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Abstract
BACKGROUND AND PURPOSE:
Many GPCRs, including the CB(1) cannabinoid receptor, are down-regulated following prolonged agonist exposure by interacting with the GPCR-associated sorting protein-1 (GASP-1). The CB(1) receptor antagonist rimonabant has also recently been described to be an agonist at GPR55, a cannabinoid-related receptor. Here we investigated the post-endocytic properties of GPR55 after agonist exposure and tested whether GASP-1 is involved in this process.
EXPERIMENTAL APPROACH:
We evaluated the direct protein-protein interaction of GPR55 with GASP-1 using (i) GST-binding assays and (ii) co-immunoprecipitation assays in GPR55-HEK293 cells with endogenous GASP-1 expression. We further tested the internalization, recycling and degradation of GPR55 using confocal fluorescence microscopy and biotinylation assays in the presence and absence of GASP-1 (lentiviral small hairpin RNA knockdown of GASP-1) under prolonged agonist [rimonabant (RIM), lysophosphatidylinositol (LPI)] stimulation.
KEY RESULTS:
We showed that the prolonged activation of GPR55 with rimonabant or LPI down-regulates GPR55 via GASP-1. GASP-1 binds to GPR55 in vitro, and this interaction was required for targeting GPR55 for degradation. Disrupting the GPR55-GASP-1 interaction prevented post-endocytic receptor degradation, and thereby allowed receptor recycling.
CONCLUSION AND IMPLICATIONS:
These data implicate GASP-1 as an important regulator of ligand-mediated down-regulation of GPR55. By identifying GASP-1 as a key regulator of the trafficking and, by extension, functional expression of GPR55, we may be one step closer to gaining a better understanding of this receptor in response to cannabinoid drugs.
LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit British Journal of Pharmacology - Volume 165, Issue 8 - Themed Section: Cannabinoids in Biology and Medicine, Part II. Guest Editors: Itai Bab and Steve Alexander - Wiley Online Library. To view Part I of Cannabinoids in Biology and Medicine visit British Journal of Pharmacology - Volume 163, Issue 7 - Cannabinoids in Biology and Medicine, Part I. Guest Editors: Itai Bab and Steve Alexander - Wiley Online Library.
Source: The GPCR-associated sorting protein 1 regulat... [Br J Pharmacol. 2012] - PubMed - NCBI
BACKGROUND AND PURPOSE:
Many GPCRs, including the CB(1) cannabinoid receptor, are down-regulated following prolonged agonist exposure by interacting with the GPCR-associated sorting protein-1 (GASP-1). The CB(1) receptor antagonist rimonabant has also recently been described to be an agonist at GPR55, a cannabinoid-related receptor. Here we investigated the post-endocytic properties of GPR55 after agonist exposure and tested whether GASP-1 is involved in this process.
EXPERIMENTAL APPROACH:
We evaluated the direct protein-protein interaction of GPR55 with GASP-1 using (i) GST-binding assays and (ii) co-immunoprecipitation assays in GPR55-HEK293 cells with endogenous GASP-1 expression. We further tested the internalization, recycling and degradation of GPR55 using confocal fluorescence microscopy and biotinylation assays in the presence and absence of GASP-1 (lentiviral small hairpin RNA knockdown of GASP-1) under prolonged agonist [rimonabant (RIM), lysophosphatidylinositol (LPI)] stimulation.
KEY RESULTS:
We showed that the prolonged activation of GPR55 with rimonabant or LPI down-regulates GPR55 via GASP-1. GASP-1 binds to GPR55 in vitro, and this interaction was required for targeting GPR55 for degradation. Disrupting the GPR55-GASP-1 interaction prevented post-endocytic receptor degradation, and thereby allowed receptor recycling.
CONCLUSION AND IMPLICATIONS:
These data implicate GASP-1 as an important regulator of ligand-mediated down-regulation of GPR55. By identifying GASP-1 as a key regulator of the trafficking and, by extension, functional expression of GPR55, we may be one step closer to gaining a better understanding of this receptor in response to cannabinoid drugs.
LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit British Journal of Pharmacology - Volume 165, Issue 8 - Themed Section: Cannabinoids in Biology and Medicine, Part II. Guest Editors: Itai Bab and Steve Alexander - Wiley Online Library. To view Part I of Cannabinoids in Biology and Medicine visit British Journal of Pharmacology - Volume 163, Issue 7 - Cannabinoids in Biology and Medicine, Part I. Guest Editors: Itai Bab and Steve Alexander - Wiley Online Library.
Source: The GPCR-associated sorting protein 1 regulat... [Br J Pharmacol. 2012] - PubMed - NCBI
