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Src promotes delta opioid receptor (DOR) desensitization by interfering with receptor recycling.

Archer-Lahlou E, Audet N, Amraei MG, Huard K, Paquin-Gobeil M, Pineyro G - J. Cell. Mol. Med. (2008)

Bottom Line: A first series of experiments demonstrated that agonist binding activates Src and destabilizes a constitutive complex formed by the spontaneous association of DORs with the kinase.Src contribution to DOR desensitization was then established by showing that pre-treatment with Src inhibitor PP2 (20 microM; 1 hr) or transfection of a dominant negative Src mutant preserved DOR signalling following sustained exposure to an agonist.This protection was afforded without interfering with endocytosis, but suboptimal internalization interfered with PP2 ability to preserve DOR signalling, suggesting a post-endocytic site of action for the kinase.

View Article: PubMed Central - PubMed

Affiliation: Département de Pharmacologie, Faculté de Médecine, Université de Montréal, Canada.

ABSTRACT
Abstract An important limitation in the clinical use of opiates is progressive loss of analgesic efficacy over time. Development of analgesic tolerance is tightly linked to receptor desensitization. In the case of delta opioid receptors (DOR), desensitization is especially swift because receptors are rapidly internalized and are poorly recycled to the membrane. In the present study, we investigated whether Src activity contributed to this sorting pattern and to functional desensitization of DORs. A first series of experiments demonstrated that agonist binding activates Src and destabilizes a constitutive complex formed by the spontaneous association of DORs with the kinase. Src contribution to DOR desensitization was then established by showing that pre-treatment with Src inhibitor PP2 (20 microM; 1 hr) or transfection of a dominant negative Src mutant preserved DOR signalling following sustained exposure to an agonist. This protection was afforded without interfering with endocytosis, but suboptimal internalization interfered with PP2 ability to preserve DOR signalling, suggesting a post-endocytic site of action for the kinase. This assumption was confirmed by demonstrating that Src inhibition by PP2 or its silencing by siRNA increased membrane recovery of internalized DORs and was further corroborated by showing that inhibition of recycling by monensin or dominant negative Rab11 (Rab11S25N) abolished the ability of Src blockers to prevent desensitization. Finally, Src inhibitors accelerated recovery of DOR-Galphal3 coupling after desensitization. Taken together, these results indicate that Src dynamically regulates DOR recycling and by doing so contributes to desensitization of these receptors.

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Src inhibitors require recycling to counter DOR desensitization. (A) The effect of PP2 on desensitization of wild-type DORs was assessed as in previous figures with the experiment being conducted in the presence of monensin (50 μM). Results are expressed as percentage of maximal cAMP inhibition obtained in non-desensitized, non-monensin-treated controls and correspond to three experiments carried out in triplicate. Statistical comparison of DPDPE dose response curves obtained from cells desensitized in presence or absence of PP2 was carried out using two-way ANOVA. DES + monensin versus PP2 + DES + monensin: P= 0.2; n= 3. Inset: representative example of DOR membrane recovery in presence or absence of monensin. Experiments were carried out as in Fig. 4 and results are expressed as percentage of maximal recovery of internalized receptor in cells that were not exposed to monensin. (B) cAMP assays were performed in transiently transfected cells co-expressing DORs and DNM-Rab11. Results are expressed as in A. DES + DNM-Rab11 versus PP2 + DES + DNM-Rab11: P= 0.7; n= 4. Inset: representative example of DOR membrane recovery in presence or absence of DNM-Rab11. Results are expressed as percentage of maximal recovery of internalized receptor in cells expressing the empty vector (pcDNA3).
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fig06: Src inhibitors require recycling to counter DOR desensitization. (A) The effect of PP2 on desensitization of wild-type DORs was assessed as in previous figures with the experiment being conducted in the presence of monensin (50 μM). Results are expressed as percentage of maximal cAMP inhibition obtained in non-desensitized, non-monensin-treated controls and correspond to three experiments carried out in triplicate. Statistical comparison of DPDPE dose response curves obtained from cells desensitized in presence or absence of PP2 was carried out using two-way ANOVA. DES + monensin versus PP2 + DES + monensin: P= 0.2; n= 3. Inset: representative example of DOR membrane recovery in presence or absence of monensin. Experiments were carried out as in Fig. 4 and results are expressed as percentage of maximal recovery of internalized receptor in cells that were not exposed to monensin. (B) cAMP assays were performed in transiently transfected cells co-expressing DORs and DNM-Rab11. Results are expressed as in A. DES + DNM-Rab11 versus PP2 + DES + DNM-Rab11: P= 0.7; n= 4. Inset: representative example of DOR membrane recovery in presence or absence of DNM-Rab11. Results are expressed as percentage of maximal recovery of internalized receptor in cells expressing the empty vector (pcDNA3).

