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.

FIGURE 1.

The DRY motif with interaction partners in rhodopsin/opsin and the B2AR. A, serpentine model of the B2AR. The interaction partners for the ArgIII:26 micro-switch for the inactive structure, AspIII:25 and GluVI:-06, and active structure, TyrVI:24, are marked in red. B, inactive structure of rhodopsin (Protein Data Bank (PDB): 1F88) displaying the ionic lock as viewed from TM-VII. Only key residues are indicated in sticks. C, active structure of opsin (PDB: 3DQB) displaying the breakage of the ionic lock and the interaction of the TyrV:24 residue to the ArgIII:26 and again from the ArgIII.26 to the Gα-peptide. D, inactive structure of the B2AR (PDB: 2RH1). E, active structure of B2AR with G_s protein (PDB: 3SN6).

FIGURE 2.

Molecular dynamics simulation studies of the salt bridge between ArgIII:26 of the DRY motif at the intracellular end of TM-III and GluVI:-06 at the intracellular extension of TM-VI in B2AR. A, The structure of the intracellular ends of TM-III, -V, and -VI in a molecular model of the B2AR in which T4-lysozyme was removed from the T4L-B2AR structure (2RH1) and the two ends joined. The structure shown in green is the starting structure, and structures after 1- and 8-ns simulation are also shown; in all structures, ArgIII:26, AspIII:25, TyrV:24, and GluVI:-06 are shown as sticks. Note that initially (0–1 ns), the backbone around GluVI:-06, which was in a loop-like structure, swings into a regular α-helical extension of TM-VI, and subsequently (1–8 ns), the side chain of GluVI:-06 swings over toward ArgIII:26 to form the salt bridge. B, the distance between the Cα of ArgIII:26 and GluVI:-06 as a function of simulation time. The horizontal red line indicates the distance between Cα of ArgIII:26 and GluVI:-06 in the rhodopsin structure (1GZM). C, the distance between the carbon atom in the guanidine and carboxylic acid groups of ArgIII:26 and GluVI:-06, respectively, during the molecular dynamics simulation. The horizontal red line indicates the distance between the carbon atom in the guanidine and carboxylic acid groups of ArgIII:26 and GluVI:-0g in the rhodopsin structure (1GZM).

FIGURE 3.

Functional consequence on G_s signaling in the B2AR of Ala substitutions of the ArgIII:26/3.50 micro-switch and its potential interaction partners. A–D, basal and agonist (pindolol)-induced cAMP production in COS-7 cells transiently transfected with either WT β2-AR (dotted line) or mutant forms of the following: AspIII:25Ala (A), GluVI:-06Ala (B), TyrV:24Ala (C), and ArgIII:26Ala (D). % of max., percentage of maximum. E and F, isoproterenol-induced cAMP production in CHO-K1 cells transiently transfected with either WT (dotted line) or mutant forms of the β2-AR: TyrV:24Ala (E) and ArgIII:26Ala (F). Cell surface receptor expression, measured by enzyme-linked immunosorbent assay, is shown in the inserted column diagrams in each panel. Error bars indicate S.E.

FIGURE 4.

Effect on G_s signaling of combining loss-of-function and gain-of-function mutants of the proposed locks for the ArgIII:26 micro-switch in B2AR. Basal and agonist (pindolol)-induced cAMP production in COS-7 cells transiently transfected with either WT (solid line) or mutant forms of B2AR is shown. A, AspIII:25Ala (dotted line), TyrV:24Ala (dotted line), and the double mutant AspIII:25Ala + TyrV:24Ala as compared with WT (solid line). % of max., percentage of maximum. B, GluVI:-06Ala (dotted line), TyrV:24Ala (dotted line), and GluVI:-06Ala+TyrV:24Ala as compared with WT (solid line). Cell surface receptor expression of the double mutants, measured by enzyme-linked immunosorbent assay, is shown in the inserted column diagrams in each panel. Error bars indicate S.D.

FIGURE 5.

Functional consequence on β-arrestin2 mobilization measured by BRET of Ala substitution of the ArgIII:26/3.50 micro-switch and potential interaction partners. A–D, agonist (isoproterenol)-induced BRET production in COS-7 cells transiently transfected using a 1:3 ratio of either WT B2AR (dotted line)/mutant forms of the receptor and β-arrestin2. The panels show AspIII:25Ala (A), GluVI:-06Ala (B), TyrV:24Ala (C), and ArgIII:26Ala (D). Error bars indicate S.D.

FIGURE 6.

Functional consequence on isoproterenol-induced internalization of Ala substitution of the ArgIII:26/3.50 micro-switch and potential interaction partners. A–D, agonist (isoproterenol)-induced internalization in HEK293 cells stably expressing either N-terminal SNAP-tagged WT B2AR (dotted line) or mutant forms of the receptor. The panels show AspIII:25Ala (A), GluVI:-06Ala (B), TyrV:24Ala (C), and ArgIII:26Ala (D). Error bars indicate S.D.