Identification of an Efficacy Switch Region in the Ghrelin Receptor Responsible for Interchange between Agonism and Inverse Agonism

Abstract

The carboxyamidated wFwLL peptide was used as a core ligand to probe the structural basis for agonism versus inverse agonism in the constitutively active ghrelin receptor. In the ligand, an efficacy switch could be built at the N terminus, as exemplified by AwFwLL, which functioned as a high potency agonist, whereas KwFwLL was an equally high potency inverse agonist. The wFw-containing peptides, agonists as well as inverse agonists, were affected by receptor mutations covering the whole main ligand-binding pocket with key interaction sites being an aromatic cluster in transmembrane (TM)-VI and -VII and residues on the opposing face of TM-III. Gain-of-function in respect of either increased agonist or inverse agonist potency or swap between high potency versions of these properties was obtained by substitutions at a number of positions covering a broad area of the binding pocket on TM-III, -IV, and -V. However, in particular, space-generating substitutions at position III:04 shifted the efficacy of the ligands from inverse agonism toward agonism, whereas similar substitutions at position III: 08, one helical turn below, shifted the efficacy from agonism toward inverse agonism. It is suggested that the relative position of the ligand in the binding pocket between this "efficacy shift region" on TM-III and the opposing aromatic cluster on TM-VI and TM-VII leads either to agonism, i.e. in a superficial binding mode, or it leads to inverse agonism, i.e. in a more profound binding mode. This relationship between different binding modes and opposite efficacy is in accordance with the Global Toggle Switch model for 7TM receptor activation. 7TM3 receptors (G-protein-coupled receptors) constitute one of the largest superfamilies of proteins, which also serve as targets for a large proportion of current medical drugs. In particular members of the family A or rhodopsin-like 7TM receptors, which also is the largest family, are considered to be rather easy drug targets. Most of these receptors are antagonist-prone, i.e. if they are screened with libraries of small organic, drug-like molecules, most if not all of the hits will be antagonists, inhibiting agonist-induced signal transduction, when they are tested in functional assays (1). However, a small proportion of the 7TM receptors, including, for example, the complement C5a receptor, the melanocortin MC4 receptor, and the ghrelin receptor, are instead agonist-prone, i.e. most of the screening hits are agonists in functional assays (2). Part of the reason for this is probably that these receptors are characterized by a rather high degree of constitutive, ligand-independent signaling activity, i.e. in the conformational equilibrium these receptors are at least partly biased for active conformation(s) (1). The ghrelin receptor (Fig. 1), for example, is among the most constitutively active receptors as it signals with ϳ50%, depending on the signal transduction pathway, of its maximal signaling capacity without the presence of any hormone (3, 4). High constitutive activity has b...

Unique Interaction Pattern for a Functionally Biased Ghrelin Receptor Agonist

“…As shown previously for the wFwLL pentapeptide, ϳ65% of the low energy structures of the wFw part of the molecule populated only two almost equally large clusters, which is very unusual for such a small peptide (22). Importantly, in contrast to the highly flexible Leu-Leu sequence of the original pentapeptide, the piperidine spacer of wFw-Isn-NH 2 could adopt only a few conformations as determined by its simple ring structure (Fig.…”

“…Conformational Analysis-Molecular dynamics simulations were performed by CHARM using the CHARM22 force field as described previously (22). Briefly, the system was minimized, heated to 310 K followed by equilibration, and simulated using Langevin dynamics for 0.1 s. The molecular dynamic trajectory was analyzed, and structures of wFw-Isn-NH 2 were clustered based on the backbone, phi and psi; and the side chain 1 dihedral angle.…”

“…Conformational analysis revealed that the N-terminal wFw peptide favored two closely related, constrained, L-shaped, low energy conformations populated with 34 and 30% of the population, respectively (22). In contrast, the two C-terminal leucine residues were highly flexible (22), and in the structure-activity relation analysis performed on the full-length [D-Arg 1 ,D-Phe 5 ,DTrp 7,9 ,Leu 11 ] substance P inverse agonist, the Leu-Leu dipeptide appeared to function mainly as a linker between the D-Trp-Phe-D-Trp motif and the C-terminal functionally important carboxyamide moiety (21). Accordingly, in the present study we replaced the C-terminal Leu-Leu dipeptide of the wFwLL pentapeptide with various non-natural amino acid linkers or spacers of variable length and flexibility (Table 1).…”

“…By the addition of a single amino acid at its N terminus this pentapeptide could, depending on the type of N-terminal residue, be converted into either a pure agonist or a pure inverse agonist (21,22). Importantly, the characteristic essential D-Trp-Phe-D-Trp (wFw) motif strongly restricts the conformational freedom of this type of peptide ligand because essentially the wFw motif is found in only two different dominating conformations (22). The structure-activity relationship analysis indicated that the more flexible C-terminal Leu-Leu dipeptide functions mainly as a spacer or linker between the wFw core and the functionally highly important C-terminal carboxyamide moiety.…”

PheVI:09 (Phe6.44) as a Sliding Microswitch in Seven-transmembrane (7TM) G Protein-coupled Receptor Activation

“…In this respect, the ghrelin receptor is particularly suited because even in the absence of its endogenous agonist, it displays almost 50% of its maximal signaling capacity, and, importantly, the ghrelin receptor is generally robust with respect to mutational changes (20,21).…”