Mentions: Results obtained with pharmacological inhibition or gene silencing of Src confirm this tyrosine kinase as a regulator of post-endocytic sorting, but they do not indicate whether the small increase in recycling might have any consequences concerning desensitization. So we reasoned that if the increase in recycling were to play a significant role in the protective effect of Src blockers, then interfering with recycling would prevent the protective effect exerted by Src inhibitors. Thus, we determined whether protective action of PP2 could be affected by interfering with recycling. In a first series of experiments, we tested the effect of monensin, a ionophore that blocks receptor recycling by trapping internalized receptors within endosomes [38, 42]. Results showed that at a concentration that reduced DOR recycling (50 μM; Inset Fig. 6A), monensin abolished PP2-mediated protection from desensitization (Fig. 6A). To confirm this observation, cells were transfected with a mutant form of Rab11 (DNM-Rab11) in which substitution of Ser 25 by Asn interferes with GTP-binding capacity of this small G protein and inhibits recycling [27, 43]. Similar to monensin, a decrease in DOR recycling by DNM-Rab11 (Inset Fig. 6B) abolished the protective effect of Src-blockers upon desensitization (Fig. 6B), confirming that recycling is essential for Src modulation of DOR signalling efficacy.


Src promotes delta opioid receptor (DOR) desensitization by interfering with receptor recycling.

Archer-Lahlou E, Audet N, Amraei MG, Huard K, Paquin-Gobeil M, Pineyro G - J. Cell. Mol. Med. (2008)

