title: The GPCR-associated sorting protein 1 regulates ligand-induced down-regulation of GPR55.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
  
  
  BACKGROUND
  This 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.
authors: Institute for Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria. julia.kargl@medunigraz.at
authors: J
authors: J
authors: Kargl
authors: N A
authors: NA
authors: Balenga
authors: W
authors: W
authors: Platzer
authors: L
authors: L
authors: Martini
authors: J L
authors: JL
authors: Whistler
authors: M
authors: M
authors: Waldhoer
mesh_terms: D015536:Down-Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008246:Lysophospholipids / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D043884:Receptor, Cannabinoid, CB1 / Q000037:antagonists & inhibitors; D043882:Receptors, Cannabinoid; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000077285:Rimonabant; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: C524885:GPR55 protein, human; C463459:GPRASP1 protein, human; D008024:Ligands; D008246:Lysophospholipids; D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D043882:Receptors, Cannabinoid; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; C025449:lysophosphatidylinositol; D005982:Glutathione Transferase; D000077285:Rimonabant