PGH1, the precursor for the anti-inflammatory prostaglandins of the 1-series, is a potent activator of the pro-inflammatory receptor CRTH2/DP2.

Prostaglandin H(1) (PGH(1)) is the cyclo-oxygenase metabolite of dihomo-γ-linolenic acid (DGLA) and the precursor for the 1-series of prostaglandins which are often viewed as "anti-inflammatory". Herein we present evidence that PGH(1) is a potent activator of the pro-inflammatory PGD(2) receptor CRTH2, an attractive therapeutic target to treat allergic diseases such as asthma and atopic dermatitis. Non-invasive, real time dynamic mass redistribution analysis of living human CRTH2 transfectants and Ca(2+) flux studies reveal that PGH(1) activates CRTH2 as PGH(2), PGD(2) or PGD(1) do. The PGH(1) precursor DGLA and the other PGH(1) metabolites did not display such effect. PGH(1) specifically internalizes CRTH2 in stable CRTH2 transfectants as assessed by antibody feeding assays. Physiological relevance of CRTH2 ligation by PGH(1) is demonstrated in several primary human hematopoietic lineages, which endogenously express CRTH2: PGH(1) mediates migration of and Ca(2+) flux in Th2 lymphocytes, shape change of eosinophils, and their adhesion to human pulmonary microvascular endothelial cells under physiological flow conditions. All these effects are abrogated in the presence of the CRTH2 specific antagonist TM30089. Together, our results identify PGH(1) as an important lipid intermediate and novel CRTH2 agonist which may trigger CRTH2 activation in vivo in the absence of functional prostaglandin D synthase.

Altered ratio of D1 and D2 dopamine receptors in mouse striatum is associated with behavioral sensitization to cocaine.

BACKGROUND: Drugs of abuse elevate brain dopamine levels, and, in vivo, chronic drug use is accompanied by a selective decrease in dopamine D2 receptor (D2R) availability in the brain. Such a decrease consequently alters the ratio of D1R:D2R signaling towards the D1R. Despite a plethora of behavioral studies dedicated to the understanding of the role of dopamine in addiction, a molecular mechanism responsible for the downregulation of the D2R, in vivo, in response to chronic drug use has yet to be identified. METHODS AND FINDINGS: ETHICS STATEMENT: All animal work was approved by the Gallo Center IACUC committee and was performed in our AAALAC approved facility. In this study, we used wild type (WT) and G protein coupled receptor associated sorting protein-1 (GASP-1) knock out (KO) mice to assess molecular changes that accompany cocaine sensitization. Here, we show that downregulation of D2Rs or upregulation of D1Rs is associated with a sensitized locomotor response to an acute injection of cocaine. Furthermore, we demonstrate that disruption of GASP-1, that targets D2Rs for degradation after endocytosis, prevents cocaine-induced downregulation of D2Rs. As a consequence, mice with a GASP-1 disruption show a reduction in the sensitized locomotor response to cocaine. CONCLUSIONS: Together, our data suggests that changes in the ratio of the D1:D2R could contribute to cocaine-induced behavioral plasticity and demonstrates a role of GASP-1 in regulating both the levels of the D2R and cocaine sensitization.

GPR55 ligands promote receptor coupling to multiple signalling pathways.

BACKGROUND AND PURPOSE: Although GPR55 is potently activated by the endogenous lysophospholipid, L-alpha-lysophosphatidylinositol (LPI), it is also thought to be sensitive to a number of cannabinoid ligands, including the prototypic CB1 receptor antagonists AM251 and SR141716A (Rimonabant). In this study we have used a range of functional assays to compare the pharmacological activity of selected cannabinoid ligands, AM251, AM281 and SR141716A with LPI in a HEK293 cell line engineered to stably express recombinant, human GPR55. EXPERIMENTAL APPROACH: We evaluated Ca(2+) signalling, stimulation of extracellular signal regulated kinase (ERK1/2) mitogen activated kinase MAP-kinases, induction of transcriptional regulators that are downstream of GPR55, including nuclear factor of activated T cells (NFAT), nuclear factor-kappaB (NF-kappaB) and cAMP response element binding protein (CREB), as well as receptor endocytosis. In addition, we assessed the suitability of a novel, label-free assay for GPR55 ligands that involves optical measurement of dynamic mass redistribution following receptor activation. KEY RESULTS: GPR55 linked to a range of downstream signalling events and that the activity of GPR55 ligands was influenced by the functional assay employed, with differences in potency and efficacy observed. CONCLUSIONS AND IMPLICATIONS: Our data help to resolve some of the issues surrounding the pharmacology of cannabinoid ligands at GPR55 and highlight some differences in effector coupling associated with distinct GPR55 ligands.

