist and biochemically. The initial inositol trisphosphate (IP3) and Ca2+ responses to TRH were normal when internalization was inhibited. The IP3 increase was sustained rather than transient, however, in cells expressing the truncated TRH receptor, implying that the C-terminal tail of the receptor may be important for uncoupling from phospholipase C. After withdrawal of TRH, cells were refractory to TRH until both ligand dissociation and resensitization of the receptor had occurred. When surface-bound TRH was removed by a mild acid wash, which did not impair receptor function, neither wild-type nor truncated receptors were able to generate full IP3 responses for about 10 min. The rate of recovery was not altered by blocking internalization. Recovery of intracellular Ca2+ responses also depended on the rate of Ca2+ pool refilling. In summary, in the continued presence of TRH, phospholipase C activity declines quickly due to receptor uncoupling; this desensitization does not take place for the truncated receptor. After TRH is withdrawn, cells are refractory to TRH. Before cells can respond, TRH must dissociate and a resensitization step, which takes place on the plasma membrane and does not require the C-terminal tail of the receptor, must occur.     

G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins

G-protein-coupled receptors (GPCRs) represent a large family of proteins that transduce extracellular signals to the interior of cells. Signalling through these receptors rapidly desensitized primarily as the consequence of receptor phosphorylation, but receptor sequestration and downregulation can also contribute to this process. Two families of serine/threonine kinases, second messenger dependent protein kinases and receptor-specific G-protein-coupled receptor kinases (GRKs), phosphorylate GPCRs and thereby contribute to receptor desensitization. Receptor-specific phosphorylation of GPCRs promotes the binding of cytosolic proteins referred to as arrestins, which function to further uncouple GPCRs from their heterotrimeric G-proteins. To date, the GRK protein family consists of six members, which can be further classified into subgroups according to sequence homology and functional similarities. The arrestin protein family also comprises six members, which are subgrouped on the basis of sequence homology and tissue distribution. While the molecular mechanisms contributing to GPCR desensitization are fairly well characterized, little is known about the mechanism(s) by which GPCR responsiveness is reestablished, other than that receptor sequestration (internalization) might be involved. The goal of the present review is to overview current understanding of the regulation of GPCR responsiveness. In particular, we will review new evidence suggesting a pleiotropic role for GRKs and arrestins in the regulation of GPCR responsiveness. GRK-mediated phosphorylation and arrestin binding are not only involved in the functional uncoupling of GPCRs but they are also intimately involved in promoting GPCR sequestration and as such likely play an important role in mediating the subsequent resensitization of GPCRs.     

A constitutively internalizing and recycling mutant of the mu-opioid receptor

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation, whereas certain other receptor types recycle constitutively, e.g., the transferrin receptor. To investigate structural domains involved in mu-opioid receptor internalization, we constructed two truncation mutants bracketing a Ser/Thr-rich domain (354ThrSerSerThrIleGluGlnGlnAsn362) unique to the C-terminus of the mu-opioid receptor (mutants Trunc354 and Trunc363). Ligand binding did not differ substantially, and G protein coupling was slightly lower for these mu-receptor constructs, in particular for Trunc363. To permit localization of the receptor by immunocytochemistry, an epitope tag was added to the N-terminus of the wild-type and mutant receptors. Both the wild-type mu-opioid receptor and Trunc363 resided largely at the plasma membrane and internalized into vesicles upon stimulation with the agonist [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin. Internalization occurred into vesicles that contain transferrin receptors, as shown previously, as well as clathrin, but not caveolin. In contrast, even without any agonist present, Trunc354 colocalized in intracellular vesicles with clathrin and transferrin receptors, but not caveolin. On blocking internalization by hyperosmolar sucrose or acid treatment, Trunc354 translocated to the plasma membrane, indicating that the mutant internalized into clathrin-coated vesicles and recycled constitutively. Despite agonist-independent internalization of Trunc354, basal G protein coupling was not elevated, suggesting distinct mechanisms for coupling and internalization. Furthermore, a portion of the C-terminus, particularly the Ser/Thr domain, appears to suppress mu-receptor internalization, which can be overcome by agonist stimulation. These results demonstrate that a mutant GPCR can be constructed such that internalization, normally an agonist-dependent process, can occur spontaneously without concomitant G protein activation.     

