title: The arginine of the DRY motif in transmembrane segment III functions as a balancing micro-switch in the activation of the β2-adrenergic receptor.
abstract: Recent high resolution x-ray structures of the β2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected "ionic lock" interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased G(s) signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of G(s) signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on G(s) signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.
authors: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, the Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.
authors: Louise
authors: L
authors: Valentin-Hansen
authors: Marleen
authors: M
authors: Groenen
authors: Rie
authors: R
authors: Nygaard
authors: Thomas M
authors: TM
authors: Frimurer
authors: Nicholas D
authors: ND
authors: Holliday
authors: Thue W
authors: TW
authors: Schwartz
mesh_terms: D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D019393:Arrestin / Q000737:chemistry; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism; D020868:Gene Silencing; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D019393:Arrestin; D018343:Receptors, Adrenergic, beta-2; D001120:Arginine; D019204:GTP-Binding Proteins; D000409:Alanine