Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

The model presented here shows a low-resolution docking of AngII and G protein heterotrimer to the AT₁ receptor. In it, the backbone of the receptor extramembrane sequences was drawn so that all their residues involved in binding by mutagenesis and other studies could be set at proper distances from the agonist and G protein structures. Nevertheless, basic structural motifs such as the two external disulphide bridges, were built rigorously according to the atomic distances and dihedral angles which have most often been found for these structures.

Regarding AngII docking, the validity of the model is supported by the fact that an active peptide conformation could be flanked by external loops of the receptor, allowing simultaneous matches between two pairs of structures: peptide C-carboxylate with receptor Lys199(561) side-chain; peptide Arg2 side-chain with receptor Asp278(709) and Asp281(712) side-chains(Figs. 3 and 4).

The structure of the AT₁ receptor is a prototype for a two-site model for peptide agonist binding to GPCRs. In fact, the model depicted in Figs. 2-4, suggests that the C-terminal and N-terminal ends of AngII can, directly and indirectly, contact two different locuses in receptor's the central cavity. The peptide's C-terminal portion contacts the receptor side-chains located at the external region of the retinal-like locus (Fig. 4). The peptide's N-terminal portion binds to a site formed by the receptor's external regions Z1, Z2, Z3 and Z7 (Fig. 1) and is seen to overlap the central cavity surrounded by helices I-II and VI-VII. Other structures such as the insertion Ins in loop VI-VII and the disulphide bridge between AT₁'s N-terminal extension and loop VI-VII, are found in other receptors (bradykinin, endothelin, chemokines and europeptides) and appear in the model not to be flanking the second site but to form an external and possibly special recognition site for these receptors.

One characteristic of large-ligand GPCRs, such as those for peptides and glycoprotein hormones, is that these receptors have to overcome two steps in order to be activated following agonist binding [44]: in a first step, external regions of the receptor bind part of the ligand to produce specific complexes; in a second step, the other part of the ligand seems to contact the retinal-like locus of the receptor to trigger the signaling mechanism.

A two-step mechanism has been proposed to accomodate experimental results concerning the acute desensitization (tachyphylaxis) of the AT₁ receptor in in intestinal smooth muscle [45]. In a first step, the C-terminal end of the peptide (specially the Phe8 aromatic ring) interacts with the receptor initiating the signal for the physiological response. This interaction probably involves the external portion of helix VI [15] similarly to the mechanisms proposed for dopamine and serotonin [46], and rhodopsin [47]. In a second step, the N-terminal portion of AngII (particularly the N-terminal ammonium and Arg2 guanidinium groups) would bind to a "tachyphylactic site" in the receptor (such as that in Fig. 3)leading to a high-affinity non-productive AngII-receptor complex which dissociates only slowly, thus blocking the action of newly added agonist.

The receptor-G protein complex model in Fig. 5 is valid for all rhodopsin-like GPCRs, since it is an attempt to explain the promiscuity in the coupling and therefore was built considering only the interactions between fully conserved structures. Nevertheless, two different contact points between receptor structures and G_alpha chain should be emphasized. The first point [left side in Fig. 5) involves the end of helix III (DRY motif) and the beginning of loop III-IV in the receptor, and the middle of alpha5-helix in the Galpha chain, which can be supposed to drive a signal towards GDP release. The second contact (right side in Fig. 5), supposedly between the C-terminus of receptor and the N-terminal alpha-helix in the G_alpha chain, might drive a process leading to stabilisation of a high-affinity receptor state, which in the model is suggested to be due to the fully conserved Tyr302(734) replacing Arg126(340) in its interaction with Asp74(224) and Asp125(339) in the receptor (Fig. 5).

Acknowledgements

We are grateful to Dr. Gert Vriend, EMBL, Heidelberg, for helpful discussions of the model and to Dr. Gregory Nikiforovich, for the coordinates of the AngII model

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