Ligand interaction with family A receptors

Rhodopsin is unique among the GPCRs in that its ligand is covalently attached to the receptor within a binding crevice formed by the transmembrane helices (Figure 93). This may be necessary to facilitate the very rapid response of rhodopsin to light.

The binding sites for the classical 'small-molecule' transmitters (biogenic amines like epinephrine, norepinephrine, dopamine, serotonin, histamine and acetylcholine)

Figure 92 Commonly performed receptor mutations to investigate ligand binding.

are located in the central pocket of their GPCRs (Figure 93). Hence, these messengers interact with amino acid residues belonging to some of the membrane-spanning domains (especially TM3, TM5, TM6 and TM7). For example, mutation studies with the p2-adrenergic receptor indicate that at least two of its membrane-spanning

Figure 93 Ligands are able to interact with extracellular and transmembrane domains of family A GPCRs. Reprinted from Biochimica Biophysica Acta, 1422, Flower, D. R., Modelling G proteincoupled receptors for drug design, 207-234. Copyright (1999), with permission from Elsevier.

Isoproterenol (agonist)

Figure 94 Ligand interactions with amino acid side chains of the p2-adrenergic receptor. Reprinted from Biochimica Biophysica Acta, 1422, Flower, D. R., Modelling G protein-coupled receptors for drug design, 207-234. Copyright (1999), with permission from Elsevier.

Isoproterenol (agonist)

Figure 94 Ligand interactions with amino acid side chains of the p2-adrenergic receptor. Reprinted from Biochimica Biophysica Acta, 1422, Flower, D. R., Modelling G protein-coupled receptors for drug design, 207-234. Copyright (1999), with permission from Elsevier.

domains (i.e. TM3 and TM5) are involved in the binding of agonists like adrenaline and isoproterenol (Figure 94 and Figure 95). The hydroxyls on the aromatic ring interact with Ser204 and Ser207 in TM5 and the secondary amine interacts with Asp113 in TM3. Phe289 and Phe290 on TM6 probably make n-stacking interactions with the catechol ring. These results provide information about the proximity of TM3 and TM5 in the messenger/agonist-bound receptor.

The binding site for a representative antagonist, alprenolol, overlaps with that of isoproterenol, but this overlap is only partial and the nature of receptor-ligand interaction is different also (Figure 94). It is of interest that Asp113 in TM3 of the P2-adrenergic receptor is conserved among the biogenic amine receptors (Asp3.32 according to the Ballesteros-Weinstein numbering) and is also thought to interact with the positively charged head group of the monoamines and related antagonists.

On the other hand, because of the relatively large size of peptide messengers, the binding site for these molecules is more likely to comprise extracellular domains

Figure 95 Molecular models of the adrenaline-p2-adrenergic receptor complex (top view) (Ostrowski et al., 1992, reproduced by permission of Annual Reviews).

Figure 95 Molecular models of the adrenaline-p2-adrenergic receptor complex (top view) (Ostrowski et al., 1992, reproduced by permission of Annual Reviews).

Adrenergic Receptors

Figure 96 Interaction between angiotensin II and the ATj receptor (Feng et al., 1995, reproduced by permission of The American Society for Biochemistry).

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Figure 96 Interaction between angiotensin II and the ATj receptor (Feng et al., 1995, reproduced by permission of The American Society for Biochemistry).

(i.e. the N-terminal part and the three extracellular loops) of GPCRs (Figure 93). As an example, mutation studies revealed that certain amino acids belonging to extracellular domains of the ATX receptor are crucial for the recognition of angiotensin II. A model for the binding of this peptide to the AT receptor is given in Figure 96. In this model, His183 and Asp281, both located in the extracellular domain of the AT receptor, are involved in binding the N-terminal Asp1 and Arg2 residues from angiotensin II, respectively. In addition, Lys199 in the TM7 of the receptor binds the C-terminal car-boxyl group of angiotensin II.

Some of the peptides may also have additional points of interaction in the TM domains and therefore, they may enter the central cleft to different degrees. Moreover, peptide receptors also recognize some small synthetic molecules. They usually behave as antagonists, but some of them also behave as agonists nothwithstanding the fact that their binding pocket may be topographically distinct from the peptide binding site. Mutation studies reveal that (similar to the binding of biogenic amines to their receptors) these small molecules rather bind within the central cleft.

For example, non-peptide antagonists of the NK-1 receptor (prototype: CP 96345) bind to residues clustering in a crevice formed by TM3 to TM6 and mutation of these residues does not affect peptide agonist binding (Figure 97). Hence, this binding pocket is most likely not occupied by substance P. Accordingly, an actual overlap in the binding sites is not required for a competitive mode of action of the non-peptide antagonists.

The protease-activated thrombin receptors also belong to family A GPCRs (Figure 98). The unique activation mechanism of the thrombin receptor involves cleavage of the N-terminal segment by thrombin. The resulting 33-amino acid N-terminus subsequently acts as tethered peptide ligand, which, through interactions with the extracellular loop regions of the receptor, is able to activate the receptor. In this vein, relatively short synthetic peptides (5-14 amino acid residues) based on the sequence of the unmasked N-terminal receptor sequence activate the receptor in the same way as thrombin.

Figure 97 Snake diagram and (left) and wheel diagram (right) of the NK-1 receptor: Yellow: the most conserved residue in each helix, green: residues involved in substance P (= messenger) binding, red: residues involved in small-molecule antagonist binding. Reprinted from Endocrine Review, 21, Gether, U., Uncovering molecular mechanisms involved in activation of G proteincoupled receptors, 90-113. Copyright 2000, The Endocrine Society.

Figure 97 Snake diagram and (left) and wheel diagram (right) of the NK-1 receptor: Yellow: the most conserved residue in each helix, green: residues involved in substance P (= messenger) binding, red: residues involved in small-molecule antagonist binding. Reprinted from Endocrine Review, 21, Gether, U., Uncovering molecular mechanisms involved in activation of G proteincoupled receptors, 90-113. Copyright 2000, The Endocrine Society.

Figure 98 Thrombin clieves the N-terminal part of its receptor. The newly formed terminus behaves as a 'tethered' agonist to activate the receptor.
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