Identification of serine 356 and serine 363 as the amino acids involved in etorphine-induced down-regulation of the mu-opioid receptor

Agonist-induced internalization of G protein-coupled receptors is influenced by many structural determinants including the carboxyl tail. To investigate the role of serine and threonine residues within the carboxyl tail, several mutants were constructed by truncating the carboxyl tail of the hemagglutinin-tagged mu-opioid receptor, thereby removing serines and threonines systematically. Neuro2A cells stably expressing the truncated receptors did not exhibit a significant alteration in the affinity of [3H]diprenorphine or etorphine for the receptor or the potency of etorphine to inhibit forskolin-stimulated adenylyl cyclase activity. Chronic etorphine treatment resulted in a time-dependent down-regulation of all the truncated receptors, except MOR1TAG355D, thus revealing the importance of the four amino acids between Ser355 and Glu359 (STIE). Surprisingly, deletion of the STIE sequence resulted in a receptor that down-regulated the same as the wild-type receptor. The involvement of multiple amino acids within the carboxyl tail was demonstrated by combining alanine substitutions of several putative G-protein-coupled receptor kinase phosphorylation sites. Systematic analysis of these receptors indicated that mutation of Ser356 and Ser363 to alanine attenuated agonist-mediated down-regulation. The magnitude of etorphine-induced phosphorylation of this mutant receptor, however, was similar to that of the wild-type mu-opioid receptor. Thus, phosphorylation of the carboxyl tail of the mu-opioid receptor is not an obligatory event for etorphine-induced down-regulation of the receptor.     

Site mutation in the rat mu-opioid receptor demonstrates the involvement of calcium/calmodulin-dependent protein kinase II in agonist-mediated desensitization

The rat mu-opioid receptor (rMOR1), expressed in human embryonic kidney 293 (HEK293) cells, shows a desensitization to the inhibitory effect of the mu agonist DAMGO on adenylate cyclase activity within 4 h of DAMGO preincubation. To investigate the role of calcium/calmodulin-dependent protein kinase II (CaM kinase II) on mu-opioid receptor desensitization, we coexpressed rMOR1 and constitutively active CaM kinase II in HEK293 cells. This coexpression led to a faster time course of agonist-induced desensitization of the mu-opioid receptor. The increase of desensitization could not be observed with a mu-opioid receptor mutant (S261A/S266A) that lacks two putative CaM kinase II phosphorylation sites in the third intracellular loop. In addition, injection of CaM kinase II in Xenopus oocytes led only to desensitization of expressed rMOR1, but not of an S261A/S266A receptor mutant. These results suggest that phosphorylation of Ser261 and Ser266 by CaM kinase II is involved in the desensitization of the mu-opioid receptor.     

Mu opioid receptor phosphorylation, desensitization, and ligand efficacy

Mu opioid receptors are subject to phosphorylation and desensitization through actions of at least two distinct biochemical pathways: agonist-dependent mu receptor phosphorylation and desensitization induced by a biochemically distinct second pathway dependent on protein kinase C activation (1). To better understand the nature of the agonist-induced mu receptor phosphorylation events, we have investigated the effects of a variety of opiate ligands of varying potencies and intrinsic activities on mu receptor phosphorylation and desensitization. Exposure to the potent full agonists sufentanil, dihydroetorphine, etorphine, etonitazine, and [D-Ala2, MePhe4, Glyol5]enkephalin (DAMGO) led to strong receptor phosphorylation, while methadone, l-alpha-acetylmethadone (LAAM), morphine, meperidine, DADL, beta-endorphin(1-31), enkephalins, and dynorphin A(1-17) produced intermediate effects. The partial agonist buprenorphine minimally enhanced receptor phosphorylation while antagonists failed to alter phosphorylation. Buprenorphine and full antagonists each antagonized the enhanced mu receptor phosphorylation induced by morphine or DAMGO. The rank order of opiate ligand efficacies in producing mu receptor-mediated functional desensitization generally paralleled their rank order of efficacies in producing receptor phosphorylation. Interestingly, the desensitization and phosphorylation mediated by methadone and LAAM were disproportionate to their efficacies in two distinct test systems. This generally good fit between the efficacies of opiates in mu receptor activation, phosphorylation, and desensitization supports the idea that activated receptor/agonist/G-protein complexes and/or receptor conformational changes induced by agonists are required for agonist-induced mu receptor phosphorylation. Data for methadone and LAAM suggest possible contribution from their enhanced desensitizing abilities to their therapeutic efficacies.     

Molecular mechanisms of G protein-coupled receptor signaling: role of G protein-coupled receptor kinases and arrestins in receptor desensitization and resensitization

Dynamic regulation of G protein-coupled receptor signaling demands a coordinated balance between mechanisms leading to the generation, turning off and re-establishment of agonist-mediated signals. G protein-coupled receptor kinases (GRKs) and arrestin proteins not only mediate agonist-dependent G protein-coupled receptor desensitization, but also initiate the internalization (sequestration) of activated receptors, a process leading to receptor resensitization. Studies on the specificity of beta-arrestin functions reveal a multiplicity of G protein-coupled receptor endocytic pathways and suggest that beta-arrestins might serve as adaptors specifically targeting receptors for dynamin-dependent clathrin-mediated endocytosis. Moreover, inactivation of the GRK2 gene in mice has lead to the discovery of an unexpected role of GRK2 in cardiac development, further emphasizing the pleiotropic function of GRKs and arrestins.     

