Lene Martini

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.

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.

GPR55 ligands promote receptor coupling to multiple signalling pathways

Background and purpose:  Although GPR55 is potently activated by the endogenous lysophospholipid, L‐α‐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.

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.—Martini, L., Waldhoer, M., Pusch, M., Kharazia, V., Fong, J., Lee, J. H., Freissmuth, C., Whistler, J. L. Ligand‐induced down‐regulation of the cannabinoid 1 receptor is mediated by the G‐protein‐coupled receptor‐associated sorting protein GASP1. FASEB J. 21, 802–811 (2007)

Two active molecular phenotypes of the Tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Gαs or Gαq and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Gαsfusion construct, whereas the lower affinity component was displayed by the tail-truncated Gαq fusion. The elusive difference between the affinity determined in heterologous versushomologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Gαs signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Gαq fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Gαs and Gαq species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or “signal-transductosomes” within the cell membrane.

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.

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 D1R∶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.

The C-terminal Tail of CRTH2 Is a Key Molecular Determinant That Constrains Gαi and Downstream Signaling Cascade Activation

Prostaglandin D2 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 Gαi and ERK1/2 activation despite enhanced constitutive and agonist-mediated internalization. Enhanced activation of Gαi proteins and downstream signaling cascades is probably due to the inability of the tail-truncated receptor to recruit β-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 Gαi 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.

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 Gαq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor–κB (NF‐κB) 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.