Differential regulation of behavioral tolerance to WIN55,212-2 by GASP1.

Cannabinoid agonists have shown some promise clinically as analgesics, in particular for cancer pain, in which they have the additional benefit of decreasing nausea. However, as for most other drugs, the long-term use of cannabinoids is limited by the development of tolerance. Several molecular mechanisms have been proposed to explain drug tolerance, including receptor downregulation. The cannabinoid 1 (CB1) receptors can be downregulated in vitro through an interaction with the G-protein-coupled receptor-associated sorting protein1, GASP1, that targets CB1 receptors for degradation after their agonist-mediated endocytosis. To investigate whether GASP1-mediated postendocytic sorting of the CB1 receptor contributes to tolerance to cannabinoid drugs in vivo, we generated a mouse with a disruption of GASP1. In wild-type mice, repeated administration of the cannabinoid agonist WIN55,212-2 promoted downregulation of CB1 receptor levels and concomitant tolerance to the effects of drug on antinociception, motor incoordination, and locomotor hypoactivity. In contrast, GASP1 knockout mice did not develop tolerance to any of these effects and showed no significant receptor downregulation. Taken together, this study provides evidence that GASP1 regulates CB1 receptor downregulation in vivo, and that postendocytic receptor trafficking has a key role in the development of tolerance to WIN55,212-2.

The G-protein coupled receptor associated sorting protein GASP-1 regulates the signalling and trafficking of the viral chemokine receptor US28.

Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.

Construction of covalently coupled, concatameric dimers of 7TM receptors.

7TM receptors are easily fused to proteins such as G proteins and arrestin but because of the fact that their terminals are found on each side of the membrane they cannot be joined directly in covalent dimers. Here, we use an artificial connector comprising a transmembrane helix composed of Leu-Ala repeats flanked by flexible spacers and positively charged residues to ensure correct inside-out orientation plus an extracellular HA-tag to construct covalently coupled dimers of 7TM receptors. Such 15 TM concatameric homo- and heterodimers of the beta(2)-adrenergic and the NK(1) receptors, which normally do not dimerize with each other, were expressed surprisingly well at the cell surface, where they bound ligands and activated signal transduction in a manner rather similar to the corresponding wild-type receptors. The concatameric heterodimers internalized upon stimulation with agonists for either of the protomers, which was not observed upon simple coexpression of the two receptors. It is concluded that covalently joined 7TM receptor dimers with surprisingly normal receptor properties can be constructed with use of an artificial transmembrane connector, which perhaps can be used to fuse other membrane proteins.

The C-terminal tail of CRTH2 is a key molecular determinant that constrains Galphai and downstream signaling cascade activation.

Prostaglandin D(2) activation of the seven-transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases, such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2. We tested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, the presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced Galpha(i) and ERK1/2 activation despite enhanced constitutive and agonist-mediated internalization. Enhanced activation of Galpha(i) proteins and downstream signaling cascades is probably due to the inability of the tail-truncated receptor to recruit beta-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits Galpha(i) signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation.

The role of mu opioid receptor desensitization and endocytosis in morphine tolerance and dependence.

