Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties

Abstract

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

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

“…As it was previouslyr eported that alanine exchanges of Val23, Leu26 and Ile30 led to significant losses in AM 1 R activation, [21] we expected (S)-2-(4-pentenyl)alanine to be generally tolerated due to the hydrophobic character,w hich poses ah igh resemblancet ot he originala mino acids. On the other hand, we also wanted to study whether an a-helicalc onformation of the ring segment does in fact represent an active conformation at the CGRPR, but not at the AM 1 R. Thus, [G 14 ,( S5F 18 , S5S 22 ) meta ]ADM (12)w as synthesized using the methodr eported by Wu et al recently. [22] While the originalphenylalanine side chain in position 18 was conserved, the threonines ide chain had to be replaced with serine, because ap entenylmodifiedt hreonineb uilding block is synthetically not available at the moment.…”

“…Experiments with RAMP2/ RAMP3 knockout-mice elucidate some of the effects, but not all. [12] Unfortunately,s uch compounds are not availables o far and there are only af ew publicationso nt he selectivity Adrenomedullin (ADM)i sapeptide hormone of the calcitonin gene-related peptide (CGRP)f amily.I ti si nvolved in the regulation of cardiovascular processes such as angiogenesis, vasodilation, and the reduction of oxidative stress. Subtype-selective analogues of ADM would be powerful tools, allowing am ore comprehensive study of the receptorp harmacology andr aising the opportunityf or the development of potent new drugs.…”

“…Subtype-selective analogues of ADM would be powerful tools, allowing am ore comprehensive study of the receptorp harmacology andr aising the opportunityf or the development of potent new drugs. [12] Unfortunately,s uch compounds are not availables o far and there are only af ew publicationso nt he selectivity Adrenomedullin (ADM)i sapeptide hormone of the calcitonin gene-related peptide (CGRP)f amily.I ti si nvolved 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 1 R/ AM 2 R), but also activates the CGRP receptor (CGRPR)w ith reduced potency.I tb inds to the extracellulard omains of the receptors with its C-terminal binding motif (residues 41-52).…”

Update on the pharmacology of calcitonin/CGRP family of peptides: IUPHAR Review 25

“…It is possible that SB‐273779 binds in a similar place. However, mutagenesis suggests that there are RAMP effects on ECL3 and so it may be possible to develop selective antagonists, which bind to this region (Watkins et al, ).…”

Adrenomedullin – new perspectives of a potent peptide hormone

“…In these models, the residues within the disulfide-bonded loop structure of the receptor-bound ADM point into differently constrained binding pockets in both receptors. Variations in the ring structure could contribute to ligand selectivity between the two ADM receptors, because the substitution of Phe 18 by Ala in ADM decreased the cAMP signaling at the AM 1 receptor to a higher extent compared with the AM 2 receptor [83]. In contrast to this very detailed view on the determinants of receptor binding affinity and selectivity within the CLR and RAMP2 ECD, much less is known about the processes in the transmembrane part of CLR after binding of ADM.…”