title: Mutations in the 'DRY' motif of the CB1 cannabinoid receptor result in biased receptor variants.
abstract: The role of the highly conserved 'DRY' motif in the signaling of the CB1 cannabinoid receptor (CB1R) was investigated by inducing single-, double-, and triple-alanine mutations into this site of the receptor. We found that the CB1R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric Go proteins (∼80% of WT CB1R (CB1R-WT)). Moreover, this mutant showed an enhanced basal β-arrestin2 (β-arr2) recruitment. More strikingly, the double-mutant CB1R-D3.49A/R3.50A was biased toward β-arrs, as it gained a robustly increased β-arr1 and β-arr2 recruitment ability compared with the WT receptor, while its G-protein activation was decreased. In contrast, the double-mutant CB1R-R3.50A/Y3.51A proved to be G-protein-biased, as it was practically unable to recruit β-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (∼70% of CB1R-WT). Agonist-induced ERK1/2 activation of the CB1R mutants showed a good correlation with their β-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and β-arr binding of the CB1R are mediated by distinct receptor conformations, and the conserved 'DRY' motif plays different roles in the stabilization of these conformations, thus mediating both G-protein- and β-arr-mediated functions of CB1R.
authors.affiliation: Department of PhysiologyFaculty of Medicine, Semmelweis University, PO Box 259, H-1444 Budapest, HungaryMTA-SE Laboratory of Molecular PhysiologyHungarian Academy of Sciences and Semmelweis University, Budapest, Hungary Department of PhysiologyFaculty of Medicine, Semmelweis University, PO Box 259, H-1444 Budapest, HungaryMTA-SE Laboratory of Molecular PhysiologyHungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.
authors.forename: Pál
authors.initials: P
authors.lastname: Gyombolai
authors.affiliation: Department of PhysiologyFaculty of Medicine, Semmelweis University, PO Box 259, H-1444 Budapest, HungaryMTA-SE Laboratory of Molecular PhysiologyHungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.
authors.forename: András D
authors.initials: AD
authors.lastname: Tóth
authors.affiliation: Department of PhysiologyFaculty of Medicine, Semmelweis University, PO Box 259, H-1444 Budapest, HungaryMTA-SE Laboratory of Molecular PhysiologyHungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.
authors.forename: Dániel
authors.initials: D
authors.lastname: Tímár
authors.affiliation: Department of PhysiologyFaculty of Medicine, Semmelweis University, PO Box 259, H-1444 Budapest, HungaryMTA-SE Laboratory of Molecular PhysiologyHungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.
authors.forename: Gábor
authors.initials: G
authors.lastname: Turu
authors.affiliation: Department of PhysiologyFaculty of Medicine, Semmelweis University, PO Box 259, H-1444 Budapest, HungaryMTA-SE Laboratory of Molecular PhysiologyHungarian Academy of Sciences and Semmelweis University, Budapest, Hungary Department of PhysiologyFaculty of Medicine, Semmelweis University, PO Box 259, H-1444 Budapest, HungaryMTA-SE Laboratory of Molecular PhysiologyHungarian Academy of Sciences and Semmelweis University, Budapest, Hungary Hunyady@puskin.sote.hu.
authors.forename: László
authors.initials: L
authors.lastname: Hunyady
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D021381:Protein Transport; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics* / Q000378:metabolism; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; C000605256:Arrb1 protein, rat; C000605257:Arrb2 protein, rat; D019390:Arrestins; D043884:Receptor, Cannabinoid, CB1; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go
keywords: G-proteins; mutations; receptors; signal transduction