The vasoactive peptide adrenomedullin 2/intermedin (AM2/IMD) has important actions in human physiology and disease such as vasodilation, physiologic and pathologic angiogenesis as well as potent protective effects in the cardiovascular and renal systems. Actions of AM2/IMD have been attributed to activation of several signaling intermediates including cAMP and Ca2+ downstream of its G protein-coupled receptor, the calcitonin receptor-like receptor (CLR). Unfortunately, there is little mechanistic insight into how AM2/IMD binds and activates CLR. CLR pharmacology is complicated because it heterodimerizes with any one of three receptor activity-modifying proteins (RAMP1, -2, or -3) that modulate its response to AM2/IMD and the related vasoactive peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). How AM2/IMD, CGRP, and AM act through shared RAMP:CLR receptor complexes to promote their unique signaling outcomes remains unclear. This limits our understanding of how AM2/IMD elicits its broad range of actions in human physiology and hinders our ability to exploit AM2/IMD signaling for drug development. I will test the hypothesis that AM2/IMD adopts a unique receptor-bound conformation and that it promotes a pattern of biased G protein activation at RAMP:CLR complexes that is distinct from those of AM and CGRP. I will test this using rigorous biochemical, pharmacological, and structural methods in two aims: 1) Define the molecular basis for AM2/IMD recognition by soluble RAMP:CLR extracellular domain (ECD) complexes, and 2) Define the G protein-coupling preferences of each full-length receptor complex promoted by AM2/IMD as compared to CGRP and AM.
For Aim 1 I purified each of the three ECD complexes as tethered RAMP ECD-CLR ECD fusion constructs and found that AM2/IMD exhibited binding preferences that were distinct from those of CGRP and AM. I solved a 2.05 resolution crystal structure that demonstrated a strikingly unique triple b-turn structure of AM2/IMD bound to the RAMP1-CLR ECD. I will determine an AM2/IMD-bound crystal structure of the RAMP3-CLR ECD to fully understand how AM2/IMD binds the different receptor ECDs, and provide crucial insights into how RAMP3 modulates CLR.
For Aim 2 we determined conditions to co-express and solubilize the three full-length RAMP:CLR complexes, which form detergent-stable ligand-free complexes. This provides a unique opportunity to study how the three peptides promote coupling of different G-proteins. We will use a native-PAGE method to determine coupling preferences to unpurified receptor complexes and we will purify the ligand-free complexes to study G-protein coupling using a fluorescence anisotropy assay. These biochemical studies will be correlated with pharmacological studies of cAMP and Ca2+ signaling bias in human cell lines that express the RAMP1:CLR (SK-N-MC) or RAMP2:CLR (HUVEC). Successful completion of these aims will provide crucial insights into AM2/IMD function that will enable AM2/IMD-based drug development.!
Adrenomedullin2/intermedin (AM2/IMD) has demonstrated therapeutic potential with protective actions in the cardiovascular system mediated by a G protein-coupled receptor (GPCR) with complex pharmacology due to association with RAMP accessory proteins. Our limited understanding of how these accessory proteins alter how AM2/IMD interacts with its receptor limits our ability to target AM2/IMD and its receptors for drug development. I propose a thorough investigation of how AM2/IMD binds and activates three RAMP:GPCR complexes using powerful structural and biochemical methods along with pharmacological approaches in human cell lines that will broaden our understanding of how RAMPs modulate GPCR pharmacology, inform drug design, and further our understanding of the unique role of AM2/IMD in human cardiovascular physiology. !
