Agonist-Regulated Internalization and Desensitization of the Human Nociceptin Receptor Expressed in CHO Cells
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In this study we examined agonist-induced internalization of the cloned human nociceptin receptor (hNOP) expressed in CHOK1 cells. Internalization was proven by receptor binding assay on viable cells and confocal microscopy. The agonists nociceptin/orphanin FQ (NC), NC-NH2, NC(1-13)-NH2, [(pF)Phe4]NC-NH2 and RO 64-6198 promote a rapid, concentration-dependent internalization of the hNOP receptor. Under the same conditions, [Phe1,ψ (CH2NH)Gly2]NC(1-13)-NH2 and [Phe1, (CH2NH)Gly2,Arg14,Lys15]NC(1-13)-NH2 failed to induce significant, concentration-dependent NOP receptor endocytosis; even when present at high concentrations (up to 1 mM) they promoted only an approximately 25-30% internalization of hNOP receptors. We also investigated hNOP receptor desensitization upon agonist challenge: ligand efficacy to inhibit forskolin-stimulated cAMP production. After 1 h exposure to NC, NC-NH2, NC(1-13)- NH2, [(pF)Phe4]NC-NH2 and RO 64-6198 (5 μM) ≉20 to 30% of receptor desensitization was observed. Moreover, we found that the blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. The noninternalizing agonists [Phe1,ψ (CH2NH)Gly2]NC(1-13)-NH2 and [Phe1, (CH2NH)Gly2,Arg14,Lys15]NC(1-13)-NH2 (100 μM) resulted in a strong (67 and 74 %, respectively) receptor desensitization which was not influenced by monensin. Finally, CHO-hNOP cells exposed to the receptor-internalizing agonists for 24 h resulted in a significantly higher cAMP accumulation (defined supersensitization) compared with the non-internalizing agonists. In addition, blocking of receptor recycling by monensin led to a decrease of the cAMP accumulation only in cells exposed to internalizing agonists. These data show that prolonged receptor signaling mediated by receptor endocytosis and recycling/reactivation might reduce the development of tolerance but can enhance compensatory mechanisms that lead to supersensitivity of specific signaling pathways. Keywords: Internalization, recycling, CHO-K1 cells, nociceptin, nociceptin receptor, desensitization, cAMPKeywords:
Internalization
Nociceptin receptor
NOP
Homologous desensitization
Regulatory elements present in the cytoplasmic carboxyl-terminal tails of G protein-coupled receptors contribute to agonist-dependent receptor desensitization, internalization, and association with accessory proteins such as beta-arrestin. The mammalian type I GnRH receptors are unique among the rhodopsin-like G protein-coupled receptors because they lack a cytoplasmic carboxyl-terminal tail. In addition, they do not recruit beta-arrestin, nor do they undergo rapid desensitization. By measuring the internalization of labeled GnRH agonists, previous studies have reported that mammalian type I GnRH receptors undergo slow agonist-dependent internalization. In the present study, we have measured the internalization of epitope-tagged GnRH receptors, both in the absence and presence of GnRH stimulation. We demonstrate that mammalian type I GnRH receptors exhibit a low level of constitutive agonist-independent internalization. Stimulation with GnRH agonist did not significantly enhance the level of receptor internalization above the constitutive level. In contrast, the catfish GnRH and rat TRH receptors, which have cytoplasmic carboxyl-terminal tails, displayed similar levels of constitutive agonist-independent internalization but underwent robust agonist-dependent internalization, as did chimeras of the mammalian type I GnRH receptor with the cytoplasmic carboxyl-terminal tails of the catfish GnRH receptor or the rat TRH receptor. When the carboxyl-terminal Tyr325 and Leu328 residues of the mammalian type I GnRH receptor were replaced with alanines, these two mutant receptors underwent significantly impaired internalization, suggesting a function for the Tyr-X-X-Leu sequence in mediating the constitutive agonist-independent internalization of mammalian type I GnRH receptors. These findings provide further support for the underlying notion that the absence of the cytoplasmic carboxyl-terminal tail of the mammalian type I GnRH receptors has been selected for during evolution to prevent rapid receptor desensitization and internalization to allow protracted GnRH signaling in mammals.
Internalization
Homologous desensitization
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In this study we examined agonist-induced internalization of the cloned human nociceptin receptor (hNOP) expressed in CHOK1 cells. Internalization was proven by receptor binding assay on viable cells and confocal microscopy. The agonists nociceptin/orphanin FQ (NC), NC-NH2, NC(1-13)-NH2, [(pF)Phe4]NC-NH2 and RO 64-6198 promote a rapid, concentration-dependent internalization of the hNOP receptor. Under the same conditions, [Phe1,ψ (CH2NH)Gly2]NC(1-13)-NH2 and [Phe1, (CH2NH)Gly2,Arg14,Lys15]NC(1-13)-NH2 failed to induce significant, concentration-dependent NOP receptor endocytosis; even when present at high concentrations (up to 1 mM) they promoted only an approximately 25-30% internalization of hNOP receptors. We also investigated hNOP receptor desensitization upon agonist challenge: ligand efficacy to inhibit forskolin-stimulated cAMP production. After 1 h exposure to NC, NC-NH2, NC(1-13)- NH2, [(pF)Phe4]NC-NH2 and RO 64-6198 (5 μM) ≉20 to 30% of receptor desensitization was observed. Moreover, we found that the blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. The noninternalizing agonists [Phe1,ψ (CH2NH)Gly2]NC(1-13)-NH2 and [Phe1, (CH2NH)Gly2,Arg14,Lys15]NC(1-13)-NH2 (100 μM) resulted in a strong (67 and 74 %, respectively) receptor desensitization which was not influenced by monensin. Finally, CHO-hNOP cells exposed to the receptor-internalizing agonists for 24 h resulted in a significantly higher cAMP accumulation (defined supersensitization) compared with the non-internalizing agonists. In addition, blocking of receptor recycling by monensin led to a decrease of the cAMP accumulation only in cells exposed to internalizing agonists. These data show that prolonged receptor signaling mediated by receptor endocytosis and recycling/reactivation might reduce the development of tolerance but can enhance compensatory mechanisms that lead to supersensitivity of specific signaling pathways. Keywords: Internalization, recycling, CHO-K1 cells, nociceptin, nociceptin receptor, desensitization, cAMP
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Nociceptin receptor
NOP
Homologous desensitization
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Homologous desensitization
Internalization
Rhodopsin-like receptors
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Evidence is presented that desensitization to cholinergic agonists in chick and leech muscle is a process involving receptor inactivation. Various possible mechanisms that could account for slow inactivation of receptors by agonists were analyzed mathematically, and expressions were obtained for the rate and extent of desensitization expected under various conditions. With the majority of agonists tested there appeared to be the same relationship between the response and the amount of desensitization produced. Certain agonists, however, were relatively more effective in causing desensitization. The kinetics of development of and recovery from desensitization were studied in chick and frog muscle. Reactivation of desensitized receptors occurred exponentially. In chick muscle the rate constant for recovery was the same (0.3 min-1) regardless of what agonist had been used to produce the desensitization. In chick muscle, tubocurarine decreased pari passu the response and the desensitization produced by carbachol or suxamethonium. Tubocurarine increased the desensitization produced by the partial agonist n-decyltrimethylammonium. These results are compatible with the cyclic model for desensitization suggested by Katz and Thesleff [J. Physiol. (London) 138, 63 (1957)], with the additional factor that certain drugs may have a preferential affinity for desensitized, compared with normal, receptors. The process of receptor activation may be very closely related to the transition from normal to desensitized receptors brought about by agonist drugs.
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The function of ATP-activated P2X3 receptors involved in pain sensation is modulated by desensitization, a phenomenon poorly understood. The present study used patch-clamp recording from cultured rat or mouse sensory neurons and kinetic modeling to clarify the properties of P2X3 receptor desensitization. Two types of desensitization were observed, a fast process (t1/2 = 50 ms; 10 μM ATP) following the inward current evoked by micromolar agonist concentrations, and a slow process (t1/2 = 35 s; 10 nM ATP) that inhibited receptors without activating them. We termed the latter high-affinity desensitization (HAD). Recovery from fast desensitization or HAD was slow and agonist-dependent. When comparing several agonists, there was analogous ranking order for agonist potency, rate of desensitization and HAD effectiveness, with 2-methylthioadenosine triphosphate the strongest and β,γ-methylene-ATP the weakest. HAD was less developed with recombinant (ATP IC50 = 390 nM) than native P2X3 receptors (IC50 = 2.3 nM). HAD could also be induced by nanomolar ATP when receptors seemed to be nondesensitized, indicating that resting receptors could express high-affinity binding sites. Desensitization properties were well accounted for by a cyclic model in which receptors could be desensitized from either open or closed states. Recovery was assumed to be a multistate process with distinct kinetics dependent on the agonist-dependent dissociation rate from desensitized receptors. Thus, the combination of agonist-specific mechanisms such as desensitization onset, HAD, and resensitization could shape responsiveness of sensory neurons to P2X3 receptor agonists. By using subthreshold concentrations of an HAD-potent agonist, it might be possible to generate sustained inhibition of P2X3 receptors for controlling chronic pain.
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Homologous desensitization
Melanocyte-stimulating hormone
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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.
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Bromoacetylalprenololmenthane was found to inhibit hormone‐induced β‐adrenergic receptor internalization in a dose‐dependent fashion in S49 lymphoma cells, besides its known ability to bind to β‐receptors irreversibly. This new found property of BAAM + was taken advantage of in studying whether receptor internalization is a necessary step in the desensitization of adenylate cyclase. BAAM‐treated cells showed functional desensitization even when receptor internalization had been blocked substantially by 50–65% This finding suggests that receptor internalization is not directly involved in desensitization.
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The glucagon-like peptide-1 receptor (GLP-1R) is an important target in the treatment of type 2 diabetes mellitus. The aim of this study was to compare the rate of agonist stimulated desensitization and internalization of GLP-1R. To this end, an N-terminally myc-tagged GLP-1R was stably expressed in HEK-293 cells. Homologous desensitization was assessed by measuring the cAMP response to agonist stimulation following pre-incubation with agonist for up to 120 min. Receptor internalization was monitored using an indirect ELISA-based method and confocal microscopy. Pre-incubation with GLP-1 resulted in a time-dependent loss of response to a second stimulation. Washing cells following pre-incubation failed to bring cAMP levels back to basal. Taking this into account, two desensitization rates were calculated: “apparent” (t1/2 = 19.27 min) and “net” (t1/2 = 2.99 min). Incubation of cells with GLP-1 also resulted in a time-dependent loss of receptor cell surface expression (t1/2 = 2.05 min). Rapid agonist-stimulated internalization of GLP-1R was confirmed using confocal microscopy. Stimulation of GLP-1R with GLP-1 results in rapid desensitization and internalization of the receptor. Interestingly, the rate of “net” desensitization closely matches the rate of internalization. Our results suggest that agonist-bound GLP-1R continues to generate cAMP after it has been internalized.
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