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Updated: Jun 27, 2025
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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
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Variable CGRP family peptide signaling durations and the structural determinants thereof
Katie M Babin1, Sandra E Gostynska1, Jordan A Karim1
1Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, United States.
Biochemical Pharmacology
|April 26, 2024
Summary
New research reveals distinct signaling durations for CGRP family peptides. Adrenomedullin 2/intermedin (AM2/IMD) fragments show prolonged cAMP signaling, offering new tools for studying these pathways.
Area of Science:
- Molecular Endocrinology
- GPCR Signaling
- Peptide Therapeutics
Background:
- Calcitonin gene-related peptides (CGRP) alpha and beta, adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) are crucial in pain, neuroimmune, and cardiovascular signaling.
- These peptides activate class B GPCRs (CLR, CTR) modulated by RAMP subunits, influencing cellular responses.
- Previous work indicated AM2/IMD(1-47) elicits long-duration cAMP signaling via CLR-RAMP3.
Purpose of the Study:
- To characterize the signaling kinetics of CGRP peptides and AM/AM2/IMD fragments using a live-cell cAMP biosensor.
- To investigate the structural basis for prolonged cAMP signaling duration mediated by AM2/IMD peptides.
- To compare the signaling duration of alpha-CGRP and beta-CGRP at different receptor complexes.
Main Methods:
- Live-cell cAMP biosensor assay to measure real-time cAMP signaling kinetics.
- Utilized various AM and AM2/IMD fragments with different N-terminal extensions and point mutants.
- Constructed AM2/IMD-AM mid-region chimeras and analyzed chimeric CGRP peptides.
Main Results:
- AM2/IMD(8-47) and AM2/IMD-53 demonstrated significantly longer cAMP signaling duration than AM2/IMD(1-47) at CLR-RAMP3.
- AM2/IMD(8-47) exhibited 8-fold longer activity than AM(13-52) at CLR-RAMP3; N-terminal extensions of AM did not affect duration.
- Beta-CGRP showed ~3-fold longer signaling duration than alpha-CGRP at CLR-RAMP1 and CTR-RAMP1 receptors, with specific residue differences contributing.
Conclusions:
- Identified key structural determinants in AM2/IMD peptides responsible for long-duration cAMP signaling.
- Discovered AM2/IMD peptides with significantly diminished signaling duration while maintaining potency, serving as potential pharmacological tools.
- Uncovered critical temporal differences in cAMP signaling among CGRP family peptides and their structural underpinnings.
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