# Receptor binding and signaling of the cardioprotective peptide Adrenomedullin 2/Intermedin.

> **NIH NIH F30** · UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR · 2020 · $47,612

## Abstract

PROJECT SUMMARY ABSTRACT
 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 Å 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...

## Key facts

- **NIH application ID:** 10020190
- **Project number:** 5F30HL142232-02
- **Recipient organization:** UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
- **Principal Investigator:** Amanda Roehrkasse
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $47,612
- **Award type:** 5
- **Project period:** 2019-08-12 → 2021-08-11

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10020190

## Citation

> US National Institutes of Health, RePORTER application 10020190, Receptor binding and signaling of the cardioprotective peptide Adrenomedullin 2/Intermedin. (5F30HL142232-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10020190. Licensed CC0.

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