Kinin-stimulated B1 receptor signaling depends on receptor endocytosis whereas B2 receptor signaling does not.

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca(2+) mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg(9)-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.

Anti-analgesic effect of the mu/delta opioid receptor heteromer revealed by ligand-biased antagonism.

Delta (DOR) and mu opioid receptors (MOR) can complex as heteromers, conferring functional properties in agonist binding, signaling and trafficking that can differ markedly from their homomeric counterparts. Because of these differences, DOR/MOR heteromers may be a novel therapeutic target in the treatment of pain. However, there are currently no ligands selective for DOR/MOR heteromers, and, consequently, their role in nociception remains unknown. In this study, we used a pharmacological opioid cocktail that selectively activates and stabilizes the DOR/MOR heteromer at the cell surface by blocking its endocytosis to assess its role in antinociception. We found that mice treated chronically with this drug cocktail showed a significant right shift in the ED50 for opioid-mediated analgesia, while mice treated with a drug that promotes degradation of the heteromer did not. Furthermore, promoting degradation of the DOR/MOR heteromer after the right shift in the ED50 had occurred, or blocking signal transduction from the stabilized DOR/MOR heteromer, shifted the ED50 for analgesia back to the left. Taken together, these data suggest an anti-analgesic role for the DOR/MOR heteromer in pain. In conclusion, antagonists selective for DOR/MOR heteromer could provide an avenue for alleviating reduced analgesic response during chronic pain treatment.

Chronic ethanol potentiates the effect of neuropeptide s in the basolateral amygdala and shows increased anxiolytic and anti-depressive effects.

Alleviating anxiety and depression is pivotal for reducing the risk of relapse in alcoholics. Currently available anxiolytic treatments are limited by side effects, including reduced efficacy in alcoholics, addiction, and sedation. We examined whether the neuropeptide S receptor (NPSR) was effective at controlling ethanol consumption and the anxiety and depression produced by forced abstinence from ethanol. We found that the anxiolytic and anti-depressant effects of NPS are enhanced in acute ethanol abstinent mice. In addition, we found that NPS reduced ethanol consumption and is not in and of itself rewarding. We also provide evidence that ethanol consumption increases the ability of NPS to modulate neuronal activity in the basolateral amygdala. Finally, we found that local injection of NPS in the basolateral amygdala promotes anxiolysis after chronic ethanol consumption, thereby providing insight into the molecular mechanism underlying the changes in behavioral response to NPS. In light of the improved anxiolytic efficacy and benign side effects of NPS in ethanol-withdrawn animals, the NPSR may prove a suitable target for reducing relapse in alcoholism.

Chronic methadone treatment shows a better cost/benefit ratio than chronic morphine in mice.

Chronic treatment of pain with opiate drugs can lead to analgesic tolerance and drug dependence. Although all opiate drugs can promote tolerance and dependence in practice, the severity of those unwanted side effects differs depending on the drug used. Although each opiate drug has its own unique set of pharmacological profiles, methadone is the only clinically used opioid drug that produces substantial receptor endocytosis at analgesic doses. Here, we examined whether moderate doses of methadone carry any benefits over chronic use of equianalgesic morphine, the prototypical opioid. Our data show that chronic administration of methadone produces significantly less analgesic tolerance than morphine. Furthermore, we found significantly reduced precipitated withdrawal symptoms after chronic methadone treatment than after chronic morphine treatment. Finally, using a novel animal model with a degrading µ-opioid receptor we showed that, although endocytosis seems to protect against tolerance development, endocytosis followed by receptor degradation produces a rapid onset of analgesic tolerance to methadone. Together, these data indicated that opioid drugs that promote receptor endocytosis and recycling, such as methadone, may be a better choice for chronic pain treatment than morphine and its derivatives that do not.

A novel knock-in mouse reveals mechanistically distinct forms of morphine tolerance.

The role of µ-opioid receptor (MOR) down-regulation in opioid tolerance remains controversial. In this study, we used a novel knock-in mouse to examine how changing the extent of MOR down-regulation alters the development of morphine tolerance. These mice express a mutant MOR, degrading MOR (DMOR), that differs from the wild-type (WT) MOR in two ways: 1) unlike the recycling WT MOR, the mutant DMOR is targeted for degradation after its internalization, thus facilitating down-regulation; and 2) unlike the WT MOR, DMOR is efficiently internalized in response to morphine activation. We found that both WT MOR and DMOR mice develop tolerance to morphine, but DMOR mice exhibit a more rapid onset of tolerance and show receptor down-regulation. WT MOR mice develop morphine tolerance more slowly but even once profoundly tolerant show no receptor down-regulation. Furthermore, WT mice show significantly more morphine dependence than DMOR mice after long-term treatment as indicated by withdrawal. Taken together these data indicate that tolerance mediated by receptor down-regulation manifests differently both at the behavioral and biochemical level than does the actual morphine tolerance that occurs in WT mice and that loss of receptor function is not a major contributor to morphine tolerance in WT MOR mice.

