The Impact of Adrenomedullin Thr22 on Selectivity within the Calcitonin Receptor‐like Receptor/Receptor Activity‐Modifying Protein System

Abstract

Adrenomedullin (ADM) is a peptide hormone of the calcitonin gene-related peptide (CGRP) family. It is involved in the regulation of cardiovascular processes such as angiogenesis, vasodilation, and the reduction of oxidative stress. ADM mediates its effects by activation of the ADM-1 and -2 receptors (AM R/AM R), but also activates the CGRP receptor (CGRPR) with reduced potency. It binds to the extracellular domains of the receptors with its C-terminal binding motif (residues 41-52). The activation motif, consisting of a disulfide-bonded ring structure (residues 16-21) and an adjacent helix (residues 22-30), binds to the transmembrane region and stabilizes the receptor conformation in the active state. While it was shown that the binding motif of ADM guides AM R selectivity, there is little information on the activation motif itself. Here, we demonstrate that Thr22 of ADM contributes to the selectivity. By using solid-phase peptide synthesis and cAMP-based signal transduction, we studied the effects of analogues in the activation motif of ADM on AM R and CGRPR activity. Our results indicate that Thr22 terminates the α-helix and orients the ring segment by hydrogen bonding. Using olefin stapling, we showed that the α-helical arrangement of the ring segment leads to decreased AM R activity, but does not affect CGRPR activation. These results demonstrate that the conformation of the ring segment of ADM has a strong impact on the selectivity within the receptor system.

Pharmacological Characterization and Investigation of N-Terminal Loop Amino Acids of Adrenomedullin 2 That Are Important for Receptor Activation

“…AM F18A has reduced E max at the AM 1 receptor while remaining a full agonist at the AM 2 receptor . When AM F18 was compromised through a peptide stapling technique, the resulting peptide again had a reduced E max at the AM 1 receptor but maintained full agonism at the CGRP receptor . Additionally, the equivalent substitution in CGRP (T4A) causes a reduction in cAMP potency but does not affect E max at the CGRP receptor .…”

“…36 When AM F18 was compromised through a peptide stapling technique, the resulting peptide again had a reduced E max at the AM 1 receptor but maintained full agonism at the CGRP receptor. 20 Additionally, the equivalent substitution in CGRP (T4A) causes a reduction in cAMP potency but does not affect E max at the CGRP receptor. 31 Thus, it seems that the residue in this position is important for stimulating cAMP production through the AM 1 receptor but has a weaker impact at other CLR-based receptors.…”

“…AM2-47 was chosen for investigation as this is the longest peptide fragment identified in vivo . AM2-40 was chosen for comparison as this length is the most similar in length to other CGRP/CT family peptides, and there are numerous examples of N-terminally truncated derivatives of AM that have a pharmacology comparable to that of AM (for example, AM 13–52 and AM 15–52 ). − Following this, individual amino acids within the AM2-40 disulfide loop were sequentially replaced with alanine (Figure ). Residues chosen for investigation were within the N-terminal disulfide loop, as work utilizing truncated peptides has shown that the disulfide loop region is critical for the activity of AM2 and the entire peptide family .…”

Targeting Adrenomedullin in Oncology: A Feasible Strategy With Potential as Much More Than an Alternative Anti-Angiogenic Therapy

“…Much effort has been made since then in order to get a molecular comprehension of the interaction of AM with its receptors for the further design and development of more selective and potent antagonists. In point of fact, nowadays it is accepted that while the C-terminal half of the AM binds the complex extracellular domain (ECD) formed by the N-terminals of CLR and RAMPs, the AM N-terminal portion -more precisely, residues 16-30interacts with the CLR 7-transmembrane domain of the receptor, so stabilizing the conformation that will induce cytoplasmic signaling (83,84,182). In this context, Moad, and Pioszak identified the AM (37-52)CONH 2 fragment as the minimal structure required to interact with the binding region of the ECD complex (178).…”

“…Potential epitope domains on AM and AM receptor components are indicated as shaded areas with the respective color of the targeting antibody. et al proved that the conformation of the ring 16-21 in AM is not only determined by the amino acid at position 22 (Thr), but also has a strong influence on the selectivity within the receptor system (182).…”