1. The GPCR‐associated sorting protein 1 regulates ligand‐induced down‐regulation of GPR55
- Author
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Kargl, J, Balenga, NA, Platzer, W, Martini, L, Whistler, JL, and Waldhoer, M
- Subjects
Substance Misuse ,Cannabinoid Research ,Drug Abuse (NIDA only) ,Underpinning research ,1.1 Normal biological development and functioning ,Aetiology ,2.1 Biological and endogenous factors ,Down-Regulation ,Glutathione Transferase ,HEK293 Cells ,Humans ,Ligands ,Lysophospholipids ,Piperidines ,Pyrazoles ,Receptor ,Cannabinoid ,CB1 ,Receptors ,Cannabinoid ,Receptors ,G-Protein-Coupled ,Recombinant Fusion Proteins ,Rimonabant ,Vesicular Transport Proteins ,GPCR ,GASP-1 ,degradation ,GPR55 ,LPI ,rimonabant ,Pharmacology and Pharmaceutical Sciences ,Pharmacology & Pharmacy - Abstract
Background and purposeMany 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 approachWe 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 resultsWe 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 implicationsThese 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 articlesThis article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.
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- 2012