The arginine of the DRY motif in transmembrane segment III functions as a balancing micro-switch in the activation of the β2-adrenergic receptor
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The arginine of the DRY motif in transmembrane segment III functions as a balancing micro-switch in the activation of the β2-adrenergic receptor. / Hansen, Louise Valentin; Groenen, Marleen; Nygaard, Rie; Frimurer, Thomas M; Holliday, Nicholas D; Schwartz, Thue W.
In: Journal of Biological Chemistry, Vol. 287, No. 38, 14.09.2012, p. 31973-82.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - The arginine of the DRY motif in transmembrane segment III functions as a balancing micro-switch in the activation of the β2-adrenergic receptor
AU - Hansen, Louise Valentin
AU - Groenen, Marleen
AU - Nygaard, Rie
AU - Frimurer, Thomas M
AU - Holliday, Nicholas D
AU - Schwartz, Thue W
PY - 2012/9/14
Y1 - 2012/9/14
N2 - 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.
AB - 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.
KW - Alanine
KW - Amino Acid Motifs
KW - Animals
KW - Arginine
KW - Arrestin
KW - CHO Cells
KW - COS Cells
KW - Cell Membrane
KW - Cercopithecus aethiops
KW - Cricetinae
KW - GTP-Binding Proteins
KW - Gene Silencing
KW - Models, Molecular
KW - Molecular Dynamics Simulation
KW - Mutagenesis, Site-Directed
KW - Protein Binding
KW - Receptors, Adrenergic, beta-2
KW - Structure-Activity Relationship
U2 - 10.1074/jbc.M112.348565
DO - 10.1074/jbc.M112.348565
M3 - Journal article
C2 - 22843684
VL - 287
SP - 31973
EP - 31982
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 38
ER -
ID: 46290242