RESUMEN
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.
Asunto(s)
Adrenomedulina/metabolismo , Proteína Similar al Receptor de Calcitonina/metabolismo , Proteína 2 Modificadora de la Actividad de Receptores/metabolismo , Proteína 3 Modificadora de la Actividad de Receptores/metabolismo , Adrenomedulina/genética , Secuencia de Aminoácidos , Proteína Similar al Receptor de Calcitonina/química , Proteína Similar al Receptor de Calcitonina/genética , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Proteína 2 Modificadora de la Actividad de Receptores/química , Proteína 2 Modificadora de la Actividad de Receptores/genética , Proteína 3 Modificadora de la Actividad de Receptores/química , Proteína 3 Modificadora de la Actividad de Receptores/genética , Receptores de Adrenomedulina/química , Receptores de Adrenomedulina/genética , Receptores de Adrenomedulina/metabolismo , Alineación de SecuenciaRESUMEN
The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.