Src inhibitors require recycling to counter DOR desensitization. (A) The effect of PP2 on desensitization of wild-type DORs was assessed as in previous figures with the experiment being conducted in the presence of monensin (50 μM). Results are expressed as percentage of maximal cAMP inhibition obtained in non-desensitized, non-monensin-treated controls and correspond to three experiments carried out in triplicate. Statistical comparison of DPDPE dose response curves obtained from cells desensitized in presence or absence of PP2 was carried out using two-way ANOVA. DES + monensin versus PP2 + DES + monensin: P= 0.2; n= 3. Inset: representative example of DOR membrane recovery in presence or absence of monensin. Experiments were carried out as in Fig. 4 and results are expressed as percentage of maximal recovery of internalized receptor in cells that were not exposed to monensin. (B) cAMP assays were performed in transiently transfected cells co-expressing DORs and DNM-Rab11. Results are expressed as in A. DES + DNM-Rab11 versus PP2 + DES + DNM-Rab11: P= 0.7; n= 4. Inset: representative example of DOR membrane recovery in presence or absence of DNM-Rab11. Results are expressed as percentage of maximal recovery of internalized receptor in cells expressing the empty vector (pcDNA3).
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fig06: Src inhibitors require recycling to counter DOR desensitization. (A) The effect of PP2 on desensitization of wild-type DORs was assessed as in previous figures with the experiment being conducted in the presence of monensin (50 μM). Results are expressed as percentage of maximal cAMP inhibition obtained in non-desensitized, non-monensin-treated controls and correspond to three experiments carried out in triplicate. Statistical comparison of DPDPE dose response curves obtained from cells desensitized in presence or absence of PP2 was carried out using two-way ANOVA. DES + monensin versus PP2 + DES + monensin: P= 0.2; n= 3. Inset: representative example of DOR membrane recovery in presence or absence of monensin. Experiments were carried out as in Fig. 4 and results are expressed as percentage of maximal recovery of internalized receptor in cells that were not exposed to monensin. (B) cAMP assays were performed in transiently transfected cells co-expressing DORs and DNM-Rab11. Results are expressed as in A. DES + DNM-Rab11 versus PP2 + DES + DNM-Rab11: P= 0.7; n= 4. Inset: representative example of DOR membrane recovery in presence or absence of DNM-Rab11. Results are expressed as percentage of maximal recovery of internalized receptor in cells expressing the empty vector (pcDNA3).
Mentions: Results obtained with pharmacological inhibition or gene silencing of Src confirm this tyrosine kinase as a regulator of post-endocytic sorting, but they do not indicate whether the small increase in recycling might have any consequences concerning desensitization. So we reasoned that if the increase in recycling were to play a significant role in the protective effect of Src blockers, then interfering with recycling would prevent the protective effect exerted by Src inhibitors. Thus, we determined whether protective action of PP2 could be affected by interfering with recycling. In a first series of experiments, we tested the effect of monensin, a ionophore that blocks receptor recycling by trapping internalized receptors within endosomes [38, 42]. Results showed that at a concentration that reduced DOR recycling (50 μM; Inset Fig. 6A), monensin abolished PP2-mediated protection from desensitization (Fig. 6A). To confirm this observation, cells were transfected with a mutant form of Rab11 (DNM-Rab11) in which substitution of Ser 25 by Asn interferes with GTP-binding capacity of this small G protein and inhibits recycling [27, 43]. Similar to monensin, a decrease in DOR recycling by DNM-Rab11 (Inset Fig. 6B) abolished the protective effect of Src-blockers upon desensitization (Fig. 6B), confirming that recycling is essential for Src modulation of DOR signalling efficacy.

Bottom Line: A first series of experiments demonstrated that agonist binding activates Src and destabilizes a constitutive complex formed by the spontaneous association of DORs with the kinase.Src contribution to DOR desensitization was then established by showing that pre-treatment with Src inhibitor PP2 (20 microM; 1 hr) or transfection of a dominant negative Src mutant preserved DOR signalling following sustained exposure to an agonist.This protection was afforded without interfering with endocytosis, but suboptimal internalization interfered with PP2 ability to preserve DOR signalling, suggesting a post-endocytic site of action for the kinase.

View Article: PubMed Central - PubMed

Affiliation: Département de Pharmacologie, Faculté de Médecine, Université de Montréal, Canada.

ABSTRACT
Abstract An important limitation in the clinical use of opiates is progressive loss of analgesic efficacy over time. Development of analgesic tolerance is tightly linked to receptor desensitization. In the case of delta opioid receptors (DOR), desensitization is especially swift because receptors are rapidly internalized and are poorly recycled to the membrane. In the present study, we investigated whether Src activity contributed to this sorting pattern and to functional desensitization of DORs. A first series of experiments demonstrated that agonist binding activates Src and destabilizes a constitutive complex formed by the spontaneous association of DORs with the kinase. Src contribution to DOR desensitization was then established by showing that pre-treatment with Src inhibitor PP2 (20 microM; 1 hr) or transfection of a dominant negative Src mutant preserved DOR signalling following sustained exposure to an agonist. This protection was afforded without interfering with endocytosis, but suboptimal internalization interfered with PP2 ability to preserve DOR signalling, suggesting a post-endocytic site of action for the kinase. This assumption was confirmed by demonstrating that Src inhibition by PP2 or its silencing by siRNA increased membrane recovery of internalized DORs and was further corroborated by showing that inhibition of recycling by monensin or dominant negative Rab11 (Rab11S25N) abolished the ability of Src blockers to prevent desensitization. Finally, Src inhibitors accelerated recovery of DOR-Galphal3 coupling after desensitization. Taken together, these results indicate that Src dynamically regulates DOR recycling and by doing so contributes to desensitization of these receptors.

Show MeSH
Related in: MedlinePlus