Kinin B2 receptor-mediated bradykinin internalization and metalloendopeptidase EP24.15-dependent intracellular bradykinin degradation.

Kinins are potent proinflammatory peptides that are produced extracellularly and are rapidly degraded by extracellular peptidases and by intracellular peptidases accessed by kinins via receptor-mediated endocytosis. In this study, we developed model cell systems expressing the kinin B(2) receptor (B(2)R) and the metalloendopeptidase thimet oligopeptidase (EC 3.4.24.15; EP24.15) either individually or together to address 1) the cellular and functional relationship between these proteins and 2) the participation of EP24.15 in the metabolism of bradykinin (BK) after BK internalization via B(2)R. B(2)R was localized almost exclusively in the plasma membrane, whereas EP24.15 was localized both intracellularly and on the cell surface and secreted in the media. Intracellular EP24.15 was present throughout the cell, both cytosolic and particulate, with less nuclear localization and no colocalization with either the endoplasmic reticulum marker calnexin or the Golgi marker GM130. No direct colocalization of B(2)R and EP24.15 was observed using immunofluorescence microscopy. However, the two proteins coimmunoprecipitated specifically, and EP24.15 attenuated maximal B(2)R responsiveness without influencing the potency of BK to stimulate phosphoinositide hydrolysis and intracellular Ca(2+) mobilization. Cell surface-bound BK remained intact in cells overexpressing EP24.15 but was degraded intracellularly in an EP24.15-dependent manner upon B(2)R-mediated endocytosis. These results show that EP24.15 acts to negatively regulate B(2)R responsiveness, and it serves as an intracellular peptidase in the degradation of BK specifically internalized via this receptor.

Kinins promote B2 receptor endocytosis and delay constitutive B1 receptor endocytosis.

Upon sustained insult, kinins are released and many kinin responses, such as inflammatory pain, adapt from a B2 receptor (B2R) type in the acute phase to a B1 receptor (B1R) type in the chronic phase. In this study, we show that kinins modulate receptor endocytosis to rapidly decrease B2R and increase B1R on the cell surface. B2Rs, which require agonist for activity, are stable plasma membrane components without agonist but recruit beta-arrestin 2, internalize in a clathrin-dependent manner, and recycle rapidly upon agonist treatment. In contrast, B1Rs, which are inducible and constitutively active, constitutively internalize without agonist via a clathrin-dependent pathway, do not recruit beta-arrestin 2, bind G protein-coupled receptor sorting protein, and target lysosomes for degradation. Agonist delays B1R endocytosis, thus transiently stabilizing the receptor. Most of the receptor trafficking phenotypes are transplantable from one receptor to the other through exchange of the C-terminal receptor tails, indicating that the tails contain epitopes that are important for the binding of protein partners that participate in the endocytic and postendocytic receptor choices. It is noteworthy that the agonist delay of B1R endocytosis is not transplanted to the B2R via the B1R tail, suggesting that this property of the B1R requires another domain. These events provide a rapid kinin-dependent mechanism for 1) regulating the constitutive B1R activity and 2) shifting the balance of accessible receptors in favor of B1R.

Dopamine responsiveness is regulated by targeted sorting of D2 receptors.

Aberrant dopaminergic signaling is a critical determinant in multiple psychiatric disorders, and in many disease states, dopamine receptor number is altered. Here we identify a molecular mechanism that selectively targets D2 receptors for degradation after their activation by dopamine. The degradative fate of D2 receptors is determined by an interaction with G protein coupled receptor-associated sorting protein (GASP). As a consequence of this GASP interaction, D2 responses in rat brain fail to resensitize after agonist treatment. Disruption of the D2-GASP interaction facilitates recovery of D2 responses, suggesting that modulation of the D2-GASP interaction is important for the functional down-regulation of D2 receptors.

Modulation of postendocytic sorting of G protein-coupled receptors.

Recycling of the mu opioid receptor to the plasma membrane after endocytosis promotes rapid resensitization of signal transduction, whereas targeting of the delta opioid receptor (DOR) to lysosomes causes proteolytic down-regulation. We identified a protein that binds preferentially to the cytoplasmic tail of the DOR as a candidate heterotrimeric GTP-binding protein (G protein)-coupled receptor-associated sorting protein (GASP). Disruption of the DOR-GASP interaction through receptor mutation or overexpression of a dominant negative fragment of GASP inhibited receptor trafficking to lysosomes and promoted recycling. The GASP family of proteins may modulate lysosomal sorting and functional down-regulation of a variety of G protein-coupled receptors.