Contrasting internalization kinetics of human and chicken gonadotropin-releasing hormone receptors mediated by C-terminal tail

The chicken gonadotropin-releasing hormone receptor (GnRH-R) is notable for having a cytoplasmic C-terminal tail, which is not present in the mammalian GnRH-Rs. We report here that the cytoplasmic tail mediates rapid agonist-promoted receptor internalization. The chicken GnRH-R mediated internalization of gonadotropin-releasing hormone (GnRH) agonist (125I[His5-D-Tyr6]GnRH) at a rate of 11.3%.min-1, compared with only 0.71 %.min-1 for the human GnRH-R. To determine whether the presence of the cytoplasmic tail was responsible for the more rapid internalization kinetics of the chicken GnRH-R we truncated the tail after the Ile336 residue (S337stop). Receptor-mediated internalization of GnRH agonist by the S337stop-chicken GnRH-R was much slower than in the wild-type chicken receptor, and was similar to the wild-type human GnRH-R (0.55 %.min-1). These data indicate that rapid agonist-promoted internalization of the chicken GnRH-R is mediated through elements in the cytoplasmic C-terminal tail, distal to or including Ser337 and suggests that elimination of the C-terminal tail during evolution of mammalian GnRH-Rs may be related to its effects on internalization.     

Identification of transmembrane domain residues determinant in the structure-function relationship of the human platelet-activating factor receptor by site-directed mutagenesis

Platelet-activating factor (PAF) is a potent phospholipid mediator that produces a wide range of biological responses. The PAF receptor is a member of the seven-transmembrane GTP-binding regulatory protein-coupled receptor superfamily. This receptor binds PAF with high affinity and couples to multiple signaling pathways, leading to physiological responses that can be inhibited by various structurally distinct PAF antagonists. We have used site-directed mutagenesis and functional expression studies to examine the role of the Phe97 and Phe98 residues located in the third transmembrane helix and Asn285 and Asp289 of the seventh transmembrane helix in ligand binding and activation of the human PAF receptor in transiently transfected COS-7 cells. The double mutant FFGG (Phe97 and Phe98 mutated into Gly residues) showed a 3-4-fold decrease in affinity for PAF, but not for the specific antagonist WEB2086, when compared with the wild-type (WT) receptor. The FFGG mutant receptor, however, displayed normal agonist activation, suggesting that these two adjacent Phe residues maintain the native PAF receptor conformation rather than interacting with the ligand. On the other hand, substitution of Ala for Asp289 increased the receptor affinity for PAF but abolished PAF-dependent inositol phosphate accumulation; it did not affect WEB2086 binding. Substitution of Asn for Asp289, however, resulted in a mutant receptor with normal binding and activation characteristics. When Asn285 was mutated to Ala, the resulting receptor was undistinguishable from the WT receptor. Surprisingly, substitution of Ile for Asn285 led to a loss of ligand binding despite normal cell surface expression levels of this mutant, as verified by flow cytometric analysis. Our data suggest that residues 285 and 289 are determinant in the structure and activation of the PAF receptor but not in direct ligand binding, as had been recently proposed in a PAF receptor molecular model.     

Phosphorylation is not required for dynamin-dependent endocytosis of a truncated mutant opioid receptor

Opioid receptors are regulated within minutes after activation by G protein-coupled receptor kinase-mediated phosphorylation and dynamin-dependent endocytosis. We addressed the question of whether phosphorylation is required for opioid receptor endocytosis by examining a functional, truncated mutant delta opioid receptor (DOR344T), which is missing phosphorylation sites located in the carboxyl-terminal cytoplasmic domain. DOR344T receptors expressed in Chinese hamster ovary cells remained predominantly in the plasma membrane, even in the presence of saturating concentrations of agonist, consistent with previous studies demonstrating strongly inhibited endocytosis of truncated receptors in this cell type. In marked contrast, DOR344T receptors expressed at similar levels in human embryonal kidney (HEK) 293 cells exhibited rapid, ligand-induced internalization either in the presence of peptide (DADLE) or alkaloid (etorphine) agonist. Quantitative assays using ELISA and flow cytometric techniques indicated that DOR344T receptors were endocytosed in HEK293 cells with similarly rapid kinetics as full-length DOR (t1/2 < 10 min), and both full-length DOR and DOR344T mutant receptors were endocytosed by a dynamin-dependent mechanism involving clathrin-coated pits. Nevertheless, DOR344T receptors failed to undergo any detectable constitutive or agonist-induced phosphorylation in the same cells in which dynamin-dependent endocytosis was observed. These findings establish the first example of a G protein-coupled receptor that does not require phosphorylation to undergo dynamin-dependent endocytosis, and they suggest that significant cell type-specific differences exist in the biochemical requirements for ligand-induced concentration of opioid receptors in clathrin-coated pits.