Following activation, most G protein coupled receptors undergo regulation by a cascade of events that promote receptor desensitization and endocytosis. Following endocytosis, receptors can then be recycled to the plasma membrane, retained in an intracellular compartment, or targeted for degradation. For receptors that are recycled, like the mu opioid receptor (MOR), endocytosis serves as the first step toward resensitizing receptors. For receptors that are degraded, endocytosis serves as the first step toward receptor downregulation. Thus, for receptors like the MOR, the desensitization-endocytosis-resensitization cycle serves as a rapid and dynamic means to titrate signaling through the receptor. However, not all agonist ligands at the MOR promote the same degree of receptor desensitization and endocytosis. For example, the endogenous peptide ligands at the MOR induce rapid desensitization, endocytosis, and recycling. By contrast, morphine induces only weak or partial desensitization and little to no endocytosis. As a consequence, signal transduction promoted by morphine is less dynamic than that induced by endogenous ligands as well as other opioid agonists that promote endocytosis. The resulting imbalance of desensitization-endocytosis-resensitization has at least two consequences: (1) in cell types where morphine induces desensitization but not endocytosis and/or resensitization, desensitization is protracted; (2) in cell types where morphine induces neither desensitization nor endocytosis, prolonged signaling through the receptor leads to multiple cellular adaptations downstream of receptor-G protein coupling. Both protracted desensitization and adaptive cellular changes probably contribute to the pronounced in vivo tolerance and dependence that occur with chronic morphine treatment. As a consequence, facilitating receptor endocytosis, using either genetic or pharmacological approaches, can restore the balance of signaling through the receptor and affect the development of tolerance and dependence.

A molecular basis of analgesic tolerance to cannabinoids.

Clinical usage of cannabinoids in chronic pain states is limited by their central side effects and the pharmacodynamic tolerance that sets in after repeated dosage. Analgesic tolerance to cannabinoids in vivo could be caused by agonist-induced downregulation and intracellular trafficking of cannabinoid receptors, but little is known about the molecular mechanisms involved. We show here that the type 1 cannabinoid receptor (CB1) interacts physically with G-protein-associated sorting protein 1 (GASP1), a protein that sorts receptors in lysosomal compartments destined for degradation. CB1-GASP1 interaction was observed to be required for agonist-induced downregulation of CB1 in spinal neurons ex vivo as well as in vivo. Importantly, uncoupling CB1 from GASP1 in mice in vivo abrogated tolerance toward cannabinoid-induced analgesia. These results suggest that GASP1 is a key regulator of the fate of CB1 after agonist exposure in the nervous system and critically determines analgesic tolerance to cannabinoids.

Ligand-induced down-regulation of the cannabinoid 1 receptor is mediated by the G-protein-coupled receptor-associated sorting protein GASP1.

The cannabinoid 1 receptor (CB1R) is one of the most abundant seven transmembrane (7TM) spanning/G-protein-coupled receptors in the central nervous system and plays an important role in pain transmission, feeding, and the rewarding effects of cannabis. Tolerance to cannabinoids has been widely observed after long-term use, with concomitant receptor desensitization and/or down-regulation depending on the brain region studied. Several CB1R agonists promote receptor internalization after activation, but the postendocytic sorting of the receptor has not been studied in detail. Utilizing human embryonic kidney (HEK293) cells stably expressing the CB1R and primary cultured neurons expressing endogenous CB1R, we show that treatment with cannabinoid agonists results in CB1R degradation after endocytosis and that the G-protein-coupled receptor-associated sorting protein GASP1 plays a major role in the postendocytic sorting process. Thus, these results may identify a molecular mechanism underlying tolerance and receptor down-regulation after long-term use of cannabinoids.

Identification of indole derivatives exclusively interfering with a G protein-independent signaling pathway of the prostaglandin D2 receptor CRTH2.

The anti-inflammatory drugs indomethacin and ramatroban, the latter showing clinical efficacy in treating allergic asthma, have been shown to act as a classic agonist and antagonist, respectively, of the G protein-coupled chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2 receptor). Here, we report the identification of two indole derivatives 1-(4-ethoxyphenyl)-5-methoxy-2-methylindole-3-carboxylic acid and N(alpha)-tosyltryptophan (hereafter referred to as 1 and 2, respectively), which are structurally related to indomethacin and ramatroban but which selectively interfere with a specific G protein-independent signaling pathway of CRTH2. In whole-cell saturation-binding assays, 1 and 2 both increase the number of [(3)H]prostaglandin D2 (PGD2)-recognizing CRTH2 sites and the affinity of PGD2 for CRTH2. Enzyme-linked immunosorbent assays show that they do not alter the total number of CRTH2 receptors on the cell surface. Analysis of their binding mode indicates that unlike indomethacin or ramatroban, 1 and 2 can occupy CRTH2 simultaneously with PGD2. On a functional level, however, 1 and 2 do not interfere with PGD2-mediated activation of heterotrimeric G proteins by CRTH2. In contrast, both compounds inhibit PGD2-mediated arrestin translocation via a G protein-independent mechanism. In human eosinophils endogenously expressing CRTH2, 1 selectively decreases the efficacy but not the potency of PGD2-induced shape change, unlike ramatroban, which displays competitive antagonistic behavior. These data show for the first time that "antagonists" can cause markedly dissimilar degrees of inhibition for different effector pathways and suggest that it may be possible to develop novel classes of specific signal-inhibiting drugs distinct from conventional antagonists.

G-protein-coupled receptor Mas is a physiological antagonist of the angiotensin II type 1 receptor.

BACKGROUND: We previously identified the G-protein-coupled receptor Mas, encoded by the Mas proto-oncogene, as an endogenous receptor for the heptapeptide angiotensin-(1-7); however, the receptor is also suggested to be involved in actions of angiotensin II. We therefore tested whether this could be mediated indirectly through an interaction with the angiotensin II type 1 receptor, AT1. METHODS AND RESULTS: In transfected mammalian cells, Mas was not activated by angiotensin II; however, AT1 receptor-mediated, angiotensin II-induced production of inositol phosphates and mobilization of intracellular Ca2+ was diminished by 50% after coexpression of Mas, despite a concomitant increase in angiotensin II binding capacity. Mas and the AT1 receptor formed a constitutive hetero-oligomeric complex that was unaffected by the presence of agonists or antagonists of the 2 receptors. In vivo, Mas acts as an antagonist of the AT1 receptor; mice lacking the Mas gene show enhanced angiotensin II-mediated vasoconstriction in mesenteric microvessels. CONCLUSIONS: These results demonstrate that Mas can hetero-oligomerize with the AT1 receptor and by so doing inhibit the actions of angiotensin II. This is a novel demonstration that a G-protein-coupled receptor acts as a physiological antagonist of a previously characterized receptor. Consequently, the AT1-Mas complex could be of great importance as a target for pharmacological intervention in cardiovascular diseases.

A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity.

Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one module. Galpha proteins modified in both modules are significantly more efficacious in channeling non-G(q) -selective receptors to G(q)-mediated signaling events compare with those containing each module alone. Additive effects of both modules were observed even if individual modules lacked an effect on GPCR-to-effector specificity. Dually modified Galpha proteins were also superior in conferring high-affinity agonist sites onto a coexpressed GPCR in the absence, but not in the presence, of guanine nucleotides. Together, our data suggest that receptor-G protein coupling selectivity involves cooperative interactions between the extreme C terminus and linker I of Galpha proteins and that distinct determinants of selectivity exist for individual receptors.

Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype.

To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization compared with wild-type receptors. This distinct phenotype of the fusion proteins can not be mimicked by coexpressing wild-type receptor with (beta)arr2. However, when the wild-type receptor was coexpressed with both (beta)arr2 and G protein-coupled receptor kinase 5, a phenotype similar to that observed for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with (beta)arr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level.

Similar activation of signal transduction pathways by the herpesvirus-encoded chemokine receptors US28 and ORF74.

The virally encoded chemokine receptors US28 from human cytomegalovirus and ORF74 from human herpesvirus 8 are both constitutively active. We show that both receptors constitutively activate the transcription factors nuclear factor of activated T cells (NFAT) and cAMP response element binding protein (CREB) and that both pathways are modulated by their respective endogenous receptor ligands. By addition of specific pathway modulators against the G protein subunit Galphai, phospholipase C, protein kinase C, calcineurin, p38 MAP kinase, and MEK1, we find that the constitutive and ligand-dependent inductions are mediated by multiple yet similar pathways in both receptors. The NFAT and CREB transcription factors and their upstream activators are known inducers of host and virally encoded genes. We propose that the activity of these virally encoded chemokine receptors coordinates host and potentially viral gene expression similarly. As ORF74 is a known inducer of neoplasia, these findings may have important implications for cytomegalovirus-associated pathogenicity.

NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.