The Ionic Lock

In the inactive state of rhodopsin, located on the intracellular side, the "ionic lock," which constrains H6 inside the helical bundle, is comprised of an extended hydrogen-bonded network involving Arg135 (H3), Glu247 (H6), Glu134 (H3), and Thr251 (H6), where Glu134 and Arg135 are the first and second residues of the conserved "D(or E)RY" motif (see Fig. 16.2a-e). The presence of the "ionic lock" is understood to characterize the inactive state and is supported by biophysical data [47, 48]. Interestingly, the antibody-

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Figure 16.2 View of the "ionic lock" region from the IC side into the core in (a) bovine rhodopsin, (b) bovine opsin with Ga-CT, (c) the human p2AR, (d) the human p1AR, and (e) the human A2a adenosine receptor.

Figure 16.2 View of the "ionic lock" region from the IC side into the core in (a) bovine rhodopsin, (b) bovine opsin with Ga-CT, (c) the human p2AR, (d) the human p1AR, and (e) the human A2a adenosine receptor.

complexed and T4L-spliced p2AR X-ray structures lack the Glu(H6)-to-Arg(H3) interaction component of the "ionic lock." One could speculate that the absence of the "ionic lock" in these constructs is a consequence of introducing the companion proteins (antibody, T4L) into the crystal structures. Because of carazolol's comparable affinity as an inverse agonist for both the native protein and the p2AR-T4L construct, one would assume inactive characteristics for the p2AR-T4L construct. Alternatively, the ligand's function as an inverse agonist (vs. antagonist) may induce a protein conformation unlike that of the inactive state. Experiments with fluorescent probes demonstrated [23] that agonists can induce protein conformational changes that are consistent with receptor activation. Moreover, considerable experimental evidence shows that p2AR has multiple conformations that correspond to different degrees of activation [7-11,13].

In the p1AR X-ray structure [28], the "ionic lock" observed in rhodopsin is also not formed. The p1AR and p2AR X-ray structures differ by the presence of a short a-helix, preserved in all four molecules of the unit cell, within ICL2 in p1AR, allowing a hydrogen bond involving Tyr149 on ICL2 and H3's Asp138 of the "D(E)RY" motif. In contrast, in the p2AR structures, this ICL2 secondary structure is not present. By analyzing mutational data, the authors conclude [28] that the p1AR X-ray structure represents the physiologically relevant conformation. The authors also conclude that the inactivity of the complex of p1AR with the antagonist cyanopindolol suggests that there is no evidence of the "ionic lock." In an alternative explanation, one may consider the Asp138-to-Tyr149 hydrogen bond as the cause of full antagonism and the lack of activity of the cyanopindolol/p1AR complex, while the two p2AR complexes with inverse agonists demonstrate residual basal activity.

The conformation of the "ionic lock" region in the A2a adenosine receptor X-ray structure [29] is similar to that of the p1AR X-ray structure, in that an ICL2 a-helix presents Tyr1123 60 for interaction with Asp101 349 in the A2a adenosine receptor X- ray structure. Thus, the ICL2 hydrogen bond can be achieved in a T4L construct. The authors therefore suggest [29] that the residual basal activity in the p2AR X - ray structures reflects the absence of the ICL2 a-helix and the associated Tyr1123 60-to-Asp101349 interaction afforded by an ICL2 